Novel Methionine Aminopeptidase 2 Inhibitor M8891 Synergizes with VEGF Receptor Inhibitors to Inhibit Tumor Growth of Renal Cell Carcinoma Models.

N-terminal processing by methionine aminopeptidases (MetAP) is a crucial step in the maturation of proteins during protein biosynthesis. Small-molecule inhibitors of MetAP2 have antiangiogenic and antitumoral activity. Herein, we characterize the structurally novel MetAP2 inhibitor M8891. M8891 is a potent, selective, reversible small-molecule inhibitor blocking the growth of human endothelial cells and differentially inhibiting cancer cell growth. A CRISPR genome-wide screen identified the tumor suppressor p53 and MetAP1/MetAP2 as determinants of resistance and sensitivity to pharmacologic MetAP2 inhibition. A newly identified substrate of MetAP2, translation elongation factor 1-alpha-1 (EF1a-1), served as a pharmacodynamic biomarker to follow target inhibition in cell and mouse studies. Robust angiogenesis and tumor growth inhibition was observed with M8891 monotherapy. In combination with VEGF receptor inhibitors, tumor stasis and regression occurred in patient-derived xenograft renal cell carcinoma models, particularly those that were p53 wild-type, had Von Hippel-Landau gene (VHL) loss-of-function mutations, and a mid/high MetAP1/2 expression score.

Preclinical Pharmacokinetics and Translational Pharmacokinetic/Pharmacodynamic Modeling of M8891, a Potent and Reversible Inhibitor of Methionine Aminopeptidase 2.

INTRODUCTION: M8891 is a selective and reversible inhibitor of methionine aminopeptidase 2 (MetAP2). We describe translational research to define the target pharmacokinetics (PK) of M8891 and associated pharmacodynamic (PD) levels, which were used to support efficacious dose selection in humans. METHODS: In vitro and in vivo PK characteristics were investigated in animal species, and data integrated using in vitro-in vivo correlation and allometric methods to predict the clearance, volume of distribution, and absorption parameters of M8891 in humans. In parallel, inhibition of MetAP2 activity by M8891 was studied in renal cancer xenografts in mice by measuring accumulation of Met-EF1α, a substrate of MetAP2. The corresponding PD effect was described by a turnover and effect compartment model. This model was used to simulate PD at the M8891 dose showing in vivo efficacy, i.e. significant tumor growth inhibition. Simulations of M8891 PK and associated PD in humans were conducted by integrating predicted human PK parameters into the preclinical PK/PD model. RESULTS: The target minimum PD level associated with efficacy was determined to be 125 µg Met-EF1α per mg protein. Integrating predicted human PK parameters into the preclinical PK/PD model defined a minimal M8891 concentration at steady-state (Ctrough) of 1500 ng/mL (3.9 µM) in humans as being required to produce the corresponding minimum target Met-EF1a level (125 µg per mg protein). CONCLUSION: The defined target PK and PD levels supported the design of the clinical Phase Ia dose escalation study of M8891 (NCT03138538) and selection of the recommended Phase II dose.

A First-in-human, Dose-escalation Study of the Methionine Aminopeptidase 2 Inhibitor M8891 in Patients with Advanced Solid Tumors.

