Patent Application
20230391828
A computer readable form of the Sequence Listing is filed herewith by electronic submission. The Sequence Listing, incorporated herewith by reference in its entirety, is contained in the ASCII text file titled “15812362.000001.US00.Sequence.Listing.revised_ST25.txt”, which was created on Aug. 11, 2023, and is 41 KB in size (as measured in Microsoft Windows®).
The present invention is defined by the claims. It relates to peptides and peptidomimetics as well as their medical use in the treatment of hyperproliferative diseases and inflammatory diseases.
In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
Ubiquitination is a reversible protein posttranslational modification that results in formation of mono- or poly-ubiquitin chains attached to substrate proteins. Ubiquitination is involved in almost all cellular processes and targets proteins to a variety of fates and functions. The most known and studied role of ubiquitination is targeting proteins for degradation by the 26S proteasome. Once ubiquitinated, proteins enter the proteasome where they are cleaved. Ubiquitination is a controlled process involving a series of steps, each involving a different type of enzyme: E1 ubiquitin-activating enzymes (also known as E1 enzymes), E2 ubiquitin-conjugating enzymes (also known as E2 enzymes), and E3 ubiquitin ligases (also known as E3 enzymes). E3 enzymes transfer one or more ubquitin molecules to the target protein, thereby labeling it for destruction. The E3 enzymes mediate transfer of one or more ubiquitin to substrate proteins and ensure substrate specificity of ubiquitination.
Within the E3 enzymes, there are three classes which can be structurally and functionally distinguished—HECT domain E3 enzymes (˜30 in humans), RING finger domain E3 enzymes (˜600 in humans) and RBR E3 enzymes (˜10 in humans). The class of RING finger domain E3 enzymes is the most abundant and is characterised by mediating the direct transfer of ubiquitin from the E2 enzyme to the substrate protein. Thus, RING finger domain E3 enzymes serve as a dynamic scaffold that brings together an E2 and a specific substrate. RING finger domain E3 enzymes can function as monomers (UBE4B), dimers (e.g. c-IAP1, TRAF2, MDM2) and as multi-subunit complexes (e.g. APC/C, SCF), one of which contains a RING finger domain. The members of this class are also referred to as RING finger E3 ligases or RING E3s.
In functional terms, RING E3s are involved in regulation of a multitude of cellular processes and functions including control of angiogenesis, apoptosis, DNA damage response and cellular proliferation which makes them key molecules involved in hyperproliferative and inflammatory diseases. Relevant RING proteins include BMI-1, BRCA1, c-CBL, c-IAP1, MDM2, TRAF2, RBX1 and APC11, the two latter being a constituent member of the Skp1/Cull/F-box (SCF) complex and anaphase-promoting complex/cyclosome (APC/C), respectively.
There are two key players in regulating cell cycle progression and genome integrity: the activity of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase and the cullin-RING E3 ubiquitin ligase Skp1/Cull/F-box (SCF) complex, which are very often mis-regulated in cancer. The APC/C consists of at least 14 subunits in human cells and utilizes two co-activators, Cdc20 and Cdh1, to recognize its substrates. There are three main causes of APC/C mis-regulation—alterations in the APC/C itself, Cdc20 and Cdh1 and in the APC/C E2 ubiquitin conjugating enzymes UBE2C and UBE2S. In a similar manner, SCF, consisting of an F-box protein and three core units, can be mis-regulated through altering its interaction with the E2 conjugating enzyme UBE2R1. Both, APC/C and SCF are targeted by inhibitory proteins, Emil and GLMN, respectively, allowing to further fine-tune their activities.
The alterations in the APC/C itself are not very frequent though present in several cancer types. Firstly, in colon cancer cells, mutations of several APC/C subunits were found—APC4, APC6 and APC8. Interestingly, all these mutations were heterozygous indicating the crucial role of the APC/C. It is thought that a quantitative or qualitative disruption of APC/C subunits is sufficient to cause a mis-regulation of APC/C activity and thereby contributes to tumorigenesis. Further, the overexpression of APC/C subunits is associated with poor prognosis and could serve as new markers for cancer evaluation. It was shown that APC3 overexpression is associated with colorectal cancer progression and APC11 overexpression with progression of lung adenocarcinoma and colorectal cancer, respectively. Conversely, downregulation of the APC7 in invasive ductal carcinoma was associated with poor prognosis indicating high variability in APC/C subunits alterations between distinct cancer types. More frequently, APC/C activity is mis-regulated via alterations in APC/C co-activators Cdc20 and Cdh1.
Cdc20 has been frequently overexpressed in a wide range of cancer such as B-cell non-Hodgkin lymphomas, bladder cancer, colorectal cancer, glioblastoma, non-small cell lung cancer and pancreatic ductal adenocarcinoma. Further, Cdc20 overexpression is associated with a poor prognosis. Overall, Cdc20 was proposed to be a new cancer prognostic marker as well as a target of cancer therapy. Interestingly, Cdc20 overexpression is likely to be associated with decrease of the tumour suppressor p53, since Cdc20 was identified as the p53-suppressive gene. Conversely, Cdh1 has been frequently downregulated in distinct cancers such as breast cancer, colorectal cancer, ovary cancer and prostate cancer. Alternatively, it was shown that also invalid Cdh1 hyperphosphorylation drives APC/C inactivation occurring in glioblastoma. As a consequence of downregulation of APC/CCdh1 activity, the levels of APC/CCdh1 substrates are elevated, some of which are oncogenes for instance Skp2 or Plk1.
Finally, the APC/C activity is often mis-regulated by overexpression of its E2 ubiquitin conjugating enzymes UBE2C and UBE2S. Overexpression of UBE2C as well as UBE2S was described in a wide range of cancer and is associated with poor prognosis. Further, UBE2C and UBE2S were suggested to be cancer prognostic markers. UBE2C overexpression was described for instance in breast cancer, bladder cancer, glioblastoma, lung cancer, ovary cancer and melanoma. UBE2C overexpression could be partially explained by its gene amplification, supported by the example of gastric adenocarcinoma in which the amplification of the chromosomal region 20q13.1, where the UBE2C gene is localized, has been described. Further, UBE2S overexpression was described for instance in breast cancer, cervical cancer, hepatocellular carcinoma, lung adenocarcinoma and pancreatic ductal adenocarcinoma.
The primary target of drugs interfering indirectly with the APC/C activity are mitotic cells and their eradication, since cancer cells proliferate faster compared to most of the healthy tissue. There are two main drug classes indirectly inhibiting the APC/C activity—(i) drugs perturbing spindle assembly and activating the spindle assembly checkpoint (SAC) such as microtubule targeting agents and (ii) drugs inhibiting the proteasome.
Microtubule targeting agents bind to tubulin, thus interfering with spindle assembly and activating the SAC. There are two main classes of microtubule targeting agents used in clinics—taxanes and vinca alkaloids. Taxanes, such as paclitaxel and docetaxel, are microtubule-stabilising drugs and vinca alkaloids, such as vinblastine, vincristine, vinolrebine, are microtubule-depolymerizing drugs. Drug-induced SAC activation leads to prolonged mitotic arrest followed in an ideal case by cell death in mitosis. However, an alternative pathway, called mitotic slippage also exists, in which cells progress slowly through mitosis despite activated SAC and form tetraploid cells entering the G1 phase. Tetraploid cells in the G1 phase have different possible fates—post-slippage cell death, entering senescence or continuing in cell cycle. Of note, the response to microtubule targeting agents is very heterogenous, and different cell fates can occur even in the related cells. Importantly, cell polyploidy is connected to drug resistance, thus with cancer relapse. Mitotic slippage is a naturally occurring process caused by slow background degradation of cyclin B, since always a small part of APC/CCdc20 is active. The cell fate decision between mitotic slippage and mitotic cell death is tightly regulated by the balance between two opposing activities, APC/C mediated cyclin B degradation and the activation of the apoptotic pathways. To eliminate mitotic slippage and prefer mitotic cell death, strategies accelerating apoptosis or delay mitotic slippage could be employed. For instance, it was shown that downregulation of p31comet prolongs a mitotic arrest and promotes apoptosis. Mitotic slippage is one of the biggest obstacles of microtubule targeting agents, causing drug resistance and cancer relapse. Further, microtubule targeting agents have strong side effects including neuropathy, limiting their application. To overcome problems with drug resistance as well as with side effects, a combinatorial therapy with lower drug doses should be considered, for example combination with drugs accelerating apoptosis or inhibiting the APC/C activity.
Cellular homeostasis is maintained by keeping balance between protein synthesis and protein degradation. Ubiquitin-proteasome pathway is essential for protein degradation including proteins involved in cell cycle, cell survival, apoptosis or DNA repair. Also, many proteasome substrates are known to be mis-regulated in cancer. Disruption of proteasome activity causes cell arrest followed by cell death due to rapid protein accumulation including APC/C substrates such as cyclins A, B, securin and geminin, key regulators of cell cycle progression.
Interestingly, contrary to predictions, cancer cells are more sensitive to proteasome inhibition than non-cancerous cells, making proteasome a rational cancer therapy target. For instance, it was shown that acute myelogenous leukaemia stem cells are more sensitive to proteasome inhibition than normal hematopoietic stem cells. Higher sensitivity towards proteasome inhibitors in cancer cells is still not fully understood. In general, since for cancer cells protein overexpression and expression of misfolded proteins are typical, they are more dependent on proteasome activity to keep cellular homeostasis.
To this date, three proteasome inhibitors, Bortezomib, Carfilzomib and Ixazomib, have received approval to be used in clinics for treatment of multiple myeloma and mantle cell lymphoma. Bortezomib is the first-generation proteasome inhibitor, it has relatively severe side effects including neuropathy, thrombocytopenia and weakness compared to the second-generation inhibitors Carfilzomib and Ixazomib. Except monotherapy, proteasome inhibitors are also tested in the combined therapy, since it was shown that proteasome inhibition sensitises cells to other chemotherapy drugs and radiation therapy. The advantage of the combination therapy is also lower doses of drugs, thereby fewer side effects.
Even though proteasome inhibitors seem to be very promising in cancer therapy, their biggest disadvantage and current challenge is a frequent development of resistance, thereby cancer relapse. Further, they were successfully used only in treatment of hematopoietic malignancies, however, they are not efficient in solid tumours. Mentioned challenges could be partially solved by the combined therapy.
The APC/C is essential for cell cycle progression. In mouse models, it has been shown that loss of the catalytic core subunit APC2 as well as loss of the APC10 subunit is lethal during early embryogenesis. Further, loss of the APC2 subunit in a mouse model leads to the cell-cycle re-entry of hepatocytes followed by cell cycle arrest in mitosis. More studies about different APC/C subunits have been performed in various cell lines. For instance, the APC3 downregulation inhibits cell growth of colon cancer cells HCT116. Specifically, APC3 downregulated cells accumulate in the G1 phase with the increased p21 protein level. Conversely, APC3 overexpression induces cell proliferation. Further, APC4 downregulation induces prolonged mitosis and metaphase arrest with increased levels of cyclin A and cyclin B in cervical cancer HeLa cells. Interestingly, not all APC/C subunits are essential for successful progression through mitosis. APC7 and APC16 are not required for mitosis in colon cancer cells HCT116 under unperturbed conditions.
Since the APC/C is a large complex with many subunits, it is easier to interfere with its co-activators Cdc20 and Cdh1 to study APC/C functions. For both co-activators, failed embryogenesis was observed in mouse knock-out models. Specifically, Cdc20 knockout mouse embryos die at the two-cell stage following metaphase arrest with high levels of cyclin B. Cdh1 knockout mouse embryos die later at E9.5-E10.5 and not due to basic cell cycle defects, however, due to defects in the endoreduplication of trophoblast cells forming placenta. Suggesting that even though Cdc20 and Cdh1 recognize partially same substrates, they are not functionally redundant and have own specific functions. Consistently with the mouse model, Cdc20 downregulation in various cell lines causes mitotic arrest followed by impaired cell proliferation and eventually cell death. Cdh1 is not essential for completion of mitosis, however, it plays a crucial role in controlling G1 phase and followed S phase. Downregulation of Cdh1 causes shortening G1 phase, due to faster accumulation of cyclin A, and conversely, prolonging S phase, due to caused replication stress and shortage of the dNTPs. Further, Cdh1 deficient cells accumulate DNA breaks and chromosomal aberrations indicating the role of Cdh1 in maintenance of genome stability. Cdh1 deficient cells also fail to maintain the DNA damage induced G2 phase arrest, thus allowing cells to enter mitosis with DNA damage, thereby causing cell death in mitosis.
As mentioned above, there are two E2 ubiquitin conjugating enzymes catalysing ubiquitination reactions together with the APC/C, UBE2C and UBE2S. In various non-cancerous and cancer cell lines, it was shown that UBE2C downregulation supresses cell growth and induces cell cycle arrest in mitosis, specifically in metaphase. Further, in glioma cells, an enhanced apoptosis was observed. Conversely, UBE2S depletion in various non-cancerous and cancer cell lines causes just a minor delay in mitosis and does not have a strong phenotype under unperturbed conditions. However, UBE2S depletion prolongs drug-induced mitotic arrest and suppresses mitotic slippage suggesting the role of UBE2S in SAC inactivation. Co-depletion of UBE2C and UBE2S prolong mitosis more and result in the accumulation of APC/C substrates in agreement with their joint role in assembling ubiquitin chains on APC/C substrates. In the absence of UBE2C and UBE2S, the E2 enzyme UBE2D, which interacts with the APC/C using the same binding site as UBE2C, takes over to support ubiquitination of APC/C subtrates.
The potential of APC/C inhibition for clinical applications is even bigger due to a phenomenon called synthetic lethality. Synthetic lethality is characterised as the setting in which inactivation of either two genes or pathways individually has little effect on cell viability but loss of function of both genes simultaneously leads to cell death. In cancer therapy, the ideal scenario is that cell death occurs only in cancer cells with no or minimum side effect on healthy cells. For instance, cells carrying K-Ras mutations are more sensitive to inhibition of the APC/C resulting in strong accumulation in prometaphase followed by cell death. K-Ras mutations are frequently found in various cancers, such as pancreatic, colon and lung cancers and are correlated with poor prognosis. Interestingly, decreased APC/C activity in patients with lung cancer carrying K-Ras mutation is associated with increased survival. Further, cells with defects in sister chromatid cohesion are more sensitive to APC/C inhibition resulting in mitotic arrest followed by cell death. Importantly, cohesion defects are reported in many cancers. Finally, lack of Cdh1 in mouse embryonic fibroblasts results in a dramatically increased sensitivity to etoposide, a topoisomerase II inhibitor. Around 70% of Cdh1 knockout cells died after treatment with etoposide compared to less than 10% of control cells. Increase sensitivity to topoisomerase II inhibitors is most probably due to the increased level of Top2α, which is an APC/CCdh1 substrate. Similarly, in a B-cell acute leukaemia mouse model and corresponding human cell lines, the lack of Cdh1 results in increased sensitivity to DNA damage due to doxorubicin, topoisomerase II inhibition, or irradiation treatment. Increased radio-sensitivity following Cdh1 downregulation has also been observed in nasopharyngeal carcinoma cells. Similarly, UBE2S downregulation sensitizes HeLa cells to etoposide and doxorubicin resulting in a suppression of cell proliferation.
To conclude, RING finger domain containing proteins such as RING E3s as well as macromolecular complexes comprising them such as the APC/C are being viewed as promising therapeutic targets for cancer therapy, but, with a few exceptions including those discussed above, difficult to target by small- to medium-sized drugs—a property which is also referred to as “non druggable”. This property applies in particular to the enzyme-enzyme interactions involved in the chain of events leading to substrate ubiquitination and thus subsequent proteasomal degradation. These enzyme-enzyme interactions include E1-E2 and, in particular, E2-E3 interactions—in the context of, for example, APC/C.
Hence, current therapeutical approaches in cancer therapy target the APC/C indirectly by activating the SAC (e.g. paclitaxel) or by inhibiting proteasomal degradation of ubiquitinated proteins (e.g. Bortezomib). What has been found so far in terms of agents, is generally associated with one or more undesirable properties or side effects. As a consequence, the technical problem underlying the present invention can be seen in the provision of improved means and methods of interfering with the function of RING E3s including the APC/C, in particular in an inhibitory manner. Given the central function of these proteins and this complex, their involvement in cell division and, accordingly, in proliferation and hyperproliferation, the technical problem underlying the present invention can also be seen in the provision of improved means and methods for the treatment of hyperproliferative and inflammatory conditions and diseases such as cancer.
As is evident from the examples which are part of this disclosure, these technical problems have been solved by the subject-matter of the attached claims.
Accordingly, in a first aspect, the present invention relates to a peptide or peptidomimetic comprising or consisting of the following sequences
The above peptides and peptidomimetics, while grouped into items (a) to (i), share significant structural properties across items. This applies even more to the subject-matter of claim 1. In more detail:
Alpha-Helix Mimetics
Items (a) and (g)-(i) of claim 1 define cyclic peptide or peptidomimetic molecules that are stabilized into alpha-helix secondary structures by introducing a side chain to side chain cross-linkage. In particular, alpha-methylated hydrocarbon, amide, ester, triazole and bis-cysteine cross-linkages are used. The mentioned chemical constraints are introduced at the following positions along their sequences: i, i+4 and i, i+7. In addition, these molecules share physicochemical properties in defined residue positions (
Items (c) to (e) of claim 1 share secondary structure and fold: claims 1(c)-(d) are directed to helix-turn-helix and claim 1(e) to helix-turn mimetics. Further common structural features are: (i) a side chain to side chain cross-linkage such as disulfide (sulfur-sulfur), carbon-sulfur, hydrocarbon and triazole linkages at positions i, i+10 and i, i+9, respectively, and (ii) turn-inducing residues, Pro-Glu and Gly-Pro being preferred for helix-turn-helix and helix-turn mimetics, respectively. Furthermore, molecules included in claims 1(c) to (e) share common properties in defined residue positions (
Beta-Hairpin Mimetics
Items (b) and (f) of claim 1 define cyclic peptide or peptidomimetic molecules that are stabilized into beta-hairpin secondary structures by introducing a cross link: (i) a side chain to side chain link such as disulfide (sulfur-sulfur), carbon-sulfur, hydrocarbon or triazole cross-linkage motifs at positions i, i+9; or (ii) a main chain to side chain or main to main chain amide linkage connecting first and last residue. In addition, a p-turn inducer is required in the sequences, such as Gly-Pro and Pro-D-Pro. Moreover, molecule sequences of claims 1(b) and (f) share common physicochemical properties in defined residue positions (
The present inventors surprisingly found short peptides and peptidomimetics (in the following also denoted “compounds of the invention” or “compounds”) to be capable of interfering with RING E3 function, in particular APC/C function, which is evidenced by the Examples. In particular, said compounds of the invention bind to RING E3 enzymes such as the APC11 subunit of APC/C. Such binding is in a manner which competes with the binding of RING E3s such as APC11 to E2 enzymes. By interfering with the recruitment of E2 enzymes by RING E3s such as the E3 ligase comprised in APC/C, said compounds provide for inhibition of mitosis, which in turn opens a new avenue for treating and preventing hyperproliferative diseases and inflammatory diseases.
Preferred RING E3s are BMI-1, BRCA1, c-CBL, c-IAP1, MDM2, RBX1, TRAF2 and APC11.
A preferred APC11 is human APC11. This applies to all proteins identified in this disclosure, i.e., a preferred species of origin is human.
The pathway leading to the ubiquitination and subsequent degradation of target proteins is well known in the art and involves enzymes generally designated as E1, E2 and E3 (said nomenclature being used above as well as in the introductory part of this disclosure).
Moreover, the inventors provided a plurality of classes of compounds which, while exhibiting similarities in terms of primary structure, preferably adopt different secondary structures. These different secondary structures are preferably defined in terms of the canonical secondary structure elements (helix, sheet, or combination of those with turns) as they are known to occur in polypeptides and proteins. The preferred secondary structures of the peptides and peptidomimetics are subject of a preferred embodiment disclosed further below. Yet, and despite differences in secondary structure, all compounds of the invention share physicochemical features in the three-dimensional space which are required for RING E3 binding and inhibition, in particular APC/C binding and inhibition. These physicochemical properties are features of the primary sequence of the compounds of the invention.
Indeed, the overarching property of the compounds of the invention is their capability to interfere with RING E3 function, including APC/C function. Given the central role of RING E3s and APC/C in cell division and proliferation, this opens the door to a host of therapeutic applications which are described in more detail below. Of note, the Examples enclosed herewith provide ample proof of this capability for a host of peptides and peptidomimetics implementing the subject-matter of this invention.
Moreover, the inventors did not only provide proof of the compounds' capability to slow down or stop cell division or induce apoptosis, but furthermore provide optimized compounds which are successfully delivered to cells. Also, this is documented in the Examples. Of note, optimization of delivery, while being preferred, is not a compulsory feature. In structural terms, delivery is optimized by N- and or C-terminal modifications. On the other hand, RING E3 and APC11 binding is governed by the sequence of amino acids.
Indeed, the compounds of the invention are composed of residues or building blocks, which generally are amino acids. These residues are designated by X and a number indicating their position within the sequence, e.g., X1, X2 etc. or generally Xi. In terms of structure, the building blocks of the peptides are preferably proteinogenic amino acids; see Table 1 below.
Yet, for the purpose of optimizing and fine-tuning RING E3 and APC/C binding and inhibition and/or delivery, the inventors also developed peptidomimetics. In such peptidomimetics, non-proteinogenic amino adds and/or non-naturally occurring amino acids take the place of proteinogenic amino acids at certain positions in the amino acid sequence of the compounds of the invention. Such amino adds are also termed “non-natural” “unusual” herein and are listed in Table 2 below. As a consequence, to the extent compounds of the invention contain such amino acids, they are referred to as “peptidomimetics” herein.
In terms of connectivity between the amino acids, in most instances this is a canonical main chain peptide bond connecting the alpha carboxylate of an amino acid with the alpha amino group of the adjacent (in direction from N- to C-terminus) amino acid. In particular, if not specified otherwise, the connection between adjacent residues Xi and Xi+1 (e.g. X1 and X2, or X2 and X3 etc.) is a main chain peptide bond (amide). In other words, if not explicitly specified otherwise, the term “peptidomimetic” as used herein does not extend to other types of connectivities between adjacent amino acids or building blocks of compounds of the invention.
In a number of instances, cross-links are either preferred or compulsory. Accordingly, these cross-links are specified either in the first aspect of the invention or in preferred embodiments thereof. These cross-links add circular elements to the compounds of the invention. This is a preferred means of stabilizing secondary- and/or three-dimensional structure, thereby facilitating binding to RING E3s such as the APC/C.
There are three preferred ways of implementing such cross-links: (i) main chain peptide bonds connecting the first with the last residue, (ii) peptide bonds (amides) which involve a main chain functional group (carboxylate or amino group) and a side chain functional group (amino group or carboxylate) or only corresponding side chain functional groups, and (iii) cross-links which are not amides.
Having said that, the invention is not limited to these particular and preferred cross-links. As known in the art, cross-links, especially in the context of peptides and peptidomimetics, generally serve to constrain the structure of said peptides and peptidomimetics. The chemical moieties involved in target binding are preferably not located in the cross-links of the compounds of the invention. Target binding is conferred by the sequence of amino acids of said compounds. In that sense, cross-links are means of enhancing, and depending on the type of cross-link used, they can also participate in binding to RING E3s such as APC11.
Class (iii) of cross-links may be implemented using functional groups of proteinogenic amino acids. An example is the S—S bond (disulfide) connecting two Cys residues within a given compound. Other implementations make use of the side chains of non-natural amino acids. This is specified in the preferred embodiments disclosed further below.
The presence of cross-links is also designated by the term “covalently connected” herein, given that cross-links involve a covalent bond. Generally speaking, and in line with the above, such cross-links are not particularly limited. Preference is given to cross-links which, in terms of structure, connect the main chain of the compounds of the invention by a chain of atoms which has a length of 20 or less atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or atoms. In one preferred embodiment, said chain of atoms consists of 3, 4 or 5 atoms. In another preferred embodiment, said cross-link comprises one or more group of atoms; for instance, 2 aromatic rings such as benzene, pyridine or triazole. In case of 2 aromatic rings, this may be implemented as a biphenyl or bipyridine moiety. In terms of counting the number of atoms forming said chain of atoms, only one edge of a given ring is counted, e.g. 4 atoms in case of benzene and pyridine. In case of a ring with an uneven number of atoms, the shorter edge is counted, e.g. 2 atoms in case of triazole. Preferred lengths of the chain of atoms in case of the presence of two aromatic rings are 10, 11, 12, 13, 14 and 15.
The term “main chain” has its usual meaning. It refers to the backbone of the peptides and peptidomimetics of the invention. As such it is defined by the following formula:
Said chain of atoms may consist of any atoms, preferably C-, N-, O- and S-atoms, at least one half of said atoms being preferably C. The chain of atoms may also consist only of carbon atoms. It is understood that, depending on the chosen chemistry of the cross-link, said chain of atoms may be substituted, but does not have to be. In case of absence of substituents at one or more or all given sites (i.e., the atoms forming said chain of atoms), the free valences are filled with hydrogen. Said chain of atoms may comprise one or more such as 2 or 3 double bonds, preferably C═C double bonds. The chain of atoms may comprise functional groups such as —S—S—, —COO— and —CONH—, wherein the carbonyl oxygens in —COO— and —CONH— and the hydrogen in —CONH— do not add to the count of atoms in said chain of atoms. Examples of said chain of atoms include —C—C—C—C— and —C—C═C—C—, i.e., chains of 4 atoms, wherein in these examples preference is given to all free valences being filled with hydrogen (not including the free valences connecting to the main chain, preferably to the alpha-carbon of the main chain at the respective positions). Further examples include —C—S—S—C— and —C—S—C—C—. Also in these cases, the preferred attachment site on the main chain is an alpha-carbon, and otherwise the free valences are filled with hydrogen.
Preferred implementations of —S—S— for items (b)-(f), —COO— for items (g)-(i) and —CONH— for items (a) and (g)-(i) connecting side chains by covalent bonds are as follows:
Preferred implementations of —C—C═C—C— for items (a)-(i), —C—C—C—C— for items (a)-(i), —C—S—S—C— for items (b)-(f) and —C—S—C—C— for items (b)-(f) connecting main and side chains by covalent bonds are as follows:
Arg mimetics with the guanidino group being derivatized may be used for positions X12 of item (a), X1 and X12 of item (b), X15 of item (c), X16 of item (d), X13 of item (e), X11 of item (f), X13 of items (g)-(i). Preferred implementations thereof are as follows:
The invention also encompasses compounds which comprise the peptides or peptidomimetics defined in the first aspect and all its preferred embodiments. This does not apply in those instances where an alpha-amino group of the first residue (X1) and/or an alpha-carboxylate of the respective last defined residue (e.g. X14 in case of part (a) of the first aspect) are involved in a cross-link. It also does not apply to those preferred embodiments detailed further below where the N- and/or the C-terminus are further derivatized (e.g., in the simplest case, by acylation, preferably acetylation of the N-terminus and/or amidation of the C-terminus).
Otherwise, there is room for flanking sequences at one or both termini, given that the APC/C-interfering functionality is provided by the sequence which is specifically spelled out herein. Such flanking sequences are preferably peptidic in nature, more preferably they comprise or consist of proteinogenic amino acids (Table 1) as building blocks. Flanking sequences may be of any length, preference being given to a length of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In case flanking sequences are present at either terminus, their length may be chosen independently.
Of note, compounds of the invention may be labelled or equipped with tags.
Labels include those which do not entail structural modification of the compounds such as isotope labels and radioactive labels. Other labels entail the attachment of a detectable moiety to the compounds of the invention, e.g. fluorescent or luminescent labels. A further type of label is a label which is a substrate of an enzyme, wherein preferably said enzyme converts said substrate into a product which is amenable to detection.
Labels confer advantages for certain methods and uses of compounds of the invention which methods and uses are disclosed further below.
Generally speaking, the compounds of the invention exhibit tolerance as regards exchange and modification of residues. Yet a distinction can be made between residues which are more tolerant and less tolerant in that respect. This is the subject of the following preferred embodiments which disclose implementations of parts (a) to (i) of the first aspect.
In a preferred embodiment of part (a) of the first aspect
In a preferred embodiment of part (b) of the first aspect
In a preferred embodiment of part (c) of the first aspect
In a preferred embodiment of part (d) of the first aspect
In a preferred embodiment of part (e) of the first aspect
In a preferred embodiment of part (f) of the first aspect
In a preferred embodiment of part (g) of the first aspect
In a preferred embodiment of part (h) of the first aspect
In a preferred embodiment of part (i) of the first aspect
In a preferred embodiment of the peptide or peptidomimetic of the invention
The term “physiological conditions” is known in the art. Preferably, for example if intracellular conditions are to be mimicked, it may be implemented by conditions comprising: 14 mM Na+, 140 mM K+, 10−7 mM Ca2+, 20 mM Mg2+, 4 mM Cl−, 10 mM HCO3−, 11 mM HPO42− and H2PO4−, 1 mM SO42−, 45 mM phosphocreatine, 14 mM carnosine, 8 mM amino acids, 9 mM creatine, 1.5 mM lactate, 5 mM ATP, 3.7 mM hexose monophosphate, 4 mM protein and 4 mM urea.
The terminology used in this embodiment to define secondary structure elements is common in the art. For example, a beta-turn is generally composed of four amino acids; see also the above residue ranges for compounds comprising beta-turns. These four positions are numbered from N- to C-terminus as positions 1 to 4. Positions 2 and 3 are particularly relevant for inducing and maintaining the turn conformation.
It is understood that the terms “alpha-helix” (or equivalently “alpha-helical”), “beta-strand/sheet” and “turn” define the main chain dihedral angles, in particular those adjacent to the alpha-carbon of the amino acid under consideration. These angles are commonly designated Φ and Ψ. They fully define the main chain conformation, given that the peptide bond is conformationally constrained. In the case of the canonical alpha-helix, typical Φ (−64°) and Ψ (−42°) dihedral angles may allowed to vary by ±35°. For beta-sheet, consisting as said of two beta strands joined by a turn, the most typical values of Φ (−120°) and Ψ (+135°) for the strand can vary by ±35°, respectively. For the joining turn, Φ and Ψ dihedral angles may vary depending on the turn-type (Table 3). Values at positions i+1 and i+2 can vary by ±30°, being possible to have one of these values with a deviation up to ±45°. Preferred are type II′ turns.
−30°
120°
30°
−30°
Means and methods for determining secondary structure are known in the art and include circular dichroism and high-resolution structure determination, the latter including NMR and X-ray crystallography. Peptide structure determination by NMR is reviewed, for example, in Williamson, Methods Mol. Biol. 1993, 17, 69-85. X-ray crystallography of peptides is described, for example, in Spencer and Nowick, Isr J Chem. 2015, 56, 678-710 (doi:10.1002/ijch.201400179).
Furthermore, in silico methods are available which permit to predict secondary structure. The use of such methods is deliberately envisaged for the purpose of verifying the features required by the above embodiment. These methods are reviewed by Pande, V. in Protein Folding Studied with Molecular Dynamics Simulation, in Roberts G. C. K. (eds.) Encyclopedia of Biophysics. Springer, Berlin, Heidelberg (2013) (doi.org/10.1007/978-3-642-16712-6_730) and Geng, H. et al. in Comput. Struct. Biotechnol. J. 2019, 17, 1162-1170 (doi: 10.1016/j.csbj.2019.07.010).
In a further preferred embodiment, alpha-amino acids located at positions 2 and/or 3 of a beta-turn are replaced by beta-turn inducing amino acids, wherein preferably said beta-turn inducing amino acids are selected from the following:
In the preferred embodiment above, use is made of beta-turn inducing moieties at one or both positions 2 and/or 3. A repertoire of beta-turn inducing moieties is available to the skilled person. This is reviewed for instance by Robinson, J. A. in Acc. Chem. Res. 2008, 41, 1278-1288 (doi.org/10.1021/ar700259k) and Nair, R. V. et al. in Chem. Commun. 2014, 50, 13874-13884 (doi.org/10.1039/C4CC03114H).
In a further preferred embodiment, said peptide or peptidomimetic is capable of interfering with ubiquitination.
As mentioned above, ubiquitination is a cellular process which targets proteins for degradation. In the course of cell division, for cell division to proceed, especially to anaphase, degradation of proteins involved in earlier stages of mitosis is key. Interfering with the ubiquitination function of RING E3s and APC/C is therefore a means of stopping mitosis.
Assays for determining such capability are known in the art and disclosed in the Examples. In a preferred embodiment, said interfering is determined by bringing said peptide or peptidomimetic into contact with APC/C, Cdc20, and E2 enzyme (UBE2C, UBE2D or UBE2S), ubiquitin, a substrate (e.g. securin or cyclin B1) and ATP, and quantifying the amount of ubiquitinated substrate in presence and absence of said peptide or peptidomimetic, wherein a decreased amount of ubiquitinated substrate in presence of said peptide or peptidomimetic is indicative of said peptide or peptidomimetic being capable of interfering with ubiquitination.
A preferred substrate is securin.
The amount of ubiquitinated substrate (in the above implementation securin or cyclin B1) may be determined by SDS-PAGE followed by fluorescence detection.
Alternatively or in addition, the capability of the compounds of the invention to bind and interfere with the ubiquitination machinery contained in APC/C or with RING E3 function may be assessed/predicted using in silico means and methods, e.g., by molecular dynamics simulation and associated calculation of free energies of binding as described by McCammon, J. A. and Harvey, S. C. in Dynamics of Proteins and Nucleic Acids, Cambridge University Press, USA (1987), Leach, A. R. in Molecular Modelling: Principles and Applications, 2nd Edition, Pearson Education Limited, UK (2001) and Wang, J. M. et al. J. Am. Chem. Soc. 2001, 123, 5221-5230 (doi:10.1021/ja003834q).
In a preferred embodiment, said interfering is reducing or abolishing the activity of an ubiquitinating enzyme and/or of a ubiquitin ligase.
In a particularly preferred embodiment, said enzyme is an E2 enzyme, preferably UBE2C, UBE2D, UBE2S, UBE2L3 or UBE2R1; or said ligase is an E3 ligase, preferably APC11, RBX1 or c-CBL.
The protein names used in this disclosure are art-established. For example, the UniProt Consortium provides curated information about proteins (Nucl. Acids. Res. 47, D1: D506-D515 (2019)).
In a further preferred embodiment, said peptide or peptidomimetic stops cell division or induces cell death.
Assays for determining such capability are known in the art and disclosed in the Examples. In a preferred embodiment, stopping or inducing is determined by bringing cells into contact with said peptide or peptidomimetic and determining whether said cells proliferate, stop dividing, or undergo cell death, wherein cells which do not divide or cell death is indicative of said peptide or peptidomimetic stopping cell division or inducing cell death. Useful cells for such assay include those used in the Examples, i.e., HeLa, hTERT RPE-1, A549, HT-1080, RKO and SW480 cells. Preferred are HeLa cells.
Also, an extension of the time required for mitosis is indicative of a beneficial effect of the compounds of the invention.
An extended duration of mitosis, arrest of cell division and cell death are all associated with morphological changes which are amenable to detection by live cell microscopy, which is a preferred means of determining these outcomes upon bringing compounds of the invention into contact with cells.
In a further preferred embodiment, said peptide or peptidomimetic is modified
It is known in the art that once proof of principle is established for a compound (here the capabilities disclosed in the preceding preferred embodiments), further optimization of such compound (generally referred to as “lead compound”) is, albeit not strictly necessary, preferred or desirable. In terms of optimizing, generally the key pharmacokinetic properties of delivery, absorption, distribution, metabolism or excretion, the latter four commonly abbreviated as “ADME”, are of interest.
Related to the notion of lead compounds, peptides and peptidomimetics of the invention may also be used in a screen. Such screens may employ the assays disclosed herein and serve to identify hits and/or peptides or peptidomimetics with particularly good performance. In view of the preferred derivatizations disclosed above and below, one may generate a focused library around one or more given peptides or peptidomimetics of the invention, wherein starting from a given compound, a variety of modifications (also referred to as derivatizations herein) may be applied independently or in conjunction. This serves to sample the sequence space (as well as the functional space) in the closer or wider proximity of said given peptide or peptidomimetic(s).
Screens may be particularly useful in those cases where binding to a RING E3 such as APC11 has been confirmed, but optimization of its inhibitory properties is desirable. On the other hand, also binding may be further optimized by performing a screen with a focused library as disclosed above.
As disclosed further below, the present inventors explored and implemented a number of optimizations in that respect. Alternatively, or in addition, use may be made of the well-established modification recited in this preferred embodiment.
In a preferred embodiment of part (a) of the first aspect and all its preferred embodiments as defined above and below, a side chain of X4 is covalently connected to a side chain of X11.
In a preferred embodiment of part (a) of the first aspect and all its preferred embodiments as defined above and below,
Items (a) and (g) define preferred modifications which increase the stability of said peptide or peptidomimetic towards enzymatic degradation from exopeptidases or useful for labelling to identify protein interactions and delivery. In particular for the last one, it includes endosome escape domains and functional moieties that target intracellular esterases.
Items (b) to (f) define alternative implementations of the X4-X11 cross-link. Each of these may be combined with (a) and/or (g).
In a preferred embodiment of part (b) of the above embodiment (said above embodiment in turn relating to part (a) of the first aspect), said bond is as follows:
In a preferred embodiment of part (c) of the above embodiment, said bond is as follows:
In a preferred embodiment of part (d) of the above embodiment, said bond is as follows:
In a preferred embodiment of part (e) of the above embodiment, said bond is as follows:
In a preferred embodiment of part (f) of the above embodiment, said bond is as follows:
In a preferred embodiment of part (b) of the first aspect and all its preferred embodiments as defined above and below, a side chain of X2 is covalently connected to a side chain of X11.
In a preferred embodiment of part (b) of the first aspect and all its preferred embodiments as defined above and below,
Items (b) and (c) are alternatives, as are sub-items a. to g. within item (c).
Items (d) and (e) are alternatives, as are sub-items a. to g. within item (d).
The most preferred connections are (c).a. and (c).d.
Any choice from these items may be combined with (a) and/or (f).
In the above preferred embodiment, there is recitation of the option that a cross-link comprises a carbon-carbon double bond. This is a result of a known process of introducing such cross-links; see, e.g., Robinson et al., Chem. Comm., 2009, 4293-4295. Such double bond may be reduced to give rise to the corresponding carbon-carbon single bond. Both options are embraced by the present invention. Preferred implementations are shown above, see the structural formulae at the end of the above embodiment.
Of note, in relation to item (b) of the above embodiment where X2 and X11 are independently selected from Trp, Phe, Tyr, Ile and Leu, such cross-link between X2 and X11 is not preferred.
In a preferred embodiment of part (c) of the first aspect and all its preferred embodiments as defined above and below,
Items (b) to (h) are alternatives. Any choice therefrom may be combined with (a) and/or (i).
In a preferred embodiment of part (d) of the first aspect and all its preferred embodiments as defined above and below,
Items (a) to (g) define alternatives. Any of these may be combined with (h).
In a preferred embodiment of part (e) of the first aspect and all its preferred embodiments as defined above and below,
Items (b) to (h) are alternatives. Any of these may be combined with (a) and/or (i).
In a preferred embodiment of part (g) of the first aspect and all its preferred embodiments as defined above and below
In a preferred embodiment of part (h) of the first aspect and all its preferred embodiments as defined above and below
In a preferred embodiment of part (i) of the first aspect and all its preferred embodiments as defined above and below
Where cross-links are implemented either as carbon-carbon double bond or involving a triazole group, a carbon-carbon double bond, e.g. as formed in a first step during preparation of the respective compound, may be reduced to a carbon-carbon single bond.
In a preferred embodiment, said peptide or peptidomimetic is conjugated, preferably via a linker, to a ligand of an E3 ligase. Generally speaking, such E3 ligase may be freely chosen. A compound of the invention, when conjugated with a ligand of an E3 ligase, provides a heterobifunctional molecule. Conjugation is preferably via main chain peptide bonds or isopeptide bonds. Conjugation may be in any orientation. Preferably, such conjugates consist of the three specified elements: peptide or peptidomimetic of the invention, linker, and E3 ligand. The location of the linker is between the other two elements. Generally speaking such bifunctional molecules are capable of recruiting E3 ligases are also known as proteolysis targeting chimeras (PROTACs). Exemplary E3 ligases as well as linker attachment point are described in, for example, Front. Chem., 5 Jul. 2021|https://doi.org/10.3389/fchem.2021.707317. Linkers are not particularly limited. Preferred are short and flexible linkers. Examples include peptidic linkers auch (Gly4Ser)3 and short PEG molecules such (PEG)n, n being 1, 2, 3, 4, 5, or 6.
In an alternative approach, a compound of the invention and said ligand of an E3 ligase are administered to a cell or formulated as a medicine (including the option of separate, i.e., subsequent in any order, administration). In such a case, both the compound of the invention and the ligand are modified by the addition of functional groups which, once compound and ligand are in the cell, react to form a covalent bond (e.g. click chemistry).
This preferred embodiment combines the capability of peptides and peptidomimetics of the invention to bind to RING E3s such as BMI-1, BRCA1, c-CBL, c-IAP1, MDM2, RBX1, TRAF2 and APC11 or further RING E3s as disclosed herein with the capability of E3 ligands to recruit another E3 ligase. Such E3 ligase in turn recruits a cognate E2 enzyme. This provides for ubiquitination and subsequent degradation of APC/C. This effect is also a means of providing the beneficial and therapeutic effects in accordance with this invention which are described in more detail further below.
The general notion of conjugating an E3 ligand to a compound (here the peptide or peptidomimetic of the invention) known to bind a target (here RING E3s such as APC/C) for the purpose of triggering degradation of said target via the ubiquitination pathway has been described, for example, in An and Fu, EBioMedicine 36: 553-562 (2018). Implementations of E3 ligands can also be found in this publication. Exemplary E3 ligands include Pomalidomide, Thalidomide, Lenalidomide and VHL-1 (the latter being a ligand of Von Hippel Lindau (VHL) E3 ligase); see Table 1 of An and Fu, loc. cit. Examplary E3 ligases are MDM2, IAP, VHL and cereblon. Useful linkers include short, peptidic and/or flexible linkers; see, e.g. Chen et al., Adv Drug Deliv Rev. 2013 Oct. 15; 65(10): 1357-1369. An example is (Gly4Ser)3.
Preferred E3 ligands are cognate or known ligands of RING E3s, for example Pomalidomide, Thalidomide, Lenalidomide, VHL-1, VHL-2 and derivatives thereof. Preferred RING E3s are MDM2, IAP, VHL, cereblon, APC/C as well as those disclosed herein above.
In a second aspect, the present invention relates to a peptide or peptidomimetic of the first aspect and of any of its embodiments for use in medicine.
Given that the compounds of the invention are capable of interfering with cell division, their medical use is evident. Preferred medical use is the subject of further aspects disclosed below.
In a third aspect, the present invention provides a medicament comprising or consisting of the peptide or peptidomimetic of any one of the preceding aspects and embodiments thereof.
The medicament (also referred to as “pharmaceutical composition”) may further comprise pharmaceutically acceptable carriers, excipients and/or diluents. Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well-known conventional methods.
These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. It is particularly preferred that said administration is carried out by injection. The compositions may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like the site of a tumour.
The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
In a preferred embodiment,
Preferred agents to be combined with compounds of the invention are those which directly or indirectly interfere with APC/C function and/or related systems (see in this respect the introductory section of this disclosure above) or with cell division in general. These agents are reviewed and defined in the introductory part of this disclosure. Further agents suitable for a combination therapy include immunotherapeutic agents such as anti-PD1 antibodies, MDM2 inhibitors such as Nutlin, histone deacetylase inhibitors such as valproic acid, cisplatin, inhibitors of MEK1 and/or MEK2, inhibitors of Hsp90 such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), BMI-1 inhibitors such as unesbulin (PTC596) and PTC-209, and anti-androgen receptor agents.
Given that the agents of part (b) on the one hand and the compounds of the invention on the other hand have distinct molecular mechanisms of action, such combinations are characterized by a synergistic effect on cell division, cell death and mitigating or curing hyperproliferative disorders.
In a fourth aspect, the present invention provides a peptide or peptidomimetic of any one of the preceding aspects and embodiments thereof or a medicament of the third aspect for use in a method of treating, ameliorating or curing a hyperproliferative disease and/or an inflammatory disease.
In a preferred embodiment, said hyperproliferative disease is a benign or malign tumour, a metastasis or cancer.
Preferably, said cancer is B-cell non-Hodgkin lymphoma, bladder cancer, breast tumour or breast cancer, cancer of the stomach, cervical cancer, colorectal cancer, esophageal squamous cell carcinoma, gliomas, glioblastoma, head and neck cancer, incasive ductal carcinoma, leukemia such as acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma, liposarcoma, liver cancer such as hepatocellular carcinoma, lung cancer such as lung adenocarcinoma and non-small cell lung cancer, melanoma, osteosarcoma, ovary cancer, pancreatic cancer such as pancreatic ductal adenocarcinoma, or prostate cancer.
Preferred inflammatory diseases include rhematoid arthritis, colitis, psoriasis, inflammatory bowel disease, inflammatory diseases of the central nervous system including the brain such as Alzheimer's disease and Parkinson's disease, and inflammatory diseases of the cardiovascular system including the heart such as cardiac hypertrophy and cardiac failure.
Such preferred medical indications are evident in view of the function of RING E3s and APC/C and the information given in the introductory section above. Having said that, and in line with the disclosure above, beneficial effects are expected for all types of hyperproliferation, inflammation, or instances or disorders where stopping cell division is desirable.
In a fifth aspect, the invention provides an in vitro or ex vivo method of interfering with or stopping cell division, said method comprising bringing one or more peptides or peptidomimetics of any one of the preceding aspects and embodiments thereof in contact with a cell.
In a preferred embodiment, the cells are cells in culture or comprised in a tissue. In a sixth aspect, the invention relates to a method of purifying APC/C, said method comprising
In a related aspect, the invention provides a method of purifying a RING E3 protein, said method comprising
In a preferred embodiment, said peptide or peptidomimetic is immobilized on a carrier or bead. Beads may be magnetic, e.g. paramagnetic beads which facilitates their handling when purifying in accordance with the method of the sixth aspect. Such carriers or beads with compounds of the invention being covalently or non-covalently immobilized thereon may be viewed as affinity matrices.
Once APC/C is bound to such carrier, bead or matrix, it may be detected by any downstream detection method such as ELISA or Biacore.
In a further preferred embodiment, said sample is a total cell extract.
In an alternative, any solution or suspension comprising APC/C may be used as sample.
In a seventh aspect, the invention provides a method of detecting APC/C, said method comprising
For the purpose of detecting, labelled forms of the compounds of the invention may be employed which labelled forms are disclosed herein further above.
In an eighth aspect, the invention provides a kit comprising or consisting of one or more peptides or peptidomimetics of the first aspect and its embodiments.
In a preferred embodiment, said kit further comprises or further consists of
In a further preferred embodiment of said kit, said peptide or peptidomimetic
In a ninth aspect, the present invention relates to a method of treating, ameliorating, curing or preventing a hyperproliferative disease and/or an inflammatory disease, said method comprising or consisting of administering one or more peptides or peptidomimetics of the first or second aspect or a medicament of the third aspect to a patient suffering from or being at risk of developing said disease.
Preferred embodiments of said disease are those disclosed further above.
In a tenth aspect, the invention provides a use of a peptide or peptidomimetic of the first aspect and of any one of its embodiments as a lead for developing a pharmaceutically active agent, wherein preferably said pharmaceutically active agent has anti-proliferative activity or anti-inflammatory activity.
While it is understood that the compounds of the invention themselves are useful as therapeutic agents, it will in many instances be possible to fine-tune pharmacokinetic properties thereof.
Related thereto, and in an eleventh aspect, the invention provides an in vitro or ex vivo method of developing a lead for the treatment of a hyperproliferative disease and/or an inflammatory disease, said method comprising:
In a preferred embodiment, said modifying is as defined in relation to the optimization of pharmacokinetic properties including delivery and ADME.
In the following, especially preferred compounds of the invention are disclosed. These compounds are not only especially preferred implementations of the first aspect, but at the same time define preferred embodiments of any of the other aspects of this invention.
Of note, the respective SEQ ID NOs define the core part of the peptide or peptidomimetic, i.e., without N- and C-terminal modifications and without information about cross-links (or, equivalently, cyclic structure). Generally speaking, and if not otherwise indicated herein above, such terminal modifications and cross-links are preferred features. Indeed, the terminal modifications are optional features. They generally provide improved delivery. Yet, sufficient activity is observed also in absence of said terminal modifications. Further information about the cross-links can be found in the description of the first aspect of the invention above.
It is evident from the numerous references to the Examples in the following that substantially all claimed classes of compounds have been reduced to practice.
Especially preferred peptides and peptidomimetics in accordance with part (a) of the first aspect: SEQ. ID. NO. 31. (designated G3-4 in the Examples)
Most preferred in accordance with part (a) of the first aspect are the above compounds comprising the sequence of SEQ ID NO: 33 or 36.
Especially preferred peptides and peptidomimetics in accordance with part (b) of the first aspect:
Most preferred in accordance with part (b) of the first aspect are the above compounds comprising the sequence of SEQ ID NO: 1 to 5, 41 to 43, 48 to 54, 94, 100, and 108 to 110, and among those the compounds comprising the sequence of SEQ ID NO: 3, 42 and 100.
Especially preferred peptides and peptidomimetics in accordance with part (c) of the first aspect:
Most preferred in accordance with part (c) of the first aspect is the above compound comprising the sequence of SEQ ID NO: 6 and 55.
Especially preferred peptides and peptidomimetics in accordance with part (d) of the first aspect:
Especially preferred peptides and peptidomimetics in accordance with part (e) of the first aspect:
Especially preferred peptides and peptidomimetics in accordance with part (f) of the first aspect:
SEQ. ID. NO. 71. cyclo(1,13)-[L 2NaI L W K W E W E K R K δOrn] (as shown in Figure below)
Most preferred in accordance with part (f) of the first aspect is the above compound comprising the sequences of SEQ ID NO: 19 and 22.
Especially preferred peptides and peptidomimetics in accordance with part (g) of the first aspect:
Most preferred in accordance with part (g) of the first aspect is the above compound comprising the sequence of SEQ ID NO: 28 and 30.
Especially preferred peptides and peptidomimetics in accordance with part (h) of the first aspect:
Most preferred in accordance with part (h) of the first aspect are the above compounds comprising the sequences of SEQ ID NO: 76, 77 and 78, respectively.
Especially preferred peptides and peptidomimetics in accordance with part (i) of the first aspect:
Most preferred in accordance with part (i) of the first aspect are the above compounds comprising the sequences of SEQ ID NO: 81, 82 and 83, respectively.
The present invention furthermore relates to the following items:
The Figures show:
(B) The amount of ubiquitinated securin, as shown in (A) was quantified and normalised to the amount of product formed by control (100%) and the mean and SD from 5 independent experiments were plotted. Statistical significance was tested using one-way Anova test, ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).
(B) The amount of ubiquitinated securin, as shown in (A) was quantified and normalized to the amount of product formed by control (100%) and the mean and SD from 3 independent experiments were plotted (left). Analogous quantification of the amount of unmodified securin was done (right), time point 0 min was set to 100%. Statistical significance was tested using one-way Anova test, ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).
(B) The amount of ubiquitinated securin, as shown in (A) was quantified and normalized to the amount of product formed by control (100%) and the mean and SD from 3 independent experiments were plotted (left). Analogous quantification of the amount of unmodified securin was done (right), time point 0 min was set to 100%. Statistical significance was tested using one-way Anova test, ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).
(B) The amount of ubiquitinated securin, as shown in (A) was quantified and normalized to the amount of product formed by control (100%) and the mean and SD from 3 independent experiments were plotted (left). Analogous quantification of the amount of unmodified securin was done (right), time point 0 min was set to 100%. Statistical significance was tested using one-way Anova test, ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).
(B) The amount of ubiquitinated securin, as shown in (A) was quantified and normalized to the amount of product formed by control (100%) and the mean and SD from 3 independent experiments were plotted (left). Analogous quantification of the amounts of unmodified securin was done (right), time point 0 min was set to 100%. Statistical significance was tested using one-way Anova test, ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).
(B) The amount of ubiquitinated securin (Securin-Ub1, Securin-Ub2 and Securin-Ub3), as shown in (A), was quantified and plotted, different colours of circles represent data from 4 independent experiments. Non-linear curve was fitted and IC50 of the G3-6 (SEQ. ID. NO. 33) was determined to 1.96 μM.
(B) The amount of ubiquitinated securin (Securin-Ub1, Securin-Ub2 and Securin-Ub3), as shown in (A), was quantified and plotted and non-linear curves were fitted. Quantification of 3 independent experiments and a summary of KM and kcat are shown. Note, difference between KM (-G3-6 (SEQ. ID. NO. 33)) and KM (+G3-6 (SEQ. ID. NO. 33)) is not significant (p=0.0886) using unpaired t test.
Based on the results of this experiment inhibitory constant (Ki) was calculated using a model for non-competitive inhibition in Prism 6.0. Ki of the lead molecule G3-6 (SEQ. ID. NO. 33) is 5 μM.
The Examples illustrate the invention.
Peptides or peptidomimetics were purchased from GenScript Biotech (Netherlands) B. V. with a >95% purity determined by analytical rpHPLC.
Peptides or peptidomimetics are preferably prepared by standard Fmoc vs. standard Boc solid-phase synthesis by using Rink Amide MBHA resin for items (a)-(e) and (g)-(i) (Scheme 1), and 2-chlorotritylchloride resin for item (f). Coupling steps involve 4 equiv. of amino acid, 4 equiv. of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as coupling reagent and 5 equiv. of diisopropylethylamine (DIPEA) in DMF. Coupling progress is monitored via Kaiser (ninhydrin) test and coupling of amino acids is repeated when results suggest yields below than 99%. Fmoc deprotections are carried out by 2×1 min treatments with excess (1:1) piperidine: DMF and the resin is sequentially washed with DMF, DCM:MeOH (1:1, v/v), and DCM upon completation. The N-terminus is acetylated by standar methods with Ac2O for 10 min. The resulting peptides or peptidomimetics are cleaved from resin by treatment with TFA/H2O/EDT/TIPS (94:2.5:2.5:1) for 2 h followed by precipitation in cold Et2O. Peptides or peptidomimetics are redisolved in water/acetonitrile and lyophilized. After freeze-drying, the peptides are purified by rpHPLC. Purity of compounds is assessed via analytical rpHPLC (solvent A is 0.065% TFA in 100% water (v/v) and solvent B is 0.05% TFA in 100% acetonitrile (v/v), Table 4).
Crosslinking reaction of unnatural olefininc amino acids is carried out by ring-dosing metathesis (RCM) (Scheme 1). The resin is swollen in dry DCM for 30 min and a solution of 4 mg/mL of Grubbs 1st generation catalyst in dry DCM is added to the resin under inert atmosphere three times for 2 h. The reaction is keeped under inert atmosphere.
Additional Synthetic Steps of Preferred Items (b)-(d) of the First Aspect.
Peptides or peptidomimetics containing Cys are prepared through Fmoc synthesis by using DIC/Oxyma or DIC/HOBt as carboxyl activators. Disulfide bridge formation can be attempted through the two following strategies. First, the Fmoc-Cys(Mmt)-OH is used. The residue is incorporated in the respective positions following the general synthetic procedure described above. The resin is washed with DCM and treated with 2% TFA in DCM (5×2 min). Cyclization is carried out on-resin using 25 mM solution of N-chlorosuccinimide (NCS) in DMF. In a second strategy, the linear peptides or peptidomimetics are converted to cyclic disulfides by dropwise addition of a saturated solution of I2 in acetic acid and repurified by rpHPLC.
Once the linear peptide is fully synthesized, the peptide is cleaved from resin with cold 0.8% TFA in DCM 5 times for one minute. The eluate is treated with DIPEA (1 mL) immediately after TFA treatment. The resin is further washed with DCM and MeOH and the collected solvents in the eluate containing the linear peptide are evaporated in high vacumm. Cyclization is performed by treatment of the resulting crude with 3 equiv. 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium (HATU), 3 equiv. 1-hydroxy-7-azabenzotriazole (HOAt) and 6 equiv. of DIPEA in DMF and stirred for 18 h. Then DMF is removed under high vacuum and the crude is further dissolved in DCM and extracted with 10% acetonitrile in water. After solvent evaporation, the crude peptide is cooled and treated with pre-cooled TFA:H2O (9:1) for 5 h at 5° C. followed by precipitation in cold Et2O.
Fmoc-Lys(Mtt)-OH and Fmoc-Asp(OPip)-OH are used for lactam cyclization. These residues are incorporated in the respective positions following the general synthetic procedure described above. The resin is washed with DCM and treated with 2% TFA in DCM (5×2 min). Cyclization is carried out on-resin using 2.5 equiv. BOP, 2.5 equiv. DIPEA in DMF 0.5 M.
aLinear gradient from 15% to 75% solvent B over 25 min.
bLinear gradient from 5% to 65% solvent B over 25 min.
Cells were cultured according to the standard mammalian tissue culture protocol and sterile technique at 37° C. in 5% CO2 and tested in regular intervals for mycoplasma.
HeLa K cells were maintained in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco), 1% (v/v) penicillin-streptomycin (Sigma-Aldrich), 1% (v/v) Glutamax (Gibco), and 0.5 μg/ml amphotericin B (Sigma-Aldrich).
hTERT RPE-1 cells were maintained in DMEM/F12 (Sigma-Aldrich,) supplemented with 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin, 1% (v/v) Glutamax, 0.26% (v/v) sodium bicarbonate (Gibco) and 0.5 μg/mL amphotericin B.
The bacmids encoding the human APC/C were a kind gift from David Barford (MRC, Cambridge, UK). SF9 cells (Expression Systems) were co-infected with two recombinant baculoviruses (ratio 2:5; the first corresponding to the virus containing Strep-tagged APC4) encoding the APC/C at a multiplicity of infection (MOI) of ˜1 and a cell density of 1 million cells/ml. SF9 cells were incubated at 27° C. for 72 h. All steps of APC/C purification were performed at 4° C. Cell pellets were re-suspended in APC/C lysis buffer (50 mM Tris-HCl pH 8.3, 250 mM NaCl, 5% Glycerol, 1 mM EDTA, 2 mM DTT, 0.1 mM PMSF, 2 mM Benzamidine, units/ml benzonase, cOmplete protease inhibitor cocktail (Roche)) and disrupted by nitrogen cavitation in a 4639 Cell Disruption Vessel (Parr Instrument Company). Cell extract was cleared by centrifugation at 48 000 g at 4° C. for 1 h. Strep-tagged APC/C was captured on Strep-Tactin Superflow resin (IBA Life Sciences), washed with APC/C wash buffer (50 mM Tris-HCl pH 8,250 mM NaCl, 5% Glycerol, 1 mM EDTA, 2 mM DTT, 2 mM Benzamidine) and eluted with Buffer E (IBA Life Sciences) supplied with additional NaCl (final concentration 250 mM), 2 mM DTT and 2 mM Benzamidine. Peak fractions were concentrated using Vivaspin 6 centrifugal concentrator (VivaProducts) and purified by size-exclusion chromatography (Superose 6 Increase 10/300 GL column; GE healthcare) in APC/C size-exclusion buffer (20 mM Hepes-NaOH pH 8, 200 mM NaCl, 2 mM DTT, 5% Glycerol). Finally, APC/C was flash-frozen in liquid nitrogen and stored at −80° C.
The bacmid encoding SBP-tagged Cdc20 was a kind gift from Jonathon Pines (ICR, London, UK). SF9 cells were infected with the recombinant baculovirus encoding Cdc20 at a multiplicity of infection (MOI) of ˜1 and a cell density of 1 million cells/ml. SF9 cells were incubated at 27° C. for 72 h. All steps of Cdc20 purification were performed at 4° C. Cell pellets were re-suspended in Cdc20 lysis buffer (250 mM NaCl, 50 mM Tris-HCl pH 8, 1 mM DTT, 5% Glycerol, cOmplete protease inhibitor cocktail, 1 mM PMSF) and disrupted by nitrogen cavitation in a 4639 Cell Disruption Vessel. Cell extract was cleared by centrifugation at 48 000 μg at 4° C. for 1 h. SBP-tagged Cdc20 was captured to Strep-Tactin Superflow resin, washed with 1× Buffer W (IBA Life Sciences) and eluted with Buffer E supplied with additional NaCl (final concentration 250 mM). Finally, glycerol was added to a final concentration of 10% and Cdc20 was flash-frozen in liquid nitrogen and stored at −80° C.
Proteins were separated by SDS-PAGE electrophoresis using precast Bolt 4-12% Bis-Tris Plus protein gels (Thermo Fisher Scientific) and Criterion XT Bis-Tris Midi Protein Gels (Bio-Rad). In different experiments, various conditions were used: in vitro APC/C ubiquitination assays—165 V, 40 min, SDS-MES running buffer (50 mM MES, 50 mM Tris base, 0.1% SDS, 1 mM EDTA, pH 7.3; Thermo Fisher Scientific); in vitro APC/C ubiquitination assays determining KM—165V, 1 h, SDS-MOPS running buffer (50 mM MOPS, 50 mM Tris base, 0.1% SDS, 1 mM EDTA, pH 7.7; Thermo Fisher Scientific); pull-down binding assay—165 V, 35 min, SDS-MES running buffer.
For the protein transfer, the 0.45 μm Immobilon-FL PVDF membrane (Merck Millipore) was used. The membrane was activated in ethanol and then transferred to the Blotting buffer (50 mM SDS-MOPS, 50 mM Tris base, 0.1% SDS, 1 mM EDTA, 20% EtOH). The proteins were transferred using a wet transfer for 1.5 h at 400 mA. Subsequently, the membrane was incubated with the blocking solution, 10% milk in PBS-T (0.02% Tween-20 in PBS), for 1 h at room temperature. To detect proteins of the interest, the membrane was incubated with primary antibodies overnight at 4° C. Followed by washing the membrane 3 times 10 min with PBS-T and incubation with IRDye fluorescently labelled secondary antibodies for 1 h. Prior detection, the membrane was twice washed with PBS-T for 10 min and once with PBS for 10 min. The detection was done using the quantitative near-infrared scanning system Odyssey (LI-COR Biosciences).
APC/C-dependent ubiquitination reactions were performed at 30° C. in 30 mM HEPES pH 7.4, 175 mM NaCl, 8 mM MgCl2, 0.05% Tween-20, 1 mM DTT and 5% glycerol and contained 20 nM recombinant APC/C (note, for testing the first generation of inhibitors, APC/C and Cdc20 immunoprecipitated from mitotic HeLa K cells was used), 340 nM Cdc20, 46 nM GST-UBA1, 340 nM UBE2C (alternatively 400 nM UBE2D1, 280 nM UBE2S), 21 μM His6-ubiquitin or if it is indicated 21 μM methylated-ubiquitin (BostonBiochem), 2.6 mM ATP, 10 mM phosphocreatine and 11 μM creatine kinase. As substrates, 35 nM fluorophore-labeled ubiquitin-cyclin B and 50 nM fluorophore-labeled securin were standardly used (fluorophore IRDye 800CW (LI-COR) was used for labelling). In case of KM and kcat determination, 50, 100, 250, 500, 750, 1000, 1250, 2500 and 5000 nM fluorophore-labelled securin was used. The reaction was done in the volume of 15 μl, it was quenched after the indicated time with LDS sample buffer (Thermo Fisher Scientific) supplemented with 100 mM DTT and subjected to SDS-PAGE. Detection of fluorescently labelled substrates was done by quantitative near-infrared scanning system Odyssey.
HeLa K cell pellet was re-suspended in the extraction buffer (30 mM HEPES pH 7.5, 175 mM NaCl, 2.5 mM MgCl2, 0.25% NP40, 10% glycerol, 1 mM DTT) supplemented with 10 μM MG132 (VWR), 1 mM PMSF (Sigma-Aldrich), complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitors (Roche) and incubated for 20 min on ice, followed by centrifugation of cell debris for 15 min at 4° C. 16 100 g. In mean time, inhibitor molecules containing Propargyl (Pra) chemical group were covalently attached to the azide agarose or azide magnetic resin (Jena Bioscience) by Cu(I)-catalysed azide-alkyne cycloaddition reaction. Specifically, the inhibitor molecules were mixed with azide agarose resin in ratio 0.125 μmol inhibitor/25 μl agarose resin and azide magnetic resin in ratio 0.018 μmol inhibitor/20 μl magnetic resin. The reaction was catalysed by 1 mM CuSO4, 0.1 mM TBTA (Sigma-Aldrich) and 1 mM Sodium ascorbate and was performed on the rotating wheel for 30 min at room temperature in the volume of 1 ml. Subsequently, the resin was washed 5 times with the extraction buffer. The inhibitor-agarose resin was added to 1 mg of HeLa K cell extract and the inhibitor-magnetic resin was added to 0.5 mg of HeLa K cell extract followed by incubation at 4° C. for 2 h. Followed by washing the resin 5 times with the extraction buffer. Pull-down proteins were eluted by boiling for 15 min with 1×LDS sample buffer supplemented with 100 mM DTT (agarose resin) or by incubation with 1×LDS sample buffer for 10 min at room temperature followed by taking supernatant that was supplemented with 100 mM DTT and boiling it for 10 min. Samples were subjected to SDS PAGE and Western blot analysis.
Prism 6.0 (Graphpad) was used, unless specified otherwise.
Question: Do molecules from items (b)-(e) of the first aspect (generation 1) inhibit APC/C activity in vitro?
Approach: in vitro APC/C ubiquitination assay
Conclusion: Molecules of items (b)-(e) of the first aspect effectively inhibit APC/C in vitro. The molecules G1-3 (SEQ. ID. NO. 3) and G1-7 (SEQ. ID. NO. 6) are the most potent and will be used for further development. [
Question: Do molecules of item (f) of the first aspect (generation 2) developed based on G1-1 (SEQ. ID. NO. 1), G1-2 (SEQ. ID. NO. 2), G1-3 (SEQ. ID. NO. 3) and G1-4 (SEQ. ID. NO. 48) inhibit APC/C activity in vitro?
Approach: in vitro APC/C ubiquitination assay
Conclusion: Molecules of item (f) of the first aspect inhibit APC/C in vitro and the molecule G2-4 (SEQ. ID. NO. 17) is the most potent. [
Question: Do optimized molecules of item (f) of the first aspect (generation 2) developed based on G1-1 (SEQ. ID. NO. 1), G1-2 (SEQ. ID. NO. 2), G1-3 (SEQ. ID. NO. 3) and G1-4 (SEQ. ID. NO. 48) inhibit APC/C activity in vitro?
Approach: in vitro APC/C ubiquitination assay
Conclusion: The optimized molecules G2-6 (SEQ. ID. NO. 19) and G2-8 (SEQ. ID. NO. 22) inhibit APC/C in vitro the most effectively compared to other molecules of item (f) of the first aspect. [
Question: Do molecules of items (a) and (g) of the first aspect (generation 3) developed based on molecules of items (b)-(f) of the first aspect inhibit APC/C activity in vitro?
Approach: in vitro APC/C ubiquitination assay
Conclusion: Molecules of items (a) and (g) of the first aspect (generation 3) inhibit APC/C in vitro the most potently compared to previous generations. [
Question: Do molecules of items (a) and (g) of the first aspect (generation 3) inhibit APC/C activity more efficiently than TAME?
Approach: in vitro APC/C ubiquitination assay
Conclusion: Molecules of items (g) G3-1 (SEQ. ID. NO. 27) and G3-3 (SEQ. ID. NO. 30) and of item (a) G3-4 (SEQ. ID. NO. 31) and G3-6 (SEQ. ID. NO. 33) of the first aspect inhibit APC/C in vitro more efficiently than TAME. [
Question: Which molecules from items (a) and (g) of the first aspect (generation 3) inhibit APC/C in vitro the most potently?
Approach: in vitro APC/C ubiquitination assay, titration of molecules of items (a) and (g) of the first aspect
Conclusion: G3-6 (SEQ. ID. NO. 33) inhibits APC/C in vitro the most potently and is the lead compound. [
Question: What is the IC50 of the lead molecule G3-6 (SEQ. ID. NO. 33)?
Approach: in vitro APC/C ubiquitination assay, titration of the G3-6 molecule (SEQ. ID. NO. 33)
Conclusion: IC50 of the lead molecule G3-6 (SEQ. ID. NO. 33) is in low micromolar range. [
Question: What is the mechanism of action of the G3-6 molecule (SEQ. ID. NO. 33) (type of the inhibition)?
Approach: in vitro APC/C ubiquitination assay, titration of substrate (securin)
Conclusion: G3-6 molecule (SEQ. ID. NO. 33) acts as a non-competitive inhibitor of APC/C in vitro. [
Question: Is APC/C inhibition by the G3-6 molecule (SEQ. ID. NO. 33) dependent on a specific E2 enzyme?
Approach: in vitro APC/C ubiquitination assay, different E2 enzymes—UBE2C, UBE2D, UBE2S
Conclusion: The lead molecule G3-6 (SEQ. ID. NO. 33) inhibits APC/C in vitro independently on employed E2 enzymes. [
Question: Is the G3-1 molecule (SEQ. ID. NO. 27) binding specifically to the APC/C?
Approach: Pull-down binding assay from HeLa K cell extract, azide agarose beads
Conclusion: The G3-1 molecule (SEQ. ID. NO. 27) binds specifically to the APC/C in HeLa K cell extract. [
Question: Is the G3-6 molecule (SEQ. ID. NO. 33) binding to the APC/C?
Approach: Pull-down binding assay in HeLa K cell extract, azide magnetic beads
Conclusion: The G3-6 molecule (SEQ. ID. NO. 33) binds to the APC/C in HeLa K cell extract. [
Question: Does the G3-6 molecule (SEQ. ID. NO. 33) bind specifically to the APC/C?
Approach: Pull-down binding assay in HeLa K cell extract, azide magnetic beads
Conclusion: The G3-6 molecule (SEQ. ID. NO. 33) binds specifically to the APC/C in cell extract. [
Question: What effect has the G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36) on mitosis of HeLa K cells?
Approach: Live cell imaging of HeLa K cells in the presence of the G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36)
Conclusion: The G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36) induces a metaphase delay and causes cell death after metaphase in HeLa K cells. [
Question: What effect has the G2-6 molecule (SEQ. ID. NO. 19) on mitosis of hTERT RPE-1 cells?
Approach: Live cell imaging of hTERT RPE-1 cells in the presence of G2-6 (SEQ. ID. NO. 19) and the G2-N2 control molecule
Conclusion: The G2-6 molecule (SEQ. ID. NO. 19) causes prolonged mitosis and mitotic or subsequent cell death in hTERT RPE-1 cells. [
Automated time-lapse microscopy was performed using ImageXpress Micro XLS wide-field screening microscope (Molecular Devices) equipped with a 10×, 0.5 NA., 20×, 0.7 NA, and 40×, 0.95 NA Plan Apo air objectives (Nikon), a laser-based autofocus and a full environmental control (5% CO2, 37° C.). Cells were grown in 96-well plastic bottom plates (clear, Greiner Bio-One) and for live cell imaging media was changed to DMEM without phenol red and riboflavin (Thermo Fisher Scientific), supplemented with 10% (v/v) FBS, 1% (v/v) Glutamax, 1% (v/v) penicillin-streptomycin, and 0.5 μg/ml amphotericin B.
Live Cell Imaging of Cells Treated with Compounds of the Invention
HeLa K cells were seeded into 96-well plates (4000-4500 cells) one day prior the treatment with compounds of the invention. Cells were treated with 50 μM of compound G3_mod1_mini PEG and cell division was monitored by live cell imaging using ImageXpress Micro XLS wide-field screening microscope, images were acquired every 3 min for time courses of 48 h. The length of mitosis was determined manually.
hTERT RPE-1 cells were seeded into 96-well plates (5000 cells) one day prior the treatment with compounds of the invention. Cells were treated with 100 μM of compound G2-6 and the negative control molecule G2-N2 and cell division was monitored by live cell imaging using ImageXpress Micro XLS wide-field screening microscope, images were acquired every 3 min for time courses of 12-24 h. The length of mitosis was determined manually.
Question: Does the G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36) prolong mitosis in different human cancer cell lines representing lung carcinoma (A549), cervical carcinoma (HeLa K), fibrosarcoma (HT-1080), colon carcinoma (RKO) and colorectal adenocarcinoma (SW480)?
Approach: Live cell imaging of A549, HeLa K, HT-1080, RKO and SW480 cells in the presence of the G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36) and the carrier (DMSO) control.
Conclusion: The G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36) prolongs mitosis in human cancer cells of different tissue origin (A549, HeLa K, HT-1080, RKO and SW480) by arresting cells in metaphase. Furthermore, G3-6_mod1_miniPEG molecules (SEQ. ID. NO. 36) cause cell death after a metaphase arrest in a subpopulation of HeLa K, HT-1080 and SW480 cells [FIG. 17]
Cells were cultured according to the standard mammalian tissue culture protocol and sterile technique at 37° C. in 5% CO2 and tested in regular intervals for mycoplasma.
A549, HeLa K and SW480 cells were maintained in DMEM (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific), 1% (v/v) penicillin-streptomycin (Sigma-Aldrich), and 1% (v/v) Glutamax (Thermo Fisher Scientific).
HT-1080 and RKO cells were maintained in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin.
Automated time-lapse microscopy was performed using ImageXpress Micro XLS wide-field screening microscope (Molecular Devices) equipped with a 10×, 0.5 NA., 20×, 0.7 NA, and 40×, 0.95 NA Plan Apo air objectives (Nikon), a laser-based autofocus and a full environmental control (5% CO2, 37° C.). Cells were grown in 96-well plastic bottom plates (clear, Greiner Bio-One) and for live cell imaging media was changed to imaging media. Imaging media for A549, HeLa K and SW480 was DMEM without phenol red and riboflavin (Thermo Fisher Scientific), supplemented with 10% (v/v) FBS, 1% (v/v) Glutamax, and 1% (v/v) penicillin-streptomycin; imaging media for HT-1080 and RKO was RPMI 1640 without phenol red (Thermo Fisher Scientific), supplemented with 10% (v/v) FBS, and 1% (v/v) penicillin-streptomycin.
Live Cell Imaging of Cells Treated with Compounds of the Invention
A549 (5000-5500 cells), HeLa K (3000-4000 cells), HT-1080 (4000 cells), RKO (5000 cells), and SW480 (7000-8000 cells) cells were seeded into 96-well plates one day prior to treatment with compounds of the invention. Cells were treated with 50 μM of compound G3_mod1_mini PEG and cell division was monitored by live cell imaging using an ImageXpress Micro XLS wide-field screening microscope and images were acquired every 3 min for time courses of 24 h. The length of mitosis was determined manually.
Question: Is the G1-3Pra molecule (SEQ. ID. NO. 100) a general RING E3 binder and hence can be used as such general binder or as a template to develop specific and/or optimized RING E3 binders?
Approach: Pull-down binding assay in HeLa K extract, azide agarose beads and detection of APC/C (APC11) and selected RING ligases involved in carcinogenesis
Conclusion: The G1-3Pra molecule (SEQ. ID. NO. 100) binds not only to the APC/C (APC11) but also other cancer relevant RING proteins (BMI-1, BRCA1, c-CBL, c-IAP1, MDM2, RBX1, TRAF2) in HeLa K cell extracts.
HeLa K cell pellet was re-suspended in the extraction buffer (30 mM HEPES pH 7.5, 175 mM NaCl, 2.5 mM MgCl2, 0.25% NP40, 10% glycerol, 1 mM DTT supplied with complete protease inhibitor cocktail (Roche), PhosSTOP phosphatase inhibitors (Roche), 1 mM PMSF, 10 μM MG132) and incubated for 20 min on ice, followed by centrifugation to clear the lysate for 15 min at 4° C. 16,100 g. In the meantime, inhibitor molecules containing the propargyl-Gly-OH (Pra) chemical group were covalently attached to the azide agarose resin (Jena Bioscience) by Cu(I)-catalysed azide-alkyne cycloaddition reaction. Specifically, the inhibitor molecules were mixed with azide agarose resin in ratio 0.125 μmol inhibitor/20 μl agarose resin. The reaction was catalysed by 0.1 mM CuSO4, 0.5 mM THPTA (Sigma-Aldrich) and 5 mM Sodium ascorbate and was performed on the rotating wheel for 30 min at room temperature in the volume of 1 ml. Subsequently, the resin was washed five times with the extraction buffer. The inhibitor-agarose resin was added to 2 mg of HeLa K cell extract and incubated at 4° C. for 2 h. Afterwards, the resin was washed four times with the extraction buffer. Pull-down proteins were eluted by incubation with pre-warmed 1×LDS sample buffer for 10 min at room temperature followed by taking supernatant supplemented with 100 mM DTT and boiling for 10 min. Afterwards the samples were halved and two SDS PAGE and Western blot analyses were performed to detect the indicated proteins.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20200499.0 | Oct 2020 | EP | regional |
This application is a US National Stage of International Application No. PCT/EP2021/077611, filed on Oct. 6, 2021, and claims the benefit of European Patent Application No. 20200499.0, filed on Oct. 7, 2020, the disclosures of which are incorporated by reference in their entirety as if fully set forth herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/077611 | 10/6/2021 | WO |
