The present invention relates to a method for identifying PI3K kinase inhibitors.
Phosphoisonitide 3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) plays a central role in the control of cell growth and proliferation. Overactivation of PI3K/mTOR pathway promotes tumor cells growth and metastasis. PI3K is therefore broadly explored as therapeutic target. Many reversible pan-PI3K inhibitors investigated in clinical trials displayed a low response rate, mainly due to adverse side effects. Inhibition of PI3K using class I pan-PI3K inhibitors triggers a rapid increase in glucose and insulin blood levels.
Isoform-selective inhibition of PI3Kα might alleviate hyperglycemia and hyperinsulinemia, but the selectivity of claimed PI3Kα-specific drugs is currently limited. To date, the specific roles of the different PI3K isoforms (mainly PI3Kα and β) in insulin signaling remain controversial. A redundant physiological role of PI3Kβ in insulin action and sensitivity is under investigation. Recent studies on liver-specific p110α knockout mice showed a minor effect on hyperglycemia suggesting the rescuing role of PI3Kβ in insulin action and metabolic control when PI3Kα is inhibited.
A strategy to achieve isoform selectivity is to exploit targeted covalent inhibitors (TCIs) binding to a cysteine in or around the ATP-binding site. Most of the explored targeted cysteines are in close proximity to a reversible scaffold (e.g. for BTK and EGFR), thus a structural optimization of linker length and flexibility has not been investigated. The method of the present invention aims to optimize targeting a cysteine in or around an ATP-binding site in a protein to be inhibited.
Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to develop optimized chemical probes that are suitable to inhibit a protein target by forming a covalent bond. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.
A first aspect of the invention relates to a method of identifying selective covalently binding inhibitors comprising the steps of
For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.
The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”
As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.
The term “enone” refers to a α,β-unsaturated carbonyl, that is a type of organic compound consisting of an alkene conjugated to a ketone. The simplest enone is methyl vinyl ketone (butenone) or CH2═CHCOCH3. They are electrophilic at both the carbonyl carbon as well as the B-carbon. Depending on conditions, either site is attacked by nucleophiles. Additions to the alkene are called Michael additions and are used in the present invention to covalently modify a target such as Cysteine 862 in PI3Kα.
The term “acrylamide” refers to an amide that is derived from acrylic acid and has the general chemical formula CH2═CHC(O)NH2. Acrylamides are used in compounds of the present invention and undergo Michael addition with Cysteine 862 in PI3Kα.
The term “PI3K” refers to phosphoinositide 3-kinase.
The term “PI3Kalpha”, “PI3Kα” or “p110a protein” relates to a subunit of PI3K that is encoded by the PI3KCA gene.
The term “irreversible” or “irreversible inhibitor” refers to an inhibitor that is able to be covalently bonded to a PI3 kinase in a substantially non- reversible manner, whereas a reversible inhibitor is able to bind to (but is generally unable to form a covalent bond with) a kinase, and therefore can be dissociated from the PI3 kinase. An irreversible inhibitor will remain substantially bound to a kinase once covalent bond formation has occurred. Methods for identifying if a compound is acting as an irreversible inhibitor are known to one of ordinary skill in the art. Such methods include, but are not limited to, enzyme kinetic analysis of the inhibition profile of the compound with the kinase, the use of mass spectrometry of the protein drug target modified in the presence of the inhibitor compound, the use of X-ray crystallography to solve the complex between the protein drug target and the inhibitor compound, discontinuous exposure, also known as “washout” experiments, as well as other methods known to one of skill in the art.
The term “warhead” or “warhead group” refers to a functional group present on a compound of the present invention wherein that functional group is capable of covalently binding to an amino acid residue (such as cysteine, lysine, histidine, or other residues capable of being covalently modified) present in the binding pocket of the target protein, thereby irreversibly inhibiting the protein. Warhead groups are essential for covalently, and irreversibly, inhibiting the protein.
The term “inhibitor” is defined as a compound that binds to and inhibits PI3 kinase with measurable affinity. In certain embodiments, the inhibitors are characterized by IC50 and/or rate constant for irreversible inactivation (Kinact).
Terms such as “compound of this invention” and “compounds of the present invention” include stereoisomers, geometric isomers, tautomers, solvates, pharmaceutically acceptable salts, and solvates of the salts thereof.
The phrase “pharmaceutically acceptable salt” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. If the compound of the invention is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
A first aspect of the invention relates to a method of identifying selective covalently binding inhibitors comprising the steps of
An alternative aspect of the invention relates to a method of identifying selective covalently binding inhibitors comprising the steps of
In certain embodiments, the fast-reacting Michael acceptor moiety of the first or second warhead is an α,β unsaturated carbonyl, wherein
wherein
one of the moieties R1 and R2 is H and
the other moiety R2 or R1 is selected from H, CH3, cyclopropyl, —F, —CH2—F, —CH2—CH2—F, —CN, —N(CH3)—CH3,
with R5 being F or CH3, R6 being C1-6-alkyl and z being 0, 1 or 2.
In certain embodiments, one of the moieties R1 and R2 H, and the other moiety R2 or R1 is H, CH3,
or —N(CH3)—CH3, particularly H or CH3.
In certain embodiments, the third warhead comprising the moderately reacting Michael acceptor moiety is selected from a moiety of formula 2a,
In certain embodiments, the first and second linker are composed of C, N, O and/or H atoms, wherein the linker has a length between 3 Å and 15 Å, particularly between 7 Å and 14 Å, more particularly between 10 Å and 13 Å, even more particularly between 10.5 Å and 12.5 Å.
In certain embodiments, the first and second linker consists of 2 to 5 moieties selected from C1-6-alkyl, —CO—, —NH—, —N(CH3)—, —O—, phenyl, and a heteroaliphatic 4-, 5- or 6-membered ring, particularly C1-6-alkyl, —CO—, —NH—, —N(CH3)—, and a heteroaliphatic 4-, 5- or 6-membered ring. In certain embodiments, the first and second linker consists of 2 to 5 moieties selected from C1-6-alkyl, —CO—, —NH—, —N(CH3)—, —O—, phenyl,
with R5 being C1-3-alkyl, F, —CH2CN or —CN and t being 0, 1 or 2, particularly 0.
In certain embodiments, the first and second linker consists of 2 to 5 moieties selected from C1-6-alkyl, —CO—, —NH—, —N(CH3)—,
with R5 being C1-3-alkyl, F, —CH2CN or —CN and t being 0, 1 or 2, particularly 0.
In certain embodiments, the first and second linker are selected from
wherein
In certain embodiments, the first and second linker are selected from L2-W2-U-(CH2)n-W1-, wherein
In certain embodiments, the first and second linker are selected from
In certain embodiments, the heteroaliphatic 4-, 5- or 6-membered ring is azetidine, pyrrolidine or piperidine, respectively.
In certain embodiments, the first and second scaffold are selected from a moiety of formula 3a, 3b, or 3c, particularly 3a,
wherein
In certain embodiments, the target protein is an enzyme that comprises a cysteine in proximity to a binding site of a reversibly binding inhibitor.
In certain embodiments, the target protein is a protein kinase, particularly a tyrosine kinase.
In certain embodiments, the target protein is selected from PI3K (phosphoinositide 3-kinase), BTK (Bruton's tyrosine kinase) EGFR (epidermal growth factor receptor).
the target protein is PI3K (phosphoinositide 3-kinase), particularly PI3Kα.
The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.
A strategy to achieve isoform selectivity is to exploit targeted covalent inhibitors (TCIs) binding to a cysteine in or around the ATP-binding site. Most of the explored targeted cysteines are in close proximity to the reversible scaffold (e.g. for BTK and EGFR,
The understanding of warhead reactivity is a pivotal parameter in the development of covalent compounds as drugs candidates and chemical probes. Herein, we designed and synthesized a set of compounds bearing nine different warheads, including enones and acrylamides (1-9,
The B-methyl substituted enone (2) and its corresponding moderately electrophilic derivative, β,β-dimethyl enone (3) were selected to explore the target space. A collection of compounds with pairwise matched linkers but either fast (2, 10-12) or moderate (3, 13-15) reacting Michael acceptors were synthesized (
Unsubstituted acrylamides could be exploited in TCIs with the advantage of avoiding off-target reactions with other nucleophiles into the cells. Therefore, nine molecules bearing an acrylamide group and different linkers (
The inventors have examined the irreversible covalent behavior of 19 and 22 in HEK293 cells expressing NanoLuc fused PI3Ks (PI3Kα, PI3Kα C862S, PI3Kβ and PI3Kδ). The reversible analogs of 19 and 22 (19-r and 22-r) were included as negative controls. In washout experiments with wild type PI3Kα, no recovery of the BRET signal was observed for both compounds 19 and 22 (
The library of acrylamide-containing compounds was evaluated for PI3K signaling in SKOV3 ovarian cancer cells. Compounds 19 and 22 displayed a good cellular activity, being twice more potent in cells than CNX-1351. Inhibition of AktSer473 phosphorylation measured by immunoblot analysis was used as an indicator of inhibition of PI3K signaling. Cells were incubated with the drugs (2.5 μM each) for 2 hours. After drug washout, the signaling was immediately recovered for PQR514 and 19-r. On the contrary, a prolonged inhibition of P-AktSer473 was observed for 19, as well as for CNX-1351 (
The metabolic stability of 19, 22 and CNX-1351 was evaluated in vitro in rat liver microsomes. Compounds 19 and 22, bearing a moderate-reactive, drug-like warhead, as well as an amide-containing linker, outperformed the rapidly metabolized CNX-1351 (
Phosphoisonitide 3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) plays a central role in the control of cell growth and proliferation. Overactivation of PI3K/mTOR pathway promotes tumor cells growth and metastasis. PI3K is therefore broadly explored as therapeutic target. Many reversible pan-PI3K inhibitors investigated in clinical trials displayed a low response rate, mainly due to adverse side effects. Inhibition of PI3K using class I pan-PI3K inhibitors triggers a rapid increase in glucose and insulin blood levels.
Isoform-selective inhibition of PI3Kα might alleviate hyperglycemia and hyperinsulinemia, but the selectivity of claimed PI3K α-specific drugs is currently limited. CNX-1351 is the only known PI3Kα inhibitor, having a limited efficiency in covalent bond form, poor in vitro and cellular potency, and chemical features not suitable for a lead optimization process. Recent studies on liver-specific p110α knockout mice showed a minor effect on hyperglycemia suggesting the rescuing role of PI3Kβ in insulin action and metabolic control when PI3Kα is inhibited.
Poor-quality and insufficiently selective molecules currently lead to misleading results, and impair reproducibility and robustness of scientific findings. The lack of selectivity of chemical probes for biological studies also applies to many marketed drugs that cannot be exploited for a detailed mechanism-based biological investigation.[3] Generation of high-quality PI3Kα chemical probes would dissect the role of PI3Ka in cancer and metabolism. Covalent inhibitors, permanently blocking target functions, have emerged as a promising strategy to enhance the ligand binding selectivity for proteins in the same family, representing ideal tools to investigate the specific roles of PI3Ka isoform. A strategy to achieve isoform selectivity is to exploit targeted covalent inhibitors (TCIs) binding to a cysteine in or around the ATP-binding site. Most of the explored targeted cysteines are in close proximity to the reversible scaffold. The inventors aim to target the non-conserved Cys862 in PI3Kα, which is located at >10 Å from the core reversible compound (
The reversible scaffold of PQR309[4] and PQR514[5] was converted into irreversible compounds. An extensive Structure Activity Relationship (SAR) study was performed using CNX-1351[1] reacting group (warhead). A collection of 12 compounds has been prepared introducing different linkers, bearing heteroaliphatic 4-,5-, and 6-member rings (
The library was tested for cellular potency on inhibition of PKB phorphorilation, considered as an indicator of inhibition of PI3K signaling. Time-resolved fluorescence resonance (TR-FRET) tracer displacement assay was used to determine the rate constant for irreversible inactivation (kinact) and the second order rate constant typically used to characterize the covalent binding of irreversible inhibitors to the target protein (kinact/Ki). The pilot chemical probes exceeded in vitro and cellular potency over CNX-1351. Compound 10 showed the best cellular activity, >40-fold compared to CNX-1351 (IC50 for pPKB=6.69 nM vs. 290 nM). The highest maximum potential rate of inactivation (kinact/KI=2.60×10−3 nM−1·s−1) was also observed for compound 10, resulting in the best irreversible inhibitor of our collection, with a clogP of 2.46 compared to CNX-1351 clogP of 4.40, providing better solubility.