Methionine aminopeptidase 2 (MetAP2) is essential to endothelial cell growth and proliferation during tumor angiogenesis. M8891 is a novel orally bioavailable, potent, selective, reversible MetAP2 inhibitor with antiangiogenic and antitumor activity in preclinical studies. The safety, tolerability, pharmacokinetics, and pharmacodynamics of M8891 monotherapy were assessed in a phase I, first-in-human, multicenter, open-label, single-arm, dose-escalation study (NCT03138538). Patients with advanced solid tumors received 7-80 mg M8891 once daily in 21-day cycles. The primary endpoint was dose-limiting toxicity (DLT) during cycle 1, with the aim to determine the maximum tolerated dose (MTD). Twenty-seven patients were enrolled across six dose levels. Two DLTs (platelet count decrease) were reported, one each at 60 and 80 mg/once daily M8891, resolving after treatment discontinuation. MTD was not determined. The most common treatment-emergent adverse event was platelet count decrease. M8891 plasma concentration showed dose-linear increase up to 35 mg and low-to-moderate variability; dose-dependent tumor accumulation of methionylated elongation factor 1α, a MetAP2 substrate, was observed, demonstrating MetAP2 inhibition. Pharmacokinetic/pharmacodynamic response data showed that preclinically defined target levels required for in vivo efficacy were achieved at safe, tolerated doses. Seven patients (25.9%) had stable disease for 42-123 days. We conclude that M8891 demonstrates a manageable safety profile, with dose-proportional exposure and low-to-moderate interpatient variability at target pharmacokinetic/pharmacodynamic levels at ≤35 mg M8891 once daily. On the basis of the data, 35 mg M8891 once daily is the recommended phase II dose for M8891 monotherapy. This study forms the basis for future development of M8891 in monotherapy and combination studies. Significance: M8891 represents a novel class of reversible MetAP2 inhibitors and has demonstrated preclinical antitumor activity. This dose-escalation study assessed M8891 treatment for patients with advanced solid tumors. M8891 demonstrated favorable pharmacokinetics, tumoral target engagement, and a manageable safety profile, and thus represents a novel antitumor strategy warranting further clinical studies.

Translational PK/PD Modeling of Tumor Growth Inhibition and Target Inhibition to Support Dose Range Selection of the LMP7 Inhibitor M3258 in Relapsed/Refractory Multiple Myeloma.

M3258 is an orally bioavailable, potent, selective, reversible inhibitor of the large multifunctional peptidase 7 (LMP7, ß5i, PSMB8) proteolytic subunit of the immunoproteasome, a component of the cellular protein degradation machinery, highly expressed in malignant hematopoietic cells including multiple myeloma. Here we describe the fit-for-purpose pharmacokinetic (PK)/pharmacodynamic (PD)/efficacy modeling of M3258 based on preclinical data from several species. The inhibition of LMP7 activity (PD) and tumor growth (efficacy) were tested in human multiple myeloma xenografts in mice. PK and efficacy data were correlated yielding a free M3258 concentration of 45 nM for half-maximal tumor growth inhibition (KC50). As M3258 only weakly inhibits LMP7 in mouse cells, both in vitro and in vivo bridging studies were performed in rats, monkeys, and dogs for translational modeling. These data indicated that the PD response in human xenograft models was closely reflected in dog PBMCs. A PK/PD model was established, predicting a free IC50 value of 9 nM for M3258 in dogs in vivo, in close agreement with in vitro measurements. In parallel, the human PK parameters of M3258 were predicted by various approaches including in vitro extrapolation and allometric scaling. Using PK/PD/efficacy simulations, the efficacious dose range and corresponding PD response in human were predicted. Taken together, these efforts supported the design of a phase Ia study of M3258 in multiple myeloma patients (NCT04075721). At the lowest tested dose level, the predicted exposure matched well with the observed exposure while the duration of LMP7 inhibition was underpredicted by the model. SIGNIFICANCE STATEMENT: M3258 is a novel inhibitor of the immunoproteasome subunit LMP7. The human PK and human efficacious dose range of M3258 were predicted using in vitro-in vivo extrapolation and allometric scaling methods together with a fit-for-purpose PK/PD and efficacy model based on data from several species. A comparison with data from the Phase Ia clinical study showed that the human PK was accurately predicted, while the extent and duration of PD response were more pronounced than estimated.

Using thrombocytopenia modeling to investigate the mechanisms underlying platelet depletion induced by pan-proteasome inhibitors.

Pan-proteasome inhibitors (pPIs) significantly improve outcomes in patients with multiple myeloma; however, their indiscriminate inhibition of multiple proteasome and immunoproteasome subunits causes diverse toxicities, including thrombocytopenia. We investigated the mechanisms underlying the platelet depletion induced by the pPIs bortezomib, carfilzomib, and ixazomib. An established thrombocytopenia model was adapted for each compound (bortezomib, ixazomib, and carfilzomib) to compare the following two pharmacodynamic mechanisms: a reversible inhibition of new progenitor cell formation (the myelosuppression model) and a reversible effect on the function of megakaryocytes to bud new platelets (platelet formation model). Bortezomib, ixazomib, and carfilzomib plasma concentration profiles and platelet counts were extracted from the literature. Pharmacokinetic (PK) and thrombocytopenia models were developed to predict the PK of these drugs and to describe their effects on proliferating cells and platelet budding. The PK models reproduced the exposure of the three compounds at steady state well compared with those reported in the literature. Both the platelet formation and myelosuppression models seemed able to describe the platelet depletion caused by bortezomib, ixazomib, and carfilzomib. Estimated structural parameters in the myelosuppression model were in the range of the values reported in the literature, whereas the mean transit time estimated with the platelet formation model was 3-fold to 10-fold higher than the highest reported value. The model of drug-induced myelosuppression yielded estimates of structural parameters in the range of those previously reported. The platelet formation model captured the temporal variation reported in clinical studies.

Identification of Clinical Candidate M2698, a Dual p70S6K and Akt Inhibitor, for Treatment of PAM Pathway-Altered Cancers.

Herein, we report the discovery of a novel class of quinazoline carboxamides as dual p70S6k/Akt inhibitors for the treatment of tumors driven by alterations to the PI3K/Akt/mTOR (PAM) pathway. Through the screening of in-house proprietary kinase library, 4-benzylamino-quinazoline-8-carboxylic acid amide 1 stood out, with sub-micromolar p70S6k biochemical activity, as the starting point for a structurally enabled p70S6K/Akt dual inhibitor program that led to the discovery of M2698, a dual p70S6k/Akt inhibitor. M2698 is kinase selective, possesses favorable physical, chemical, and DMPK profiles, is orally available and well tolerated, and displayed tumor control in multiple in vivo studies of PAM pathway-driven tumors.

Discovery of Covalent Bruton's Tyrosine Kinase Inhibitors with Decreased CYP2C8 Inhibitory Activity.

Bruton's tyrosine kinase (BTK) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage. Evidence has shown that inhibition of BTK has clinical benefit for the treatment of a wide array of autoimmune and inflammatory diseases. Previously we reported the discovery of a novel nicotinamide selectivity pocket (SP) series of potent and selective covalent irreversible BTK inhibitors. The top molecule 1 of that series strongly inhibited CYP2C8 (IC50 =100 nM), which was attributed to the bridged linker group. However, our effort on the linker replacement turned out to be fruitless. With the study of the X-ray crystal structure of compound 1, we envisioned the opportunity of removal of this liability via transposition of the linker moiety in 1 from C6 to C5 position of the pyridine core. With this strategy, our optimization led to the discovery of a novel series, in which the top molecule 18 A displayed reduced CYP inhibitory activity and good potency. To further explore this new series, different warheads besides acrylamide, for example cyanamide, were also tested. However, this effort didn't lead to the discovery of molecules with better potency than 18 A. The loss of potency in those molecules could be related to the reduced reactivity of the warhead or reversible binding mode. Further profiling of 18 A disclosed that it had a strong hERG (human Ether-a-go-go Related Gene) inhibition, which could be related to the phenoxyphenyl group.

Optimization of a Screening Hit toward M2912, an Oral Tankyrase Inhibitor with Antitumor Activity in Colorectal Cancer Models.

Constitutive activation of the canonical Wnt signaling pathway, in most cases driven by inactivation of the tumor suppressor APC, is a hallmark of colorectal cancer. Tankyrases are druggable key regulators in these malignancies and are considered as attractive targets for therapeutic interventions, although no inhibitor has been progressed to clinical development yet. We continued our efforts to develop tankyrase inhibitors targeting the nicotinamide pocket with suitable drug-like properties for investigating effects of Wnt pathway inhibition on tumor growth. Herein, the identification of a screening hit series and its optimization through scaffold hopping and SAR exploration is described. The systematic assessment delivered M2912, a compound with an optimal balance between excellent TNKS potency, exquisite PARP selectivity, and a predicted human PK compatible with once daily oral dosing. Modulation of cellular Wnt pathway activity and significant tumor growth inhