Patent Application
20230167058
The present disclosure provides, inter alia, compounds to modulate GPX4 activity. Also provided are pharmaceutical compositions containing same compounds, as well as methods of using such compounds and compositions.
This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing XML file “CU21144-seq26.xml”, file size of 22 KB, created on Sep. 11, 2022. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).
Cancer cells are dependent on their lipid composition for establishing and modulating membrane structural integrity, morphology, metabolism, migration, invasiveness, and other functions. For example, among the thousands of lipid species that compose eukaryotic cell membranes, the abundance and localization of polyunsaturated-fatty-acid-(PUFAs)-containing phospholipids (PUFA-PLs) is a major factor in determining the fluidity of cell membranes (Agmon et al. 2017). Since the cis conformation of double bonds in PUFA-PLs hinders efficient stacking of fatty acyl tails, elevated levels of PUFA-PLs contribute to increasing membrane fluidity and thinning (Agmon et al. 2018). PUFA-PLs are, however, susceptible to peroxidation via iron-catalyzed reaction with molecular oxygen at bis-allylic positions, catalyzed by lipoxygenases and labile iron (Yang et al. 2016). Thus, some cancer cells depend on a critical network of proteins to eliminate their PUFA-PL peroxides; a key protein at the center of this defense network is the selenoprotein glutathione peroxidase 4 (GPX4). When GPX4 activity is compromised, lipid peroxidation can cause ferroptosis (Stockwell et al. 2017), an oxidative, iron-dependent form of non-apoptotic cell death (Dixon et al. 2012). Ferroptosis acts as a natural tumor suppressive and immune surveillance mechanism, and can be induced by exogenous agents in cells that are addicted to GPX4 (Dixon and Stockwell, 2019). Cancer cells from tissues of diverse origins have been screened for their sensitivity to ferroptosis-inducing compounds (Viswanathan et al. 2017). It has been found that ferroptosis inducers, including GPX4 inhibitors, selectively target cancers with a mesenchymal or otherwise drug-resistant signature (Viswanathan et al. 2017). Consistent with the mesenchymal state being associated with drug resistance, an independent study on persister cancer cells, which are proposed to escape from conventional cytotoxic treatment through a dormant state and then revive to cause tumor relapse, revealed a similar selective dependency on GPX4 (Hangauer et al. 2017).
Examination of persister cells also revealed upregulation of mesenchymal markers and downregulation of epithelial markers (Hangauer et al. 2017). Overexpression of mesenchymal state genes is associated with epithelial-mesenchymal transition (EMT). Since EMT increases motility of tumor cells and enables the invasion of primary tumors to distant sites, EMT is a key step in metastasis. EMT also renders cancer cells resistant to apoptosis and chemotherapy (Viswanathan et al. 2017). EMT requires plasma membrane remodeling to increase fluidity, which is associated with elevated biosynthesis of PUFA-PLs. Given that PUFA-PLs are more susceptible to peroxidation than saturated or mono-unsaturated fatty acid PLs, cells in an EMT state have increased dependency on GPX4 to remove these lipid peroxides (Viswanathan et al. 2017). Therefore, cancer cells undergoing EMT that acquire resistance to apoptosis become vulnerable to lipid peroxidation and ferroptosis induced by GPX4 inhibition (Viswanathan et al. 2017). As cancer cells evolve into a high-mesenchymal drug-resistant state and become resistant to apoptosis, one may selectively target such cells through ferroptosis; the most effective compounds in this context are GPX4 inhibitors (Viswanathan et al. 2017). For example, in-vivo xenografts of GPX4-knockout high-mesenchymal therapy-resistant melanoma regressed after cessation of ferrostatin-1 (a lipophilic antioxidant discovered in the Stockwell Lab that suppresses the loss of GPX4) and did not relapse after ceasing dabrafenib and trametinib treatment, while wt GPX4 xenografts continued to grow in both experiments (Viswanathan et al. 2017). GPX4 inhibitors are selectively lethal to persister and EMT cancer cells, with minimal effects on parental cells and non-transformed cells, suggesting that addiction to GPX4 creates a large therapeutic window.
Accordingly, there is a need for developing GPX4 inhibitors for the treatment of aggressive drug-resistance cancers and other GPX4-associated diseases. This disclosure is directed to meeting these and other needs.
One of the most pressing problems in oncology is metastatic, drug-resistant cancers. Indeed, most deaths of cancer patients are caused by aggressive, metastatic, drug-resistant cancers. A surprising finding is that as cancers evolve into aggressive and drug-resistant forms, they acquire an exquisite sensitivity to GPX4 inhibition. These data provide the tantalizing possibility that the most aggressive neoplastic diseases can be treated through the use of GPX4 inhibitors, and that the ideal patients for treatment with these inhibitors are end-stage patients that have exhausted other therapeutic options. In 2012, we reported the existence of a new form of tumor suppressive cell death, ferroptosis (Dixon et al. 2012). In 2014, we discovered that the key negative regulator of ferroptosis was the lipid repair enzyme GPX4, demonstrating that GPX4 functions in ferroptosis in manner analogous to how the oncogene Bcl-2 functions in apoptosis (Yang et al. 2014). We discovered the first GPX4 inhibitor—the nanomolar potency small molecule RSL3 (Yang et al. 2014). In 2017, we reported that cancer cells that have undergone epithelial-to-mesenchymal (EMT) transition become hypersensitive to ferroptosis, and to GPX4 inhibitors (Viswanathan et al. 2017). We also discovered how RSL3 inhibits GPX4, obtaining a co-crystal structure of RSL3 bound to GPX4, which revealed a novel drug-binding site on GPX4. We propose, inter alia, to exploit this finding to discover drug-like GPX4 inhibitors with favorable ADMET properties that can be developed as a first in class GPX4 inhibitors for drug-resistant cancers having a high EMT gene expression signature.
Accordingly, one embodiment of the present disclosure is a compound according to formula (1):
wherein:
R1, R2, and R3 are independently selected from the group consisting of H, D, O, N, halo, ether, ester, amide, C(O), (O)C(R), C(O)O, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl, wherein the ether, ester, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
wherein R is selected from the group consisting of H, D, O, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof, with the proviso that the compound is not
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a compound according to formula (2):
wherein:
a dashed line indicates the presence of an optional double bond;
X and Y are independently selected from the group consisting of C, N, S and O;
R1, R2, R3, and R4 are independently selected from the group consisting of no atom, H, D, O, N, halo, ether, ester, amide, C(O), (O)C(R), C(O)C, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl, wherein the ether, ester, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, O, Sn, halo, C1-4alkyl, CF3, and combinations thereof,
wherein R is selected from the group consisting of H, D, O, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
with the proviso that the compound is not
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
In some embodiments, the compound has a structure selected from the group consisting of:
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a compound according to formula (3):
wherein:
a dashed line indicates the presence of an optional double bond;
X1, X2, X3 and Y are independently selected from the group consisting of C, N, S and O;
R1, R2, R3, R4, R5, and R6 are independently selected from the group consisting of no atom H, D, O, N, halo, ether, ester, amide, C(O), (O)C(R), C(O)O, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl, wherein the ether, ester, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
wherein R is selected from the group consisting of H, D, O, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
with the proviso that the compound is not
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a compound according to formula (4):
wherein:
a dashed line indicates the presence of an optional double bond;
X1, X2, and X3 are independently selected from the group consisting of C, N, S and O;
R1 and R2 are independently selected from the group consisting of no atom, H, D, —OH, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C2-6alkenyl, C2-6alkenyl-aryl, and C2-6alkenyl-heteroaryl, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C2-6alkenyl, C2-6alkenyl-aryl, and C2-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, —OH, halo, C1-4alkyl, CF3, and combinations thereof, or R1 and R2 may together form a C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, and combinations thereof;
R3 is selected from the group consisting of H, D, O, N, halo, ether, ester, amide, amino, C(O), (O)C(R), C(O)O, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl, wherein the ether, ester, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
R4 and R5 are independently selected from the group consisting of no atom, H, D, —OH, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C2-6alkenyl, C2-6alkenyl-aryl, and C2-6alkenyl-heteroaryl, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C2-6alkenyl, C2-6alkenyl-aryl, and C2-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, —OH, halo, C1-4alkyl, CF3, and combinations thereof, or R4 and R5 may together form a C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, and combinations thereof;
R6 is selected from the group consisting of H, D, O, N, halo, ether, ester, amide, amino, C(O), (O)C(R), C(O)O, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl, wherein the ether, ester, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
wherein R is selected from the group consisting of H, D, O, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
with the proviso that the compound is not
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a composition, including pharmaceutical compositions, comprising one or more compounds disclosed herein and a pharmaceutically acceptable carrier, adjuvant or vehicle.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or the compositions disclosed herein.
Another embodiment of the present disclosure is a method for modulating the activity of glutathione peroxidase 4 (GPX4) in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or the compositions disclosed herein.
Another embodiment of the present disclosure is a method for increasing the level of peroxide in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or the compositions disclosed herein.
A further embodiment of the present disclosure is a method for inducing ferroptosis in a cell, comprising contacting the cell with an effective amount of one or more compounds disclosed herein or the compositions disclosed herein.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, which is one or more compounds disclosed herein or the compositions disclosed herein, and ii) an effective amount of a second anti-cancer agent.
An additional embodiment of the present disclosure is a kit for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising an effective amount of one or more compounds disclosed herein or the compositions disclosed herein, packaged with its instructions for use.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds having a structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of a compound having the structure of:
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a method for increasing the level of peroxide in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds having a structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a method for inducing ferroptosis in a cell, comprising contacting the cell with an effective amount of one or more compounds having a structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, and ii) an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is one or more compounds having a structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, and ii) an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is a compound having the structure of:
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
The application file contains at least one photograph executed in color. Copies of this patent application with color photographs will be provided by the Office upon request and payment of the necessary fee.
In the present disclosure, GPX4 inhibitors were developed, inter alia, for cancers with a high-mesenchymal-state gene expression signature. All known GPX4 inhibitors (
Accordingly, one embodiment of the present disclosure is a compound according to formula (1):
wherein:
R1, R2, and R3 are independently selected from the group consisting of H, D, O, N, halo, ether, ester, amide, C(O), (O)C(R), C(O)O, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl, wherein the ether, ester, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
wherein R is selected from the group consisting of H, D, O, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
with the proviso that the compound is not
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a compound according to formula (2):
wherein:
a dashed line indicates the presence of an optional double bond;
X and Y are independently selected from the group consisting of C, N, S and O;
R1, R2, R3, and R4 are independently selected from the group consisting of no atom, H, D, O, N, halo, ether, ester, amide, C(O), (O)C(R), C(O)O, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl, wherein the ether, ester, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, O, Sn, halo, C1-4alkyl, CF3, and combinations thereof,
wherein R is selected from the group consisting of H, D, O, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
with the proviso that the compound is not
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a compound according to formula (3):
wherein:
a dashed line indicates the presence of an optional double bond;
X1, X2, X3 and Y are independently selected from the group consisting of C, N, S and O;
R1, R2, R3, R4, R5, and R6 are independently selected from the group consisting of no atom H, D, O, N, halo, ether, ester, amide, C(O), (O)C(R), C(O)O, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl, wherein the ether, ester, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
wherein R is selected from the group consisting of H, D, O, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
with the proviso that the compound is not
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a compound according to formula (4):
wherein:
a dashed line indicates the presence of an optional double bond;
X1, X2, and X3 are independently selected from the group consisting of C, N, S and O;
R1 and R2 are independently selected from the group consisting of no atom, H, D, —OH, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C2-6alkenyl, C2-6alkenyl-aryl, and C2-6alkenyl-heteroaryl, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C2-6alkenyl, C2-6alkenyl-aryl, and C2-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, —OH, halo, C1-4alkyl, CF3, and combinations thereof, or R1 and R2 may together form a C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, and combinations thereof;
R3 is selected from the group consisting of H, D, O, N, halo, ether, ester, amide, amino, C(O), (O)C(R), C(O)O, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl, wherein the ether, ester, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
R4 and R5 are independently selected from the group consisting of no atom, H, D, —OH, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C2-6alkenyl, C2-6alkenyl-aryl, and C2-6alkenyl-heteroaryl, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C2-6alkenyl, C2-6alkenyl-aryl, and C2-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of N, epoxy, —OH, halo, C1-4alkyl, CF3, and combinations thereof, or R4 and R5 may together form a C3-12carbocycle that may be optionally substituted with an atom or a group selected from the group consisting of O, N, halo, C1-4alkyl, CF3, and combinations thereof;
R6 is selected from the group consisting of H, D, O, N, halo, ether, ester, amide, amino, C(O), (O)C(R), C(O)O, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl, wherein the ether, ester, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, and C1-6alkenyl-heteroaryl may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
wherein R is selected from the group consisting of H, D, O, N, halo, C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle, wherein the C1-6alkyl, C1-6alkyl-aryl, C1-6alkyl-heteroaryl, C1-6alkenyl, C1-6alkenyl-aryl, C1-6alkenyl-heteroaryl and C3-12carbocycle may be optionally substituted with an atom or a group selected from the group consisting of halo, C1-4alkyl, CF3, and combinations thereof,
with the proviso that the compound is not
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a composition, including pharmaceutical compositions, comprising one or more compounds disclosed herein and a pharmaceutically acceptable carrier, adjuvant or vehicle.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or the compositions disclosed herein.
In some embodiments, the GPX4-associated disease is selected from the group consisting of a cancer, a neurotic disorder, a neurodegenerative disorder, spondylometaphyseal dysplasia, mixed cerebral palsy, pontocerebellar hypoplasia, and male infertility.
In some embodiments, the GPX4-associated disease is a cancer. Non-limiting examples of cancer include hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma. In some embodiments, the cancer is hepatocellular carcinoma.
In some embodiments, the subject is a mammal. In some embodiments, the mammal is selected from the group consisting of humans, veterinary animals, and agricultural animals. In some embodiments, the subject is a human.
In some embodiments, the cancer is metastatic. In some embodiments, the cancer is under epithelial-to-mesenchymal (EMT) transition. In some embodiments, the cancer is hypersensitive to ferroptosis and/or addicted to GPX4. In some embodiments, the cancer is refractory to standard cancer treatment. Non-limiting examples of standard cancer treatment include chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof.
Another embodiment of the present disclosure is a method for modulating the activity of glutathione peroxidase 4 (GPX4) in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or the compositions disclosed herein. In some embodiments, the modulation comprises inhibiting GPX4 activity.
Another embodiment of the present disclosure is a method for increasing the level of peroxide in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds disclosed herein or the compositions disclosed herein. Non-limiting examples of peroxide include hydrogen peroxide, organic hydroperoxide, lipid peroxide, and combinations thereof.
A further embodiment of the present disclosure is a method for inducing ferroptosis in a cell, comprising contacting the cell with an effective amount of one or more compounds disclosed herein or the compositions disclosed herein.
In some embodiments, the cell has abberant lipid accumulation. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is selected from the group consisting of hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma. In some embodiments, the cancer is hepatocellular carcinoma.
In some embodiments, the cell is a human cell. In some embodiments, wherein the cancer cell is metastatic. In some embodiments, the cancer cell is under epithelial-to-mesenchymal (EMT) transition. In some embodiments, the cancer cell is hypersensitive to ferroptosis and/or addicted to GPX4. In some embodiments, the hypersensitivity to ferropotosis is identified by NADPH abundance, GCH1 expression, NF2-YAP activity, EMT signature, and GPX4 expression. In some embodiments, the cancer cell is refractory to standard cancer treatment. Non-limiting examples of standard cancer treatment includes chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, which is one or more compounds disclosed herein or the compositions disclosed herein, and ii) an effective amount of a second anti-cancer agent.
In some embodiments, the second anti-cancer agent is selected from the group consisting of chemotherapy, radiation therapy, targeted therapy, immunotherapy, and combinations thereof. In some embodiments, the second anti-cancer agent is an immunotherapy, such as checkpoint inhibitor therapy including PD-1 and CTLA-4 inhibitor therapy. Non-limiting examples of immunotherapy include ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, ofatumumab, blinatumomab, daratumumab, elotuzumab, obinutuzumab, talimogene laherparepvec, necitumumab, lenalidomide, dinutuximab, and combinations thereof.
In some embodiments, the subject is a human.
In some embodiments, the cancer is metastatic. In some embodiments, the cancer is under epithelial-to-mesenchymal (EMT) transition. In some embodiments, the cancer is hypersensitive to ferroptosis and/or addicted to GPX4. In some embodiments, the cancer is refractory to standard cancer treatment.
In some embodiments, the cancer is selected from the group consisting of hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colon cancer, diffuse large B cell lymphoma, leukemia, lung cancer, clear-cell carcinoma, and non-small cell lung carcinoma. In some embodiments, the cancer is hepatocellular carcinoma.
In some embodiments, the first anti-cancer agent is administered to the subject before, concurrently with, or after the second anti-cancer agent.
An additional embodiment of the present disclosure is a kit for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising an effective amount of one or more compounds disclosed herein or the compositions disclosed herein, packaged with its instructions for use.
The kits may also include suitable storage containers, e.g., ampules, vials, tubes, etc., for each compound of the present disclosure (which, e.g., may be in the form of pharmaceutical compositions) and other reagents, e.g., buffers, balanced salt solutions, etc., for use in administering the active agents to subjects. The compounds and/or pharmaceutical compositions of the disclosure and other reagents may be present in the kits in any convenient form, such as, e.g., in a solution or in a powder form. The kits may further include a packaging container, optionally having one or more partitions for housing the compounds and/or pharmaceutical compositions and other optional reagents.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds having a structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a glutathione peroxidase 4 (GPX4)-associated disease in a subject in need thereof, comprising administering to the subject an effective amount of a compound having the structure of:
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a method for increasing the level of peroxide in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds having a structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a method for inducing ferroptosis in a cell, comprising contacting the cell with an effective amount of one or more compounds having a structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, and ii) an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is one or more compounds having a structure selected from the group consisting of:
and combinations thereof, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
Another embodiment of the present disclosure is a method for treating or ameliorating the effects of a cancer in a subject in need thereof, comprising administering to the subject i) an effective amount of a first anti-cancer agent, and ii) an effective amount of a second anti-cancer agent, wherein the first anti-cancer agent is a compound having the structure of:
or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof.
As used herein, the terms “treat,” “treating,” “treatment” and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.
As used herein, the terms “ameliorate”, “ameliorating” and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.
As used herein, a “subject” is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present disclosure include, for example, agricultural animals, veterinary animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of veterinary animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. in the context of the present disclosure, the phrase “a subject in need thereof” means a subject in need of treatment for a GPX4-associated disorder, such as, e.g., a cancer. Alternatively, the phrase “a subject in need thereof” means a subject diagnosed with a GPX4-associated disorder, such as, e.g., a cancer.
As used herein, “lipid peroxidation” means the oxidative degradation of fats, oils, waxes, sterols, triglycerides, and the like. Lipid peroxidation has been linked with many degenerative diseases, such as atherosclerosis, ischemia-reperfusion, heart failure, Alzheimer's disease, rheumatic arthritis, cancer, and other immunological disorders. (Ramana et al., 2013).
As used herein, “ferroptosis” means regulated cell death that is iron-dependent. Ferroptosis is characterized by the overwhelming, iron-dependent accumulation of lethal lipid reactive oxygen species. (Dixon et al., 2012) Ferroptosis is distinct from apoptosis, necrosis, and autophagy. (Id.)
As used herein, the terms “modulate”, “modulating”, “modulator” and grammatical variations thereof mean to change, such as increasing or decreasing the activity of GPX4. In this embodiment, “contacting” means bringing the compound and optionally one or more additional therapeutic agents into close proximity to the cells in need of such modulation. This may be accomplished using conventional techniques of drug delivery to the subject or in the in vitro situation by, e.g., providing the compound and optionally other therapeutic agents to a culture media in which the cells are located.
As used herein, a “pharmaceutically acceptable salt” means a salt of the compounds of the present disclosure which are pharmaceutically acceptable, as defined herein, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.
In the present disclosure, an “effective amount” or “therapeutically effective amount” of a compound or pharmaceutical composition is an amount of such a compound or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of the subject, and like factors well known in the arts of, e.g., medicine and veterinary medicine. In general, a suitable dose of a compound or pharmaceutical composition according to the disclosure will be that amount of the compound or composition, which is the lowest dose effective to produce the desired effect with no or minimal side effects. The effective dose of a compound or pharmaceutical composition according to the present disclosure may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
A suitable, non-limiting example of a dosage of a compound or pharmaceutical composition according to the present disclosure or a composition comprising such a compound, is from about 1 ng/kg to about 1000 mg/kg, such as from about 1 mg/kg to about 100 mg/kg, including from about 5 mg/kg to about 50 mg/kg. Other representative dosages of a compound or a pharmaceutical composition of the present disclosure include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg.
A compound, composition, or pharmaceutical composition of the present disclosure may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a compound, composition, or pharmaceutical composition of the present disclosure may be administered in conjunction with other treatments. A compound, composition, or pharmaceutical composition of the present disclosure may be encapsulated or otherwise protected against gastric or other secretions, if desired.
The compositions or pharmaceutical compositions of the disclosure are pharmaceutically acceptable and comprise one or more active ingredients in admixture with one or more pharmaceutically-acceptable carriers or diluents and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the compounds/compositions/pharmaceutical compositions of the present disclosure are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.). More generally, “pharmaceutically acceptable” means that which is useful in preparing a composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.
Pharmaceutically acceptable carriers and diluents are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable carrier or diluent used in a composition of the disclosure must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Carriers or diluents suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers or diluents for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
The compositions or pharmaceutical compositions of the disclosure may, optionally, contain additional ingredients and/or materials commonly used in such compositions. These ingredients and materials are well known in the art and include (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (11) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monosterate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.
Compounds, compositions or pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.
Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers or diluents and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.
Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.
Compositions for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier or diluent. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.
Compositions suitable for parenteral administrations comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.
The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier or diluent, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.
We propose to target hepatocellular carcinoma as a first indication for several reasons. First, we showed that the FDA-approved multi-targeted kinase inhibitor sorafenib is one of the very few kinase inhibitors that induce ferroptosis (
Hepatocellular carcinoma (HCC) is an important unmet medical need, as it is one of the most aggressive, heterogeneous, and difficult to treat cancers (Medavaram and Zhang, 2018), causing >700,000 annual deaths globally (Singh et al. 2018). HCC affects children and adults of all ages, and has genetic and environmental origins. In addition, patients with hereditary tyrosinemia due to inactivation of the last step of the tyrosine catabolism pathway often develop HCC; such patients accumulate 4-fumarylacetoacetate (FAA), which, is an electrophilic Michael acceptor that depletes liver glutathione, a central inhibitor of ferroptosis (Yang et al. 2014). Exogenous glutathione suppresses much of the toxicity of FAA in cell culture and in a genetic mouse model of the disease (Singh et al. 2018), suggesting that GSH depletion can drive HCC, but also collateral sensitivity to ferroptosis (Yang et al. 2014). FAA is converted into succinylacetone, which inhibits porphyrin biosynthesis, leading to accumulation of liver iron and contributing to HCC pathogenesis (Singh et al. 2018), driving collateral sensitivity to ferroptosis (Dixon et al. 2012). Thus, GSH depletion and iron overload, key drivers of sensitivity to ferroptosis (Stockwell et al. 2017), are genetically linked to HCC through hereditary tyrosinemia patients and mouse models. Moreover, as noted above, the first targeted therapy approved for advanced HCC was the multi-targeted kinase inhibitor sorafenib, in 2007 (Medavaram and Zhang, 2018), which induces ferroptosis by inhibiting system xc− (Dixon et al. 2014). Many other therapies that don't induce ferroptosis have been explored in HCC, with few positive clinical results (Medavaram and Zhang, 2018). This suggests that ferroptosis may be a uniquely effective therapeutic approach to HCC.
In addition to GSH depletion and iron accumulation seen in familial-tyrosinemia-driven HCC, additional factors that predispose to HCC drive sensitivity to ferroptosis: excess alcohol metabolism in the liver depletes GSH (Zhou et al. 2019), and fatty liver in NASH patients is reported to trigger ferroptosis (Tsurusaki et al. 2019). These observations suggest that HCC tumors may harbor an intrinsic sensitivity to ferroptosis inducers, which we found to be the case in HCC cell lines (
Additional indications where there is evidence for sensitivity to GPX4 inhibitors and/or ferroptosis include sarcomas, gliomas, renal cell carcinomas, ovarian cancers, DLBCLs (lymphomas), lung cancers, and breast cancers, as well as drug-resistant metastatic and EMT cancers from additional lineages (Hangauer et al. 2017).
We identified several biomarkers that predict sensitivity to ferroptosis inducers in general and GPX4 inhibitors specifically, including NADPH abundance (Shimada et al. 2015), GCH1 expression (Kraft et al. 2020), NF2-YAP activity (Wu et al. 2019), EMT signature (Viswanathan et al. 2017), and GPX4 expression itself (unpublished). We will examine each of these biomarkers and determine which combination of biomarkers is the best predictor of sensitivity to the GPX4 inhibitors we create for HCC and other target indications.
Starting from deciphering the nanomolar potency of the proof-of-concept GPX4 inhibitors, we solved the co-crystal structure of GPX4 with its covalent inhibitors RSL3 and ML162, which revealed that RSL3 binds to an alternative allosteric site rather than the expected active site as ML162 does (
The x-ray structure of the potent GPX4 inhibitor RSL3 showed that this compound binds to an allosteric site distinct from the active site. Thus, we will, in parallel to the above efforts with the validated binders, develop a screening assay focused on the RSL3-binding site.
We will perform a high-throughput screen of compounds from our library of ˜10,000 lead-like compounds (selected from 3.3 million compounds for those with favorable lead-like properties) for those that can compete with the binding of a fluorescently-labeled RSL3 probe to its allosteric binding site on GFP-GPX4; activity will be measured by FRET between the probe and the GFP fused to GPX4. Our hypothesis is that this library contains additional binders to the RSL3-binding site, but that these compounds were not detected in the thermal shift assay we used for our previous GPX4 screen—this assay is not that sensitive and can miss many compounds. Positives will be processed through the funnel described above to obtain validated hits. As an additional backup, we will also screen an 8,000 compound covalent fragment library and the Sigma DyNAbind DNA-encoded library of 370,000 fragment compounds. This will provide a diverse set of additional compounds that may be able to target the RSL3-binding site.
As the leading causes of deaths in cancer patients, metastatic and drug-resistant cancers stand among the most pressing problems in oncology (Dillekas et al. 2019; Wang et al. 2019). As a key step in metastasis, the epithelial-to-mesenchymal transition (EMT), which increases motility of tumor cells and enables the invasion of primary tumors to distant sites, has been reported to render cancer cells resistant to apoptosis and chemotherapy (Fischer et al. 2015). During the transition, the elevated level of polyunsaturated-fatty-acid-(PUFA)-containing phospholipids (PUFA-PLs) is a major factor to increase the fluidity of cell membranes, as the cis conformation of double bonds in PUFA-PLs hinders efficient stacking of fatty acyl tails (Agmon and Stockwell, 2017). However, as PUFA-PLs are inherently susceptible to peroxidation at bis-allylic positions, cancer cells that have undergone EMT inevitably became more dependent on a key protein, glutathione peroxidase 4 (GPX4), which is the only peroxidase in mammals capable of reducing phospholipid hydroperoxides within cell membranes (Thomas et al. 1990; Kuhn and Borchert, 2002; Zou et al. 2020). When GPX4 activity is compromised, lipid peroxidation can cause ferroptosis, a new form of tumor suppressive cell death (Dixon et al. 2012; Yang et al. 2014). Indeed, as cancers evolve into aggressive and drug-resistant forms with a mesenchymal or other signatures, they simultaneously acquire an exquisite sensitivity to GPX4 inhibition, which indicated a tantalizing possibility that the most aggressive neoplastic diseases can be treated through the use of GPX4 inhibitors (Viswanathan et al. 2017; Hangauer et al. 2017; Zou et al. 2019).
Inhibition of GPX4 with small molecules has been limited by the characteristic flat surface surrounding its active site and the lack of known allosteric regulatory sites (Borchert et al. 2018; Sakamoto et al. 2017). To date, all known GPX4 inhibitors are assumed to be covalent inhibitors that react with the active site selenocysteine of GPX4 (Yang and Stockwell, 2008; Yang et al. 2014; Weiwer et al. 2012; Yang et al. 2016; Eaton et al. 2019; Eaton et al. 2020; Eaton et al. 2020). The top inhibitors, (1S, 3R)-RSL3 (hereinafter referred to as “RSL3”) and ML162, featured nanomolar potencies to induce ferroptosis to cancer cells, but they are limited by poor drug-like properties associated with their chloroacetamide warheads (Yang and Stockwell, 2008; Yang et al. 2014; Weiwer et al. 2012; Yang et al. 2016; Allimuthu and Adams, 2017). Additionally, developing drug-like compounds selectively binding to the flat active site of GPX4 over selenocysteine of other glutathione peroxidases in the GPX family may be challenging. Although extensive medicinal-chemistry efforts have been devoted to the current inhibitors, the potencies have not been further improved (Eaton et al. 2019; Eaton et al. 2020; Eaton et al. 2020). This indicated the necessity of a deeper understanding of the nanomolar potency of current inhibitors or a search for alternative binding site before the adaption of a structure-based approach for the development of improved GPX4 inhibitors.
In addition to its peroxidase function, as a moonlighting protein under specific condition, GPX4 can polymerize into an enzymatically inactive, oxidatively cross-linked, insoluble structural element of the mitochondrial sheath of the midpiece of mature spermatozoa (Ursini et al. 1999; Conrad et al. 2005). This unusual feature may derive from the less restricted dependence on glutathione as reducing substrate and the expression of multiple non-conserved surface cysteines, which are distinct from other members of the GPX family (Conrad et al. 2005; Scheerer et al. 2007). The structural and mechanism basis for this dual function and whether it is involved in the regulation of ferroptosis by GPX4 have not been clearly demonstrated. We hypothesized that further mechanism study of the enzymatically inactive dual function would benefit the discovery of novel approach to GPX4-specific inhibition.
Here, via deciphering the nanomolar potency of known GPX4 inhibitors, we identified an allosteric binding site on GPX4 that allows inactivation of GPX4 without targeting the active site selenocysteine. We then found this site is involved in the dual function of GPX4, based on which we proposed an alternative GPX4 catalytic cycle. After confirmation of the druggability of this allosteric binding sites with multiple proof-of-concept inhibitors, we screened a library of lead-optimized compounds and found a hit compound binding in the allosteric site to inhibit GPX4, which provided a novel approach for therapeutic strategies targeting GPX4.
Starting from deciphering the nanomolar potency of the proof-of-concept GPX4 inhibitors, we solved the co-crystal structure of GPX4U46C with its covalent inhibitors RSL3 and ML162, which revealed that RSL3 and ML162 both bind to cys66, an alternative site rather than the expected active site (
Moreover, overexpression of tag-free GPX4C66S also protected HT-1080 cells from ferroptosis induced by RSL3 and ML162 to a greater extent than overexpression of tag-free GPX4WT, excluding the possibility of GFP tag interference (
GPX4 contains one seleno-cysteine at the active site and seven other cysteines, which are all potentially reactive with electrophiles (
In our previous work, sepharose beads coupled with anti-fluorescein antibodies were not able to pull down a detectable amount of allCys(−) A66C GPX4 from G401 allCys(−) A66C cells treated with RSL3-fluorescein (Yang et al. 2016). We hypothesized this could be because RSL3 bound to cys66 of GPX4 and subsequently induced the degradation of allCys(−) A66C GPX4 in the cellular context (Shimada et al. 2016). In line with this hypothesis, we found that, while allCys(−) GPX4 cannot be covalently modified by RSL3 and therefore was resistant to RSL3-induced degradation observed with native WT GPX4, allCys(−) A66C GPX4 with the cys66 binding site is vulnerable to the RSL3-induced degradation, based on the Western-blotting of G401 overexpressing allCys(−) or allCys(−) A66C GPX4 after treatment with RSL3 (
In addition to cys66, other surface cysteines (cys10, cys107, and cys148) of GPX4 may also be amenable to electrophilic attacks by RSL3 and ML162. Moreover, sec46 at the active site is expected to be more reactive towards electrophiles because of its lower pKa values than cysteines (Yang et al. 2016). To test whether sec46 and other surface cysteine residues on GPX4 also contribute to RSL3 binding, we applied G401 cell lines stably expressing corresponding revertants of allCys(−) GPX4 (Yang et al. 2016). Five of such G401 cell lines (A10C, A46C, A46U, A107C, and A148C) were therefore included in the test, along with G401 overexpressing WT, allCys(−), or allCys(−) A66C GPX4, for RSL3 and ML162 sensitivities (
It is noteworthy that overexpression of allCys(−) A46C GPX4 didn't exhibit a significant protection against RSL3 and only a slight degradation was observed. This further confirmed the reported importance of selenocysteine for the binding of RSL3 to GPX4 active site and explained the absence of inhibitor at the active site of GPX4U46C co-crystal structure (Yang et al. 2016; Gao et al. 2018). Additionally, allCys(−) A46U GPX4 showed the most significant protection and degradation, which indicated a preference of inhibitor binding to sec46, potentially due to its higher activity against electrophiles, and explained why tagged RSL3 pulled down a larger amount of allCys(−) A46U than allCys(−) with any other reverted cysteines (Yang et al. 2016; Gao et al. 2018).
To validate selenocysteine 46 and cysteine 66 as inhibitor binding sites on GPX4 in a cellular context, we stably overexpressed either GFP-tagged GPX4U46C_C66S a double mutant which lacks both binding sites, or GFP-tagged GPX4U46C as control, in HT-1080 cells. Using HT-1080 cells transfected with empty vector (pBabe-puro) as a control, we found that GPX4U46C and GPX4U46C_C66S, both of which have a much lower enzymatic activity than WT, exhibited no effects against treatment with FIN56 or FINO2 (
Besides from the three classes of ferroptosis inducers that we have tested, imidazole ketone erastin (IKE), an inhibitor of cystine/glutamate antiporter system xc−, represents another class of ferroptosis inhibitors (Feng and Stockwell, 2018). IKE prevents cystine import, which will lead to the depletion of GPX4 cofactor GSH and a loss of GPX4 activity thereafter, to induce ferroptosis (Zhang et al. 2019). When we tested G401 cells overexpressing WT, allCys(−), or individual revertants of allCys(−) GPX4 (A10C, A46C, A46U, A66C, A107C, and A148C) for IKE sensitivities, we found overexpression of enzymatically inactive AllCys(−) A66C GPX4 significantly protected G401 cells from IKE, the protection effect of which is even comparable with the overexpression of enzymatically active WT GPX4 and AllCys(−) A46U GPX4 (
Previous studies revealed that at low GSH concentrations, GPX4 acted as a protein thiol peroxidase to utilize specific protein thiols as the reductants in its catalytic cycle and structurally crosslink proteins, which is also known as the dual function of GPX4 (Ursini et al. 1999; Conrad et al. 2005). The reported low GSH conditions coincidentally matched the IKE-treated cellular conditions in our experiments (Ursini et al. 1999).
We therefore propose that, in addition to the canonical glutathione-dependent catalytic cycle of GPX4 (I), GPX4 may utilize the thiol of cys10 on a second GPX4 protein as reductant to form a dimer intermediate, which will then be regenerated with the thiol of cys66 on a third GPX4 protein to complete the catalytic cycle (II,
In accordance with the model, we found that overexpression of enzymatically inactive AllCys(−) A66C GPX4 in G401 cells boosted the enzymatic activity of endogenous WT GPX4, while AllCys(−) A10C GPX4 significantly suppressed the activity (
To further verify the potential role of cys66 and cys10 in modulating GPX4 activity, especially at limited GSH levels, we stably overexpressed GFP-tagged GPX4C10S_C66S, which is devoid of the cys66 and cys10 cross-linking sites, in HT-1080 cells and then tested the cells against IKE. Using HT-1080 cells either overexpressing GFP-tagged GPX4WT or transfected with empty vector (pBabe-puro) as controls, we found that overexpression of GPX4C10S_C66S, which lacks the linking sites, still protected HT1080 cells from ferroptosis induced by IKE, but to a significantly lower extent than the overexpression of GPX4WT, suggesting a role of cys10 and cys66 in modulating GPX4 function at low GSH concentrations (
As our results demonstrated that RSL3, the proof-of-concept GPX4 inhibitor with nanomolar potency, not only blocked sec46 at the active site, but also specifically bound to cys66, a positive modulator of GPX4 activity, and subsequentially induced degradation of GPX4 in a cellular context, the potential of taking advantage of cys66 binding site for the design of novel drugs targeting GPX4 was indicated. To specifically examine the potency of cys66 as an inhibitor binding site, in vitro purified GPX4U46C protein was applied, as our cellular data and co-crystal structure indicated RSL3 solely bound to cys66 of GPX4U46C. To further verify this selective binding to cys66, we conducted intact protein MALDI MS analysis of GPX4U46C preincubated with an excess amount of RSL3, which showed 1:1 covalent binding of RSL3 to GPX4U46C (
To demonstrate the single covalent modification by RSL3 is not on cys46, we expressed and purified tag-free GPX4allCys(−) A46C protein, in which ala46 in allCys(−) GPX4 was reverted to cys46 (
To evaluate the impact on GPX4 after RSL3 binds to cys66, we measured the enzymatic activity of GPX4U46C after an incubation with RSL3 or DMSO, which revealed that binding of RSL3 to cys66 inhibited the capability of GPX4 to reduce phospholipid hydroperoxides (
Since we demonstrated that cys66 is an allosteric binding site of RSL3 on GPX4, we then initiated a search for additional compounds that also bind to this site, to validate the druggability of the site and provide foundations for the future design of novel therapeutics targeting GPX4. We started with two fragments of ML162, CDS9 and TMT10, which share similar warheads and structures as RSL3 and ML162. As intact protein MALDI MS analysis of GPX4U46C preincubated with CDS9 and TMT10 suggested covalent bindings, we proceeded to solve the co-crystal structures of GPX4U46C with each of the compounds, which showed both compounds selectively bound to the cys66 site (
To fully utilize the versatility shown by the cys66 site to accommodate structurally diverse compounds, we applied thermal shift assay to screen 9,719 compounds in the Lead-Optimized-Compound (LOC) library for in vitro binders of GPX4U46C, which would shift the melting temperature of GPX4U46C (|ΔTm|>2° C.,
The LOC library was assembled in-house via stringently filtering a database of 3,372,615 commercially available small molecules for structurally diverse compounds with desired drug-like properties and suitability for lead development (Kaplan et al. 2015). Top hits from the screening were then tested by the 1H, 15N-HSQC-NMR assay that we developed to examine their individual binding modes. Accordingly, we found that LOC1886, a thermal shift screening top hit compound (|ΔTm|=3° C.), strongly interacted with the cys66 binding site, based on the significant chemical shifts of the residues around the site, and resulted in a global conformational change of GPX4U46C (
Together, these data suggested that we identified a druggable allosteric site on GPX4, and compounds that bind in this site.
Our investigation into the binding modes of RSL3 and ML162 revealed that they not only interact with the active site selenocysteine, but also selectively bind to cysteine 66 of GPX4. With the U46C and AllCys(−)-A66C GPX4 constructs, which excluded binding of inhibitors to active site, we specifically showed that binding of inhibitor to cys66 site resulted in the inactivation of GPX4. In particular, using RSL3 as an example, we showed that its binding to cys66 caused a global conformational change, loss of activity, and also a subsequent degradation in the cellular context. The additional proof-of-concept compounds binding to this allosteric site further validated its druggability. Moreover, since cysteine 66 is not conserved across the GPX isoforms and, unlike selenocysteine, is unique for GPX4, we expected inhibitors designed for cys66 allosteric sites to be selective towards GPX4 (Scheerer et al. 2007).
In line with all known GPX4 inhibitors, the proof-of-concept inhibitors that we identified in this study also form covalent bonds with GPX4. This prevalence might derive from the lack of deep, well-defined pockets on the surface of GPX4, even in the co-crystal structures (Eaton et al. 2018). Although covalent inhibitor used to be intentionally avoided during pharmaceutical development due to selectivity concerns, multiple covalent inhibitors constituted important medicines (such as penicillin and aspirin) (Sutanto et al. 2020; Baillie, 2016). As more covalent inhibitors entered clinical trials recently, the interest in applying the power of covalent inhibitors has been growing, especially for the targets undruggable with reversible inhibitors (Janes et al. 2018; Bar-Peled et al. 2017; Backus et al. 2016). Here, we reported the first co-crystal structure of the top GPX4 inhibitor RSL3, along with structural determination of GPX4 in complex with another four structurally diverse compounds, which would provide structural foundations for the development of improved inhibitor. Additionally, the novel warheads in MAC-5576 (2-pyridylester) and LOC1886 (N-acyl imidazole) represent alternative substituent of the promiscuous chloroacetamide warhead. We envision that larger scales of screening followed by structure-based optimization of the binders would lead to the first drug-like allosteric GPX4-specific inhibitors.
HT-1080 cells were obtained from ATCC and grown in DMEM with glutamine and sodium pyruvate (Corning 10-013) supplemented with 10% FBS (Gibco), 1% non-essential amino acids (Invitrogen) and 1% penicillin-streptomycin mix (Invitrogen). G401 cells were obtained from ATCC and grown in McCoy's 5A medium (Thermo Fisher, #16600108) supplemented with 10% FBS and 1% penicillin-streptomycin mix.
Bacterial expression vectors pET15b-His-tagged-c-GPX4U46C and pET15b-His-tagged-c-GPX4AllCys(−)A46C were prepared in the previous work (Yang et al. 2016). The His-tagged-c-GPX4U46C protein was expressed in E. coli and purified according to a published protocol with minor modifications (Scheerer et al. 2007). Isolated colonies of BL21-Gold (DE3) competent cells (Agilent, #230132) with each plasmid were separately transferred to 8 mL of LB medium with 100 μg/mL ampicillin, and the inoculated culture was incubated while being shaken (225 rpm) at 37° C. for 16 h. 3 mL of the starter culture was added to 1 L of fresh LB medium with 100 μg/mL ampicillin. The culture was incubated while being shaken at 37° C. and 225 rpm until the OD600 reached 0.9. The temperature was then decreased to 15° C. Cells were incubated with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) while being shaken at 15° C. and 225 rpm overnight. The next day, the bacteria were harvested by centrifugation at 4000 g for 20 min at 4° C. and the pellet obtained was ready for purification or stored at −20° C. The pellet was resuspended in 25 mL of chilled lysis buffer (100 mM Tris pH 8.0, 300 mM NaCl, 20 mM imidazole, 3 mM TCEP, and protease inhibitor cocktail (Roche-Sigma, #11836170001)). The bacteria were lysed by sonication on ice for 6 min, and the lysate was centrifuged at 10000 rpm for 20 min at 4° C. to remove cell debris. The clarified lysate was incubated with Ni Sepharose 6 Fast Flow beads (GE Life Sciences, via Cytiva #17-5318-01) on a rotator at 4° C. for at least 1 h. The beads were washed with wash buffer (100 mM Tris pH 8.0, 300 mM NaCl, 50 mM imidazole, and 3 mM TCEP) to remove nonspecific binding. The protein was eluted with 100 mM Tris pH 8.0, 300 mM NaCl, 100 mM imidazole, and 3 mM TCEP. The protein was further purified using a gel filtration Superdex 200 column in FPLC buffer containing 100 mM Tris pH 8.0, 300 mM NaCl, and 3 mM TCEP. The fractions containing GPX4 were pooled together and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
GPX4U46C or GPX4AllCys(−)-A46C protein was pre-incubated with DMSO control or the inhibitor to be tested prior to MALDI MS analysis: 50 μM GPX4 protein was incubated with 500 μM inhibitors in FPLC buffer (100 mM Tris pH 8.0, 300 mM NaCl, and 3 mM TCEP) with 5% DMSO at RT for 1 hour before transferring to 4° C. overnight.
1 μl of the ligand-free protein (pre-incubated with DMSO) or protein-inhibitor complex (pre-incubated with the inhibitor to be tested) was mixed with 9 μl of 10 mg/ml sinapinic acid in the matrix solution (70:30 water/acetonitrile, with 0.1% TFA). 1.0 μl of the final mix was deposited onto the target carrier and allowed to air dry. MALDI spectrum was recorded using Bruker ultrafleXtreme MALDI-TOF instrument. The range of m/z detection and suppression was adjusted to accommodate the molecular weight of target protein. 2000 Hz and 50% intensity was applied for the laser setting. For each sample, five cumulative spectra were collected and the sum was recorded for analysis. All MALDI spectra of protein-inhibitor complex were compared with ligand-free protein to determine the mass shift. Mass shifts were aligned with the mass of potential staying group of each inhibitor to conclude covalent binding.
GPX4U46C was pre-incubated with the covalent inhibitors before crystallization using the following specifically optimized condition.
RSL3 and ML162 condition: 50 μM GPX4U46C incubated with 150 μM RSL3 or ML162 in the reaction buffer (20 mM Tris pH 9.0, 100 mM NaCl, 3 mM TCEP, 1.5% DMSO) at 15° C. for 1 hour before transferring to 4° C. overnight. CDS9 condition: 25 μM GPX4U46C incubated with 2 mM CDS9 in the reaction buffer (100 mM Tris pH 9.0, 300 mM NaCl, 1 mM TCEP, 2% DMSO) at 37° C. for 2 hours before transferring to 4° C. overnight. TMT10 condition: 25 μM GPX4U46C incubated with 2 mM TMT10 in the reaction buffer (100 mM Tris pH 9.0, 300 mM NaCl, 2% glycerol, 2% DMSO) at 37° C. for 4 hours before transferring to 4° C. overnight. MAC-5576 condition: 50 μM GPX4U46C protein was incubated with 500 μM MAC-5576 in the reaction buffer (100 mM Tris pH 9.0, 300 mM NaCl, and 3 mM TCEP, 5% DMSO) at RT for 1 hour before transferring to 4° C. overnight.
After confirmation of covalent binding using intact protein MALDI MS analysis, the protein-inhibitor complex were exchanged into crystallization buffer (10 mM Tris pH 8.0, 5 mM TCEP) and concentrated to 5 mg/ml.
Molecular surface figures and 2D inhibitor-receptor interaction diagram of co-crystal structures were generated with Maestro (Schrodinger, suite 2020-3), while zoom-in figures with protein backbone and inhibitor electron densities were prepared with Pymol.
The pBabe-puro vectors incorporated with the cDNA of GFP-tagged-cyto-GPX4WT, tag-free-cyto-GPX4WT, or GFP-tagged-cyto-GPX4U46C were prepared in previous work (Yang et al. 2014). With the vectors as template, the following mutagenesis primers were designed using the Agilent QuikChange Primer Design application: C10S (5′-GGA GCG CGC ACT GCG CCA GTC G-3′ (SEQ ID NO: 1), 5′-ACG ACT GGC GCA GTG CGC GCT C-3′ (SEQ ID NO: 2)) and C66S (5′-CCC GAT ACG CTG AGA GTG GTT TGC GGA TC-3′ (SEQ ID NO: 3), 5′-GAT CCG CAA ACC ACT CTC AGC GTA TCG GG-3′ (SEQ ID NO: 4)). Primers were purchased from Integrated DNA Technologies. Site-directed mutagenesis kit (QuickChange II, Agilent 200521) was then used to acquire pBP-GFP-cGPX4C66S, pBP-GFP-cGPX4U46C_C66S, and pBP-GFP-cGPX4C10S_C66S. All mutations and the resulted plasmids were confirmed by sequencing at GENEWIZ.
HT-1080 cells were seeded into a 6-well dish at a density of 300,000 cells/well the night before lipofection. 2.5 μg DNA (empty pBabe-puro vector and the above GPX4 expressing pBabe-puro vectors, separately), 7.5 μL Lipofectamine 3000 (Invitrogen, L3000015), and 250 μL Opti-MEM were incubated for 5 min at room temperature before adding to the HT-1080 cells. Following transfection, cells were passaged several times in HT1080 media supplemented with 1.5 mg/mL puromycin and grown in this media for all experiments performed. Expression of the exogenous GFP-tagged-GPX4 was confirmed with fluorescence microscope and Western Blot with both GFP and GPX4 antibodies.
The G401 cells overexpressing WT, allCys(−), allCys(−) A10C, allCys(−) A46C, allCys(−) A46U, allCys(−) A66C, allCys(−) A107C, or allCys(−) A148C GPX4 were reported in the previous work (Yang et al. 2016). The G401 cells were cultured in G401 media supplemented with 1.5 mg/mL puromycin.
1000 cells of each HT-1080 or G401 cell line were plated 36 μL per well of a 384-well plate on day 1. The remaining cells were immediately tested for Western Blot (to monitor GPX4 protein overexpression level). For dose response curves, compounds were dissolved in DMSO and a 12-point, twofold dilution series was prepared. The compounds were then diluted 1:50 in media and 4 μL was added to each well of the plates on day 2. Final concentrations of the compounds on the 384-well plate started from 2 μM for RSL3/ML162 and 20 μM for IKE/FIN56/FINO2. For single-point ferroptosis test, HT1080 cells were treated either with 12.5 μM RSL3 or 125 μM LOC1886, with or without supplementation of 2 μM Fer-1. After 48 h of treatment, the viability of cells was measured using 1:1 dilution of the CellTiter-Glo luminescent reagent (Promega G7573) with media, which was read on a Victor 5 plate reader after 10 min of shaking at room temperature on day 4. The intensity of luminescence was normalized to that of DMSO control. Results were quantified using GraphPad Prism 9. Based on the dose-response curve of viability, area under curves were calculated and reported in bar graph formats with standard errors using GraphPad Prism 9.
For the GPX4 degradation study, G-401 cells were seeded at 800,000 per well in a 60-mm plate and allowed to adhere overnight. Cells were then treated with 10 μM Fer-1 and 0 (vehicle), 2 or 4 μM RSL3 for 10 h. Cells were harvested with trypsin (Invitrogen, 25200-114), pelleted, and lysed with RIPA buffer.
For the quantification of GPX4 protein level for cellular viability and GPX4-specific activity assay, each cell line subject to the cellular viability and GPX4-specific activity was tested by Western Blot in technical duplicates. In particular, cells were harvested with trypsin (Invitrogen, 25200-114), pelleted, and lysed by LCW lysis buffer (0.5% TritonX-100, 0.5% sodium deoxycholate salt, 150 mM NaCl, 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 30 mM Na-pyrophosphate, and cOmplete protease inhibitor cocktail). While part of the cell lysates was blotted for protein quantification, the other part of lysates was used for the GPX4-specific activity assay. For both experiments, cell lysates were blotted and imaged as previously described (Yang et al. 2014). Antibodies used were GPX4 (Abcam, ab125066, 1:250 dilution) and actin (Cell Signaling, D18C11, 1:3,000 dilution). Results were quantified using a LI-COR Odyssey CLx IR scanner, ImageJ, and GraphPad Prism 9.
We applied a NADPH-coupled cellular GPX4 enzymatic activity assay as previously reported with minor modifications (Roveri et al. 1994). Oxidized glutathione, generated by GPX4 during reducing its specific phospholipid hydroperoxides substrate, was reduced by Glutathione Reductase at the expense of NADPH, the decrease in the characteristic absorbance of which at 340 nm was monitored and quantified as GPX4 activity. The GPX4-specific substrate PCOOH was prepared by enzymatic hydroperoxidation of phosphatidylcholine by soybean lipoxidase type IV: 22 mL of 0.2 M Tris-HCl, pH 8.8, containing 3 mM sodium deoxycholate and 0.3 mM phosphatidylcholine was incubated at room temperature, under continuous stirring, for 30 min with 0.7 mg of soybean lipoxidase type IV. The mixture was loaded on a Sep-Pak CI8 cartridge (Waters-Millipore) washed with methanol and equilibrated with water. After washing with 10 volumes of water, phosphatidylcholine hydroperoxides were eluted in 2 mL of methanol. 50 millions of particular G401 or HT-1080 cells were harvested and lysed by LCW lysis buffer (0.5% TritonX-100, 0.5% sodium deoxycholate salt, 150 mM NaCl, 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 30 mM Na-pyrophosphate, and complete protease inhibitor cocktail). The concentration of protein in the lysate was determined using BCA assay kit using BSA as standards. Then, on a 96-well plate, 250 μL 1.5 μg/μL cell lysate was incubated in the GPX4 activity assay buffer (0.1% Triton X-100, 100 mM Tris-HCl pH 7.4, 10 mM NaN3, 5 mM EDTA, 0.6 IU/mL Glutathione reductase, 0.5 mM NADPH, and 3 mM GSH unless otherwise noted) at 37° C. for 10 min. For evaluation of GPX4 inhibitors, DMSO, 50 μM RSL3, or 500 μM LOC1886 was added to the 250 μL cell lysate in GPX4 activity assay buffer before the 10 min incubation. PCOOH was then added to the mixture to initiate GPX4 reaction. Absorbance of NADPH at 340 nm was determined kinetically at 1 min interval over the 20 min time. Experiments using lysis buffer instead of cell lysate and controls without addition of PCOOH were also done to measure the particular activity of GPX4 to reduce phospholipid hydroperoxides. Total GPX4 activity of each sample were normalized to their specific GPX4 level based on Western Blot for unit GPX4 enzymatic activity for comparison. Results were quantified using GraphPad Prism 9.
Similar to the determination of cellular GPX4-specific activity, by coupling the oxidation of NADPH to NADP+ by oxidized glutathione produced by GPX4 in the presence of glutathione reductase, GPX4U46C activity was assessed by measuring the decrease in NADPH (absorbance at 340 nm). GPX4 reaction buffer was prepared by adding 0.05 U/mL glutathione reductase, 210 μM GSH, 250 μM NADPH, 50 μM Cumene-OOH into 50 mM Tris HCl, pH 8.0, 0.5 mM EDTA. 15 μM His-tagged-GPX4U46C pre-incubated with DMSO (vehicle) or 20 μM RSL3 was added to the GPX4 reaction buffer and the absorbance at 340 nm was monitored for the following 10 min. Results were quantified using GraphPad Prism 9.
Since the binding of small molecules may alter the thermostability of protein, we applied thermal shift assay to screen the LOC library, which determined binding of ligand from the change of the unfolding transition temperature (ΔTm) obtained in the presence of ligands relative to that obtained in the absence of ligands (Kaplan et al. 2015; Lo et al. 2004). For higher throughput purpose, we adapted the assay on 384-well PCR plate. With the assistance of Biomek Automated Liquid Handler (Beckman), 2 μl 10 mM stock solution (in 100% DMSO) of the each compound was transferred from 384-well library plates to 38 μl FPLC buffer (100 mM Tris pH 8.0, 300 mM NaCl, 3 mM TCEP) and mixed in each well of a 384-well mother plate. The compound solution was then dispensed, 12 μl per well, into three 384-well PCR plates. GPX4U46C protein and Sypro orange were manually added to the plate, with the final concentrations of 5 μM and 5×, respectively, to make a volume of 20 μl per well right before thermal shift analysis of the specific plate. The thermal shift assay was performed on the ViiA 7 Real-Time PCR system (Thermo Fisher) with the thermal protocol: 25° for 15 s, Increase temp to 99° at a rate of 0.05°/sec, 99° for 15 sec. The fluorescence was recorded and analyzed by Protein Thermal Shift™ software. The mean |ΔTm| value for the biological triplicates were calculated and used for hit identification.
1H, 15N-HSQC-NMR Spectrum for 15N Isotope-Labeled GPX4U46C
Uniformly 15N-labeled GPX4U46C protein with N-terminal His6 tag was prepared. The GPX4U46C construct was expressed in E. coli BL21-Gold (DE3) cells (Stratagene) growing at 37° C. in M9 minimal medium supplemented with 100 mg/mL ampicillin, 2 mM MgSO4, 100 mM CaCl2, 1× trace metals, 1×RPMI 1640 vitamin stock (Sigma-Aldrich, #R7256), 10 mg/mL biotin, 10 mg/mL thiamine hydrochloride, 4 g/L glucose, and 3 g/L 15NH4Cl as the sole nitrogen source. The following induction, lysis, and protein purification was the same as for the non-isotope-labeled His-tagged GPX4U46C described above, except the N-terminal His6 tag was removed by adding 5 U/mg thrombin before FPLC purification. The thrombin reaction was after a buffer exchange to remove imidazole, which otherwise would inhibit thrombin, and was allowed to proceed overnight at 4° C.
For HSQC of GPX4 with RSL3, since solubility of RSL3 in aqueous solution is low, 10 μM 15N-labeled GPX4 was preincubated with 100 μM RSL3 in buffer (100 mM MES, 5 mM TCEP, pH 6.5) for 12 h at 4° C. Then the protein solution was concentrated to 50 μM before testing. For HSQC of GPX4 with other inhibitors, 50 μM 15N-labeled GPX4 was preincubated with 800 μM inhibitor to be tested for κ h at room temperature in buffer (100 mM MES, 5 mM TCEP, pH 6.5). D20 (10%) was then added for the field frequency lock. The 1H-15N HSQC spectra were collected on Bruker Avance III 500 Ascend (500 MHz) spectrometers (Columbia University) at 298K. The 1H carrier frequency was positioned at the water resonance. The 15N carrier frequency was positioned at 115 ppm. The spectral width in the 1H dimension was 7,500 Hz and the width in the 15N dimension was 1,824.6 Hz. Suppression of water signal was accomplished using the WATERGATE sequence. Heteronuclear decoupling was accomplished using GARP decoupling scheme.
3D Protein NMR Experiments for 13C, 15N Isotope-Labeled GPX4U46C
Uniformly 13C, 15N-labeled GPX4U46C protein with N-terminal His6 tag was prepared. The GPX4U46C construct was expressed in E. coli BL21-Gold (DE3) cells (Stratagene) growing at 37° C. in M9 minimal medium supplemented with 100 mg/mL ampicillin, 2 mM MgSO4, 100 mM CaCl2), 1× trace metals, 1×RPMI 1640 vitamin stock (Sigma-Aldrich, #R7256), 10 mg/mL biotin, 10 mg/mL thiamine hydrochloride, 4 g/L U-13C6-glucose as the sole carbon source, and 3 g/L 15NH4Cl as the sole nitrogen source. The following induction, lysis, protein purification, and tag removal were the same as for uniformly 15N-labeled GPX4U46C protein.
HNCACB and CBCA(CO)NH NMR spectrum of 50 μM uniformly 13C, 15N-labeled GPX4U46C protein in 100 mM MES pH 6.5, with 5 mM TCEP and 5% D20 were collected on 700 MHz Avance III/TS 3.5.6 at NYSBC and applied for HSQC backbone resonance assignments. We performed the assignment based on standard triple resonance backbone assignment (Cα and Cβ) in the practical guide on protein NMR (https://www.protein-nmr.org.uk) and used CCCPNmr Analysis software (v3). To verify the assignment, we also collected HNCO, HN(CA)CO, HNCA, and HN(CO)CA NMR spectrum, and performed Cα and C′ (carbonyl caron) based assignments in parallel as a cross-check. Of the expected 168 amino acid residues of our GPX4U46C construct (175 total residues excluding 7 prolines which has no H attached to its N and therefore does not give signal in HSQC), backbone resonances of 147 residues were assigned. The unassigned residues included a 10-residue N-terminal linker sequence, which was also not visible in the crystal structure of GPX4 (PDB: 2OBI) (Scheerer et al. 2007). In the sequence of our construct which is corresponding to D6-F170 of human GPX4 (NCBI Reference Sequence: NP_001354761.1), DDWRC ARSMHEFSAK DIDGHMVNLD KYRGFVCIVTNVASQCGKTE VNYTQLVDLH ARYAECGLRI LAFPCNQFGK QEPGSNEEIK EFAAGYNVKF DMFSKICVNG DDAHPLWKWM KIQPKGKGIL GNAIKWNFTK FLIDKNGCW KRYGPMEEPL VIEKDLPHYF (SEQ ID NO: 5), the assigned residues were underlined (154/165). The inability to detect and/or assign the remaining backbone peaks may reflect exchange broadening owing to conformational dynamics and mobility inherent to GPX4 in solution, which notably included cysteine 46 at the active site and other residues on the loop surrounding it (Vadrevu et al. 2003).
Spondylometaphyseal dysplasia (SMD), first described in 1967 by Kozlowski, was defined as a form of congenital bone dysplasia comprising various types of chondrodystrophy (Nural et al. 2006). Sedaghatian-type spondylometaphyseal dysplasia (SSMD) is a rare neonatal lethal disorder characterized by severe metaphyseal chondrodysplasia with mild limb shortening, platyspondyly, cardiorespiratory defects, and central nervous system abnormalities (Elcioglu and Hall, 1998). Most infants with SSMD succumb within days after birth due to respiratory distress (Ipek and Akin, 2016). So far four SSMD-related GPX4 variants have been described: two biallelic splice site variants (c.587+5G>A and c.588-8_588-4del) in one patient, biallelic homozygous p.Tyr127* in the second patient, and biallelic homozygous variant c.153_160del in the third patient (Smith et al. 2014; Fedida et al. 2020). To date, only recessive truncating mutations in the GPX4 gene have been reported to induce SSMD. The severity of biallelic truncation mutations on GPX4 function are likely due to significant loss of enzymatic functioning, but the exact physiological validation is lacking. Furthermore, whether less involved disease exist and possible genotype-phenotype correlations are unknown. Understanding GPX4 mechanisms may expand the actual overall phenotype by elucidating less involved disease states.
In this study, we identified three individuals from 2 unrelated families who were found to harbor a recurrent homozygous point mutation in the GPX4 gene, c.647 G>A (p. R216H), by whole exome sequencing. One child (patient 1, family 1) has typical skeletal features of SSMD in addition to auditory neuropathy, optic nerve hypoplasia, hypotonia and severe motor delays. A second family (patients 2 and 3, family 2) was identified through a rare disease organization (Curegpx4.org) with similar neurological features but with atypical skeletal findings for SSMD as they lacked platyspondyly and rhizomelia. These findings raise the question of whether missense mutations in GPX4 are pathological or not. Therefore, we sought to further examine the potential impact of this variant.
GPX4 is a selenoprotein and a member of the glutathione peroxidase family of enzymes, which share an antioxidant function of reducing peroxides through the use of the co-substrate glutathione (Brigelius-Flohe and Maiorino, 2013). Despite the structural and functional similarities between these enzymes, GPX4 is distinct from other GPx enzymes, being the only enzyme in mammals capable of reducing esterified phospholipid hydroperoxides and cholesterol hydroperoxides within the context of cell membranes (Brigelius-Flohe and Maiorino, 2013). Although oxygenated lipids can serve as signal molecules regulating inflammatory processes, they disrupt membrane architecture and are prone to decompose into reactive species, which can damage biomolecules, such as proteins and nucleic acids (Imai and Nakagawa, 2003). Therefore, when GPX4 activity is compromised, lipid peroxidation products can accumulate, resulting in cell death through ferroptosis, an iron-dependent form of non-apoptotic cell death (Yang et al. 2014). Accordingly, an essential role for GPX4 during embryogenesis and early development has been demonstrated by the failure of GPX4 homozygous knockout mice to survive past early gestation, and by the neonatal lethality of neuronal-specific GPX4 knockout in mice (Seiler et al. 2008; Yant et al. 2003).
Here, we sought to investigate the impact of a human GPX4 variant missense mutation on the structure and function of GPX4 protein to determine potential pathological features of this variant, and guide SSMD patients towards precise treatments at the earliest opportunity. Additionally, GPX4 has been reported to be a promising therapeutic target for drug-resistant and metastatic cancers, based on the elevated dependency of these cancers on the GPX4 lipid peroxide repair pathway during epithelial-mesenchymal transition (EMT) and in the drug-resistant persister and epithelial to mesenchymal transition cell states (Viswanathan et al. 2017; Hangauer et al. 2017; Liu et al. 2018). We hypothesized that greater insight into the basic biochemical mechanisms of GPX4 might emerge from studying a patient-derived variant, providing us with opportunities to modulate GPX4 for therapeutic benefit in clinical contexts.
Large-scale expression of selenocysteine-containing proteins in recombinant systems is challenging, due to inefficient selenocysteine-incorporating machinery; thus, the selenocysteine to cysteine (U to C, inserting a thiol group in place of the selenol group) mutant of GPX4 is widely used for structural studies, despite its lower enzymatic activity compared to the wild-type protein (Scheerer et al. 2007; Sakamoto et al. 2017; Janowski et al. 2016; Li et al. 2019). Since recent studies on selenocysteine-containing GPX4 confirmed the relevance of the catalytic triad and other structural properties discovered in the context of the U to C mutant, we used a GPX4U46C construct for in vitro structural studies and structure-based computational analysis, and simultaneously examined the selenocysteine-containing cytosolic GPX4 in human cells via enzymatic assays and cellular assays (Borchert et al. 2018; Ingold et al. 2018; Yu et al. 2014). In the mature short form GPX4 construct that we used for both in vitro and the cell-based study, the patient-derived variant is denoted R152H on the GPX4 protein (Arai et al. 1996; Liang et al. 2009).
We found that the missense R152H variant resulted in a change in the protein structure, caused substantial loss of enzymatic function, and thus is likely the basis for the pathological features in patients, considering the essential role of gpx4 in the early development of mice. The functional analysis was validated in patient fibroblasts. Our further structural inspection into the origin of reduced enzymatic activity revealed K48 has an essential role in modulating GPX4 function, in addition to the previously reported catalytic triad (Sec46/Gln81/Trp136) and Asn137 (Tosatto et al. 2008). We also found that the R152H variant alters the degradation of GPX4, revealing for the first time the degradation mechanism of GPX4 protein.
To investigate the impact of the R152H variant on GPX4 structure and function, we began with computational modeling of the GPX4R152H protein structure (
Comparing GPX4R152H with GPX4R152R, we found that the R152H variant significantly altered the surface around residue 152, as evidenced by the loss of a hydrophobic pocket centered on Arg152 in the wild-type protein (
Accordingly, the average distance between the active site catalytic residue Cys46 and its catalytic partners Gln81/Trp136 was significantly increased in GPX4R152H and presented a considerably wide distribution throughout timescale of the dynamics simulated, which indicates a predicted weaker interaction among the catalytic triad (
Following the results from computational modeling, we established a cell model of the R152H mutation by stably overexpressing either GFP-tagged GPX4WT or GFP-tagged GPX4R152H in HT-1080 fibrosarcoma cells, in which GPX4 functions to protect cells from ferroptosis, in order to experimentally determine the impact of this variant in a human cell context. Using HT-1080 cells transfected with empty vector (pBabe-puro) as a control, we measured the enzymatic activity of the transfected GFP-GPX4 protein via its ability to reduce a phospholipid hydroperoxide in an NADPH-coupled assay, as reported previously (Roveri et al. 1994). By normalization of the enzyme activity to the protein level, as measured by western blot, we found that one unit of GPX4R152H exhibited 36% of the activity of GPX4WT (
We then predicted that the reduction in enzyme activity in R152H would result in impaired resistance to ferroptosis: as the primary enzyme capable of reducing phospholipid hydroperoxides, overexpression of GPX4 protects cells from ferroptosis (Yang et al. 2014).
Accordingly, we tested the ferroptosis sensitivity of HT-1080 cells overexpressing comparable level of GFP-GPX4WT or GFP-GPX4R152H. While both cell lines were more resistant to ferroptosis induced by the GPX4 inhibitors RSL3, ML162 and by the system xc− inhibitor IKE, as compared to the cell line transfected with empty vector, overexpression of GPX4R152H was less protective against ferroptosis inducers than GPX4WT, which was consistent with lower activity of the mutant protein to reduce lipid hydroperoxides (
To further understand why the alteration of a single amino acid residue distant from the active site caused a significant loss of enzymatic function in GPX4, we expressed, purified, and solved the x-ray crystal structure of His-tagged GPX4U46C and GPX4U46C_R152H proteins at 1.5 Å resolution. The structure of GPX4U46C that we solved is consistent with the previously reported structure (PDB: 2OBI). While the backbone of GPX4U46C_R152H aligned well with GPX4U46C, the most significant change was in the loop between Pro125 and Ala132, which was intrinsically disordered in GPX4R152H, as evidenced by the loss of electron density corresponding to these residues; this indicates the lack of fixed or ordered three-dimensional structure in this part of the protein (
Moreover, since Trp136 is also on this disordered loop, an examination on the catalytic triad at the active site showed that distances between each pair of the catalytic triad residues were increased, as expected from modeling (
Point Mutation Reveals that Lys48 has an Essential Role in Modulating the Catalytic Activity of GPX4
To further investigate the role of Lys48 in the enzymatic function of GPX4, we generated HT-1080 cells stably overexpressing GFP-GPX4K48A. Using HT-1080 cells transfected with GFP-GPX4WT or empty vectors (pBabe-puro) as a control, we measured the enzymatic activity of GFP-GPX4K48A. Strikingly, we found that GPX4K48A had an almost complete loss of activity to reduce phospholipid hydroperoxides (
To illuminate the mechanism for this essential function, we expressed, purified and solved the crystal structure of His-tagged GPX4U46C_K48A protein.
While the GPX4U46C_K48A protein is stable and superimposes well upon the structure of GPX4U46C (
To further study the role of Lys48 in the context of the canonical catalytic cycle of the GPX4 enzymatic reaction, in which the first step is the oxidation of GPX4 by its hydroperoxides substrates, we solved the crystal structure of fully oxidized GPX4U46C, where the sulfur of the Cys46 was oxidized to sulfonic acid (SO3H). We found that Lys48 is positioned in a close proximity (3.6 Å distance) to the oxidized Cys46, even closer than it was to the reduced Cys46 (5.2 Å distance) in the crystal structure of reduced GPX4U46C (
The second step of the canonical catalytic cycle of GPX4 is the incorporation of its cofactor GSH via the formation of a Se—S bond, which prepares oxidized GPX4 for regeneration to the reduced form. Therefore, we covalently docked GSH into the crystal structures of GPX4U46C and GPX4U46C_K48A in silico using Maestro, and attained better covalent-docking affinities with GPX4U46C (Zhu et al. 2014). This is in line with a previously reported modelling work that proposed a role of Lys48 in interacting with GSH (Mauri et al. 2003). Additionally, the flexible and open active site of the oxidized GPX4U46C, as indicated by its MD simulation in comparison with GPX4U46C_K48A, might also be more accessible for GSH to incorporate and reduce oxidized GPX4.
In summary, these data depict the multi-pronged mechanism of the modulation of GPX4 enzymatic activity by Lys48: to stabilize the active site to be in a more compact and functional state, to modulate the oxidized active site for cycling, and to facilitate the incorporation of the cofactor GSH. Together, this provides an explanation for the loss of activity when this residue was mutated to Ala.
To further dissect the interaction between Lys48 and the active-site residues, we prepared and solved the crystal structure of GPX4U46C_K48L protein, for which the mutation only removed the positively charged E-amino group on the side chain, as compared to the wild-type. While this mutant was stable and superimposes well with the structure of GPX4U46C (
The diverse catalytic activities and structural characteristics of the wild-type, K48L (a mutant without the positive charge), and K48A GPX4 (a mutant without both charge and side chain) highlighted that both the positive charge and the long flexible side chain are involved in the function of Lys48. Accordingly, we concluded that the Lys48 has an unexpected and essential role in modulating the enzymatic activity of GPX4 via multiplex interactions with the active site throughout its catalytic cycle. Additionally, we found that in both crystal structures of K48A and K48L, there is a conformational change in the 124-133 loop, as compared to wild-type (
When we tested the ferroptosis sensitivities of HT-1080 cells overexpressing GFP-GPX4R152H or GFP-GPX4WT, we were surprised to find that GPX4R152H provided comparable protection as GPX4WT to the ferroptosis inducer FIN56, despite the insufficient protection against RSL3, ML162 and IKE (
The mechanism of GPX4 degradation induced by RSL3 and FIN56 is not clear, despite some efforts to understand this phenomenon (Shimada et al. 2016; Wu et al. 2019).
Based on our finding that the loop between Pro125 and Ala132, which contains Lys125 and Lys127, became intrinsically disordered in the context of GPX4R152H, we developed a hypothesis that RSL3-induced or FIN56-induced GPX4 degradation involved a proteasome-dependent mechanism, with Lys125 and Lys127 as potential ubiquitination sites on GPX4; in this model, the highly mobile lysines hinder ubiquitination and prevent degradation. We tested this hypothesis by examining GPX4 degradation in HT-1080 cells stably overexpressing GFP-GPX4K125R_K127R, where the proposed sites of ubiquitin ligation were removed by point mutations. While the endogenous GPX4 was degraded upon treatment with RSL3, ML162, or FIN56, we found that GFP-GPX4K125R_K127R was resistant to degradation, even more so than in the context of the R152H mutation (
The results described above demonstrated that the patient-derived R152H variant of GPX4 caused partial loss of its function in phospholipid peroxidase activity and therefore is pathological. However, the data also suggested that the overexpression of the partially-active but degradation-resistant GPX4R152H did partially protect cells from ferroptosis induced by lipid peroxidation, though less effectively than wild-type GPX4. Therefore, we proposed a hypothesis that boosting the expression level of the R152H variant of selenoprotein GPX4 via selenium supplementation should be able to compensate the partial loss of function and be helpful to patients. This is also based on the observation that a subtoxic selenium supplementation can significantly increase the expression level of GPX4 (up to 48-fold), as well as another anti-oxidant selenoprotein GPX1 (up to 40-fold), in human cells (Romanowska et al. 2007). Additionally, selenium is readily available from dietary source or as OTC supplement tablets (Bodnar et al. 2016).
We tested the hypothesis on a HT-1080 OE GFP-GPX4R152H cell model, which was transfected to overexpress GFP-GPX4R152H and only produced a relatively negligible amount of endogenous GPX4WT after pro-longed selections. Sodium selenite, selenomethione, and (methyl-)selenocysteine were tested as sources of selenium, while N-acetyl-cysteine was included as a control, which might also benefit the patients as a metabolic precursor of GPX4 cofactor GSH (Yang and Stockwell, 2016). After a pre-supplementation of selenium-containing compounds or cysteine at titrated concentrations for 24 h, HT-1080 OE GFP-GPX4R152H cells were then uniformly treated with RSL3 at IC10 for 48 h to simulate a patient-related scenario, featuring a suppressed GPX4 activity and a vulnerability to phospholipid peroxidation and ferroptosis (
We also tested the protective effect of selenium-containing compounds and cysteine against other classes of ferroptosis inducers, which indirectly suppressed GPX4 activity via diverse pathways (
We repeated the above analysis of sodium selenite at an extended range of concentrations, and determined its EC50 value of rescue effect against RSL3 (19 nM), IKE (66 nM), FIN56 (34 nM), and FINO2 (12 nM,
In summary, selenium supplementation, as a proof-of-concept treatment, was shown to be effective at nanomolar concentration in a cell model of a patient-derived GPX4 variant and therefore may benefit such patients.
To validate these observations on molecular and cellular models, fibroblasts developed from a patient with homozygous mutations (RAG01, GPX4R152H/R152H) and his parent as a healthy control with heterozygous mutations (RAG02, GPX4R152H/WT) were tested for GPX4 protein level and enzymatic activity. Although the two human fibroblast cell lines expressed an equivalent level of GPX4 protein based on western blot analysis, RAG01 exhibited a significantly lower level of GPX4 enzymatic activity than RAG02, confirming that the patient-derived R152H variant in GPX4 caused substantial loss of function (
As fibroblast data validated the pathology analysis on the HT-1080 cell model, we further tested selenium supplementation and other proof-of-concept therapeutic treatments on fibroblasts. In contrast to the HT-1080 cell model, since patient fibroblasts already exhibited comprised GPX4 activity and a poor viability in the regular media, we didn't add ferroptosis inducers to the fibroblasts for these rescue experiments, but instead we directly treated fibroblast cells with various concentrations of selenium supplementation or antioxidants and then monitored the increase of cell numbers over control fibroblast cells treated with DMSO only as a readout. Accordingly, we found that selenium supplementation with sodium selenite, seleno-methione, and methyl-seleno-cysteine can boost the viability of RAG01 to 138.0%, 137.9%, and 136.3%, with an EC50 value of 0.8 nM, 0.5 nM, and 0.8 nM, respectively (
Additionally, we found that the antioxidants α-Tocopherol, CoQ10 and idebenone (a soluble analog of CoQ10) can also boost the viability of RAG01 to 250%, 197%, and 245%, with an EC50 value of 115 nM, 5 μM, and 228 nM, respectively (FIGS. 20F-20H). We also found that dimethyl fumarate, an Nrf2 activator that promotes an antioxidant response, boosted the viability of RAG01 to 127% (Wang et al. 2015) (
To further evaluate the potency of the proof-of-concept treatments and compare effects on GPX4R152H to a mock control solely expressing wild-type GPX4 (instead of RAG02 expressing both variants), we tested all treatments on Gpx4-knockout Pfa-1 cells that are transfected to overexpress human/murine GPX4WT or GPX4R152H (
In summary, the observation that selenium supplementation, ferroptosis inhibitors, and antioxidants can increase the cell number and viability of patient fibroblasts and our R152H cell models further confirmed our pathological mechanism analysis, whereby the partial loss-of-function mutation in GPX4 sensitize the patient's cells to lipid peroxidation and ferroptosis. Therefore, we would expect that a combinational treatment of selenium supplementation to boost GPX4 level and antioxidants to suppress lipid peroxidation (α-Tocopherol and CoQ10) might be the most effective treatment, which are all widely available as OTC supplements.
An outstanding phenomenon from the immunofluorescence imaging of the fibroblast cells might further support the potency of these proof-of-principle therapeutic treatments for patients, as we found cells undergoing mitosis exhibited a much higher fluorescence intensity of GPX4 than non-mitotic cells (
Prior to this study, only three previously reported infants with SSMD were identified to have predicted loss of function variants based on in silico data, although these variants were not studied in vitro (Smith et al. 2014; Fedida et al. 2020). Our report of the patient with biallelic missense variants from an unrelated family with typical findings of SSMD, combined with the functional characterization of the R152H missense variant, provided further evidence of the association of biallelic variants in GPX4 with SSMD. In addition, this report extends the phenotype associated with bi-allelic variants in GPX4 beyond SSMD to include long-term survival beyond infancy and other skeletal and neurological findings.
This study began by examining the effect of the R152H missense mutation on GPX4, which adversely changed the protein structure and caused a partial loss of its antioxidant activity. As a hypomorphic allele, we therefore hypothesize that there is sufficient enzymatic function to allow for survival beyond infancy but results in increased susceptibility to ferroptosis culminating in cell death, tissue damage, and neurodegeneration. This finding would be in keeping with observations of the adult condition Gpx4 knockout mouse which developed seizures, ataxia and progressive neuronal loss (Yoo et al. 2012).
Interestingly, we also reported a second family harboring the R152H missense variant in a homozygous state with concordant neurological phenotypes but the affected individuals (patients 2 and 3) had atypical skeletal findings for SSMD. The reasons for the disparate skeletal phenotypes despite the identical genotype are unclear. We speculate that the individuals in family 2 may harbor other genetic variants that are protective against the impact of GPX4 variation on developing chondrocytes. Identification and study of other individuals with biallelic variants in GPX4, regardless of the skeletal phenotype, as well as genotyping of other reported individuals with typical SSMD may improve our understanding of the phenotypic spectrum of GPX4 associated disease.
Through real-time communications with patients along our research timeline, living patients with the identified mutation have begun taking selenium supplementation and antioxidant treatments, including vitamin E, N-acetyl-cysteine, and CoQ10. With 50 mg BID CoQ10, 100 mcg selenium per day, and 150 mg BID N-acetyl-cysteine, patient 1 has developed enough strength to lift his head from a prone position for a brief time, and his skeletal X-ray indicated his bone is normalizing.
During this analysis, we used a structure-based computational modeling approach to study the effect of the variant on protein structure in silico, the predictions from which were confirmed by protein crystal structures and cellular assays. These experiments suggested that structure-based modeling of variant protein structures is an important and reliable approach to dissect the impact of patient-derived variants. The low-cost and high-throughput of computational modeling would potentially benefit more patients with orphan disease or variation in key genes.
This variant unexpectedly revealed the structural basis of GPX4's enzymatic activity and the regulation of its degradation: Lys48 was found to modulate GPX4 enzymatic activity and Lys125/Lys127 were revealed as sites of ubiquitin ligation. In addition, this also suggested Arg152 as an allosteric site indirectly regulating GPX4 activity. Since recent studies highlighted GPX4 protein as an Achilles's heel of drug-resistant and metastatic cancers, which are exceptionally dependent on the GPX4 lipid peroxide repair pathway, we would expect biochemical therapeutic strategies targeting the essential residue Lys48 and allosteric site Arg152 or taking advantage of its degradation mechanism, to be developed as a high priority and benefit additional patients (Viswanathan et al. 2017; Hangauer et al. 2017).
HT-1080 cells were obtained from ATCC and grown in DMEM with glutamine and sodium pyruvate (Corning 10-013) supplemented with 10% FBS, 1% non-essential amino acids (Invitrogen) and 1% penicillin-streptomycin mix (Invitrogen). Human fibroblast cell line RAG01 and RAG02 were developed from patient with homozygous R152H variant and his parent with heterozygous R152H variant. RAG01 and RAG02 were grown in DMEM with glutamine and sodium pyruvate (Corning 10-013) supplemented with 15% FBS, 1% non-essential amino acids (Invitrogen) and 1% penicillin-streptomycin mix (Invitrogen). RAG01 and RAG02 cell lines are available for both commercial and academic use through CureGPX4.org, a patient organization dedicated to finding a treatment for SSMD disease. Cells were maintained in a humidified environment at 37° C. and 5% CO2 in a tissue incubator.
In silico residue mutation analysis and molecular dynamics (MD) simulations were performed in Maestro (Schrödinger Suite).
The crystal structure of GPX4U46C (PDB: 2OBI) was imported into Maestro, preprocessed to remove water and add hydrogens, optimized at pH 7 for H-bond assignment, and minimized in the OPLS3e force field using Protein Preparation Wizard. Substitution of the Arg152 residue in the structure by His was followed by a global minimization of the whole structure in an implicit solvent to generate a GPX4R152H model. As a control, GPX4R152R was also generated by a synonymous mutation of Arg152 to Arg using the same algorithm and minimization method. SiteMap was then run under the default setting. A structural comparison between GPX4R152H and GPX4R152R was performed thereafter.
For MD simulation, each of the above structures was set up in an orthorhombic box with 0.15 M NaCl in SPC solvent and OPLS3e force field. MD simulations of the system for 100 ns with 4.8 ps per step at 300 K and 1.01325 bar were performed with random seeding in Desmond Molecular Dynamics. Simulation quality and event analysis were also done by Desmond. Videos for representative simulation process were exported.
A pBabe-puro vector incorporated with the cDNA of GFP-tagged-cyto-GPX4WT was prepared in previous work (Yang et al. 2014). With the vector as template, the following mutagenesis primers were designed using the Agilent QuikChange Primer Design application: R152H (5′-CTG CGT GGT GAA GCA CTA CGG ACC CAT GG-3′ (SEQ ID NO: 6), 5′-CCA TGG GTC CGT AGT GCT TCA CCA CGC AG-3′ (SEQ ID NO: 7)), K48A (5′-GGC CTC CCA GTG AGG CGC GAC CGA AGT AAA CTA C-3′ (SEQ ID NO: 8), 5′-GTA GTT TAC TTC GGT CGC GCC TCA CTG GGA GGC C-3′ (SEQ ID NO: 9)), K125R_K127R (5′-TGG ATG AAG ATC CAA CCC AGG GGC AGG GGC ATC CTG-3′ (SEQ ID NO: 10), 5′-CAG GAT GCC CCT GCC CCT GGG TTG GAT CTT CAT CCA-3′ (SEQ ID NO: 11)). Primers were purchased from Integrated DNA Technologies. Site-directed mutagenesis kit (QuickChange II, Agilent 200521) was then used to acquire pBP-GFP-cGPX4R152H, pBP-GFP-cGPX4K48A, and pBP-GFP-cGPX4K125R_K127R. All mutations and the resulted plasmids were confirmed by sequencing at GENEWIZ.
HT-1080 cells were seeded into a 6-well dish at a density of 300,000 cells/well the night before lipofection. 2.5 μg DNA (empty pBabe-puro vector and the above four GFP-GPX4 expressing pBabe-puro vectors, separately), 7.5 μL Lipofectamine 3000 (Invitrogen, L3000015), and 250 μL Opti-MEM were incubated for 5 min at room temperature before adding to the HT-1080 cells. Following transfection, cells were passaged several times in media containing 1.5 mg/mL puromycin and grown in this media for all experiments performed. Expression of the exogenous GFP-tagged-GPX4 was confirmed with fluorescence microscope and Western Blot with both GFP and GPX4 antibodies.
For the quantification of GPX4 protein level, the biological duplicates of each transfected HT-1080 cell lines subject to the GPX4-specific activity were tested by Western Blot in technical duplicates, which made a total of 4 samples for each cell line. In particular, cells were harvested with trypsin (Invitrogen, 25200-114), pelleted, and lysed by LCW lysis buffer (0.5% TritonX-100, 0.5% sodium deoxycholate salt, 150 mM NaCl, 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 30 mM Na-pyrophosphate, and complete protease inhibitor cocktail). While part of the cell lysates was blotted for protein quantification, the other part of lysates was used for the GPX4-specific activity assay.
For the GPX4 degradation study, HT-1080 cells were seeded at 800,000 per well in a 60-mm plate and allowed to adhere overnight. Cells were then treated with 100 μM α-tocopherol and either 1 μM RSL3, 1 μM ML162, 10 μM IKE, 10 μM FIN56, or vehicle for 10 h. Cells were harvested with trypsin (Invitrogen, 25200-114), pelleted, and lysed with RIPA buffer.
For both experiments, cell lysates were blotted and imaged as previously described (Yang et al. 2014). Antibodies used were GPX4 (Abcam, ab125066, 1:250 dilution), actin (Cell Signaling, D18C11, 1:3,000 dilution), and GAPDH (Santa Cruz, sc-47724, 1:10,000 dilution). Results were quantified using a LI-COR Odyssey CLx IR scanner and GraphPad Prism 7.
Human fibroblast cells RAG01 and RAG02 were separately seeded on poly-lysine-coated coverslips (Sigma Aldrich P4832) in a 24-well plate (100,000 per coverslip and three coverslips for each cell line) and allowed to grow overnight. Medium was removed and the cells were gently washed with PBS2+ (PBS with 1 mM CaCl2 and 0.5 mM MgCl2) gently twice. The cells were fixed and permeabilized by adding 200 μL/well of 4% paraformaldehyde (PFA) in PBS with 0.1% Triton X-100 (PBS-T), and incubated at room temperature for 20 min. The cells were then washed with PBS-T three times. Then the cells were blocked with 5% goat serum (ThermoFisher 50197Z) in PBS-T for 1 h at room temperature. The cells were then incubated with monoclonal mouse GPX4 antibody (Santa Cruz, sc-166570, 1:500 dilution) in PBS-T with 1% BSA and 5% goat serum overnight at 4° C. The cells were washed with PBT for 5 min three times. The cells were incubated with goat anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific Cat #A-11032, RRID:AB_2534091, 1:200 dilution) at room temperature for 1 h. The cells were washed with PBS-T for 5 min three times. ProLong Diamond anti-fade mountant with DAPI (ThermoFisher P36962) was added to stain the nucleus. All images were captured on a Zeiss LSM 800 confocal microscope at Plan-Apochromat 63×/1.40 oil DIC objective with constant laser intensity for all samples. When applicable, line-scan analysis was performed on representative confocal microscopy images using Zeiss LSM software to qualitatively visualize fluorescence overlap.
The quantification of the intensity of antibodies was analyzed using CellProfiler 3.1.8 (Carpenter et al. 2006) (CellProfiler Image Analysis Software, RRID:SCR_007358). Nuclei were first identified as primary objects using global minimum cross entropy strategy, based on DAPI fluorescence signal. The whole cells were then identified as secondary objects based on primary objects by propagation using global minimum cross entropy strategy, based on GPX4 fluorescence (Alexa Fluor 594) signal. The cytoplasm were then identified as the tertiary objects as the part of each cell excluding the nucleus. Then mean intensities of GPX4 fluorescence of nuclei, cytoplasm and whole cell were measured and reported. Graphs were created in Prism 8.
We applied a NADPH-coupled cellular GPX4 enzymatic activity assay as previously reported (Roveri et al. 1994). Oxidized glutathione, generated by GPX4 during reducing its specific phospholipid hydroperoxides substrate, was reduced by Glutathione Reductase at the expense of NADPH, the decrease in the characteristic absorbance of which at 340 nm was monitored and quantified as GPX4 activity. The GPX4-specific substrate PCOOH was prepared by enzymatic hydroperoxidation of phosphatidylcholine by soybean lipoxidase type IV: 22 mL of 0.2 M Tris-HCl, pH 8.8, containing 3 mM sodium deoxycholate and 0.3 mM phosphatidylcholine was incubated at room temperature, under continuous stirring, for 30 min with 0.7 mg of soybean lipoxidase type IV. The mixture was loaded on a Sep-Pak CI8 cartridge (Waters-Millipore) washed with methanol and equilibrated with water. After washing with 10 volumes of water, phosphatidylcholine hydroperoxides were eluted in 2 mL of methanol. 48 million HT-1080 cells were harvested and lysed by LCW lysis buffer (0.5% TritonX-100, 0.5% sodium deoxycholate salt, 150 mM NaCl, 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 30 mM Na-pyrophosphate, and complete protease inhibitor cocktail). The concentration of protein in the lysate was determined using BCA assay kit using BSA as standards. Then, on a 96-well plate, 250 μL 1.5 μg/μL cell lysate was incubated in the GPX4 activity assay buffer (0.1% Triton X-100, 100 mM Tris-HCl pH 7.4, 10 mM NaN3, 5 mM EDTA, 0.6 IU/mL Glutathione reductase, 0.5 mM NADPH) at 37° C. for 10 min. PCOOH was then added to the mixture to initiate GPX4 reaction. Absorbance of NADPH at 340 nm was determined kinetically at 1 min interval over the 20 min time. Experiments using lysis buffer instead of cell lysate and controls without addition of PCOOH were also done to measure the particular activity of GPX4 to reduce phospholipid hydroperoxides. Total GPX4 activity of each sample were normalized to their specific GPX4 level based on Western Blot for unit GPX4 enzymatic activity for comparison. Results were quantified using GraphPad Prism 7.
For dose response curves, 1000 cells of each transfected HT-1080 cell lines subject to the GPX4-specific activity were plated 36 μL per well of a 384-well plate on day 1. The remaining cells were immediately tested for Western Blot and activity assay. Compounds were dissolved in DMSO and a 12-point, twofold dilution series was prepared. The compounds were then diluted 1:50 in media and 4 μL was added to each well of the plates on day 2. Final concentrations of the compounds on the 384-well plate started from 2 μM for RSL3/ML162 and 20 μM for IKE/IN56. After 48 h of treatment, the viability of cells was measured using 1:1 dilution of the CellTiter-Glo luminescent reagent (Promega G7573) with media, which was read on a Victor 5 plate reader after 10 min of shaking at room temperature on day 4. The intensity of luminescence was normalized to that of DMSO control. Results were quantified using GraphPad Prism 7.
Bacterial expression vector pOE30-His-tagged-c-GPX4U46C was described in the previous work (Yang et al. 2016). With the vector as template, the following mutagenesis primers were designed using the Agilent QuikChange Primer Design application: R152H (F: 5′-CTG CGT GGT GAA GCA CTA CGG ACC CAT GG-3′ (SEQ ID NO: 12), R: 5′-CCA TGG GTC CGT AGT GCT TCA CCA CGC AG-3′ (SEQ ID NO: 13)), K48A (F: 5′-GGC CTC CCA GTG TGG CGC GAC CGA AGT AAA CTA C-3′ (SEQ ID NO: 14), R: 5′-GTA GTT TAC TTC GGT CGC GCC ACA CTG GGA GGC C-3′ (SEQ ID NO: 15)), K48L (F: 5′-CGT GGC CTC CCA GTG TGG CCT AAC CGA AGT AAA CTA CAC TC-3′ (SEQ ID NO: 16), 5′-GAG TGT AGT TTA CTT CGG TTA GGC CAC ACT GGG AGG CCA CG-3′ (SEQ ID NO: 17)). Primers were purchased from Integrated DNA Technologies. Site-directed mutagenesis kit (QuickChange II, Agilent 200521) was then used to acquire pOE30-c-GPX4U46C_R152H, pOE30-c-GPX4U46C_K48A, and pOE30-c-GPX4U46C_K48L. All mutations and the resulted plasmids were confirmed by sequencing at GENEWIZ.
Isolated colonies with each plasmid were separately transferred to 8 mL of LB medium with 100 μg/mL ampicillin, and the inoculated culture was incubated while being shaken (225 rpm) at 37° C. for 16 h. 3 mL of the starter culture was added to 1 L of fresh LB medium with 100 μg/mL ampicillin. The culture was incubated while being shaken at 37° C. and 225 rpm until the OD600 reached 0.9. The temperature was then decreased to 15° C. Cells were incubated with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) while being shaken at 15° C. and 225 rpm overnight. The next day, the bacteria were harvested by centrifugation at 4000 g for 20 min at 4° C. and the pellet obtained was ready for purification or stored at −20° C. The pellet was resuspended in 25 mL of chilled lysis buffer (100 mM Tris pH 8.0, 300 mM NaCl, 20 mM imidazole, 3 mM TCEP, and Roche protease inhibitor cocktail). The bacteria were lysed by sonication on ice for κ min, and the lysate was centrifuged at 10000 rpm for 20 min at 4° C. to remove cell debris. The clarified lysate was incubated with Ni Sepharose 6 Fast Flow beads (GE Life Sciences) on a rotator at 4° C. for at least 1 h. The beads were washed with wash buffer (100 mM Tris pH 8.0, 300 mM NaCl, 50 mM imidazole, and 3 mM TCEP) to remove nonspecific binding. The protein was eluted with 100 mM Tris pH 8.0, 300 mM NaCl, 100 mM imidazole, and 3 mM TCEP. The protein was further purified using a gel filtration Superdex 200 column in FPLC buffer containing 100 mM Tris pH 8.0, 300 mM NaCl, and 3 mM TCEP. The fractions containing GPX4 were pooled together and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Protein sample of GPX4U46C was initially screened at the High-Throughput Crystallization Screening Center (Luft et al. 2003) of the Hauptman-Woodward Medical Research Institute (HWI) (https://hwi.buffalo.edu/high-throughput-crystallization-center/). The most promising crystal hits were reproduced using under oil micro batch method in a COY anaerobic glove box at 23° C. Plate-like crystals of GPX4U46C were grown using a crystallization reagent comprising 0.056 M sodium phosphate monobasic monohydrate, pH 8.2 and 1.344 M potassium phosphate with protein to crystallization reagent ratio of 2:1 μl. All crystals were subsequently transferred into a similar crystallization reagent that was supplemented by 20% (v/v) glycerol and flash-frozen in liquid nitrogen in the glove box. A native dataset was collected on a crystal of GPX4U46C at the NE-CAT24-ID-C beam line of Advanced Photon Source in Lemont, Ill. Crystals of GPX4U46C were subsequently used as seeds for growing crystals of GPX4U46C-R152H, GPX4U46C-K48L, GPX4U46C-K48A, and GPX4U46C-sulfone outside of the glove box, albeit different crystallization conditions were used for growing these crystals. Crystals of GPX4U46C-R152H were grown in a crystallization condition comprising 0.2 M sodium thiocyanate, pH 6.9, and 20% (w/v) PEG 8000, while those of K48L and K48A were grown in a condition consisting of 0.1 M sodium chloride, 0.1 M MES, pH 6, and 40% (w/v) PEG 8000. Crystals of the fully oxidized GPX4U46C-sulfone were grown in 0.1 M potassium thiocyanate, 0.1 M sodium acetate, pH 5, and 20% (w/v) PEG 8000, and were harvested after one month. In each case, crystals were transferred into the respective crystallization reagent, which was supplemented by 20% (v/v) ethylene glycol.
Crystals of GPX4U46C, GPX4U46C-R152H, GPX4U46C-K48L, GPX4U46C-K48A, and GPX4U46C-sulfone diffracted X-ray at NE-CAT24-ID-C beam line to resolution 1.40 Å, 1.61 Å, 2.10 Å, 1.57 Å, and 1.58 Å, respectively. The images were respectively processed and scaled in space group P21, P21, P21, P212121, and P21, using XDS (Kabsch, 2010). The structure of each protein was determined by molecular replacement method using MOLREP (Vagin and Teplyakov, 2010) program and the crystal structure of GPX4U46C (PDB id: 2OBI) was used as a search model for structure determination of GPX4U46C. The latter structure was used as the search model for subsequent structure determination of other crystal structures. The geometry of each crystal structure was fixed using programs XtalView (McRee, 1999) and COOT (Emsley et al. 2010), and refined by Phenix (Adams et al. 2010). There is one protomer of GPX4 in the asymmetric unit (ASU) of the crystal with space group P21. While the ASU of GPX4-U46C-K48A with space group P212121 contains two protomers. The crystallographic statistics is shown in Table 3.
All documents cited in this application are hereby incorporated by reference as if recited in full herein.
Although illustrative embodiments of the present disclosure have been described herein, it should be understood that the disclosure is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the disclosure.
The present application is a continuation of PCT International Application No. PCT/US2021/022143, filed Mar. 12, 2021, which claims benefit of U.S. Provisional Patent Application Ser. No. 63/122,143, filed on Dec. 7, 2020, and U.S. Provisional Patent Application Ser. No. 62/989,425, filed on Mar. 13, 2020, which applications are incorporated by reference herein in their entireties.
This disclosure was made with government support under grant no. CA209896, awarded by National Institutes of Health. The government has certain rights in the disclosure.
| Number | Date | Country | |
|---|---|---|---|
| 63122143 | Dec 2020 | US | |
| 62989425 | Mar 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/022143 | Mar 2021 | US |
| Child | 17931341 | US |