The covalent bond adducts between selected compounds and the targeted cysteine residue was confirmed by X-ray crystallography (
The library of enone-containing compounds was evaluated for PI3K signaling in SKOV3 ovarian cancer cells. Compounds 1, 7 and 9 displayed a good cellular activity, being 4-, 9- and 10-fold more potent in cells than CNX-1351 (Table 1), respectively. Inhibition of AktSer473 phosphorylation measured by immunoblot analysis was used as an indicator of inhibition of PI3K signaling. Cells were incubated with the drugs (2.5 μM each) for 2 hours. After drug washout, the signaling was immediately recovered for PQR514. On the contrary, a prolonged inhibition of P-AktSer473 was observed for compounds 1, 7 and 9, as well as for CNX-1351 (
Phosphoinositide-3-kinase (PI3K) signaling is a key regulator of cellular processes such as cell growth, proliferation and metabolism. Constitutively activated PI3K is frequent in tumors and drives cancer progression. PI3K is therefore broadly explored as therapeutic target, but many pan-PI3K inhibitors displayed a low response rate in clinical trials. Inhibition of PI3K using class I pan-PI3K inhibitors triggers a rapid increase in blood glucose and insulin. Recent studies on liver-specific p110α knockout mice showed a minor effect on hyperglycemia suggesting the rescuing role of PI3Kβ in insulin action and metabolic control when PI3Kα is inhibited. The inventors developed a rational approach to increase target selectivity by exploiting a covalent binding of inhibitors targeting non-conserved Cys862 residue on PI3Kα.
Non-conserved C862 residue in PI3Kα is targeted to irreversibly inhibit PI3Kα activity by exploiting covalent inhibition strategy. The X-ray crystallographic structure of PI3Kαin complex with one of our covalent inhibitor was solved, proving the covalent bond between the thiol group of Cys862 and the warhead. Moreover, to confirm the covalent nature of the PI3Kα inhibitors, the inventors have synthesized the corresponding reversible analogs and analyzed irreversible/reversible analogs using a wide range of biochemical and cellular assay platforms. TR-FRET assay was designed to measure drug-target engagement kinetics on the recombinant protein. Only the irreversible inhibitor presented an IC50-shift but not its reversible analog, which is a typical feature of TCIs (
Establishment of a NanoBRET cellular assay for quantification of drug-target engagement kinetics inside live cells introduces a new platform to get physiologically relevant drug-target engagement profile. NanoBRET assay platform exploiting the PI3Kα C862S mutation confirmed the covalent modification of Cys862. In addition, it has been shown that only the targeted PI3Kα is covalently modified by the compound 1 among class IA PI3K isoforms (
Class IA PI3K isoform signaling activities are deconvoluted in terms of phosphorylation of its downstream effector, Akt (S473) using isoform selective reversible PI3K inhibitors. Accordingly, drug washout studies using 1 indicated novel targeting strategies by promoting prolonged PI3Kα inhibition in cancer cells. Irreversible elimination of PI3Kα activity was efficacious to downregulate PI3K aberrant signaling in PI3Kα-driven cancer cells. In the PTEN-deficient cancers, residual Akt phosphorylation after drug washout indicated redundant roles of class IA PI3K isoforms. (
In order to study dynamics of PI3K/Akt downstream activity at the single cell level, stable cell lines expressing a fluorescent reporter fused-Forkhead box protein O1 (FOXO1) has been incorporated, which translocates from the nucleus to the cytoplasm in response to Akt stimulation. Dynamic translocation probes based on the FOXO1 transcription factor enabled measurement of signaling downstream of Akt and revealed on/off dynamics of the PI3K/Akt signaling axis. (
PI3Kα-selective covalent inhibition strategy introduced gain in potency and prolonged inhibitory activity in terms of growth inhibition of PI3Kα-constitutively active luminal breast cancer cell lines. In PTEN deficient melanoma cell line (A2058), pan-PI3K/PI3Kα-selective covalent inhibition strategy using 1 showed superior inhibitory activity compared to PI3Kα-selective reversible inhibition using BYL719. (
Number | Date | Country | Kind |
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21167756.2 | Apr 2021 | EP | regional |
This is the U.S. National Stage of International Patent Application No. PCT/EP2022/059550, filed Apr. 9, 2022, which claims the benefit of European Patent Application No. 21167756.2, filed Apr. 10, 2021.
Filing Document | Filing Date | Country | Kind |
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PCT/EP2022/059550 | 4/9/2022 | WO |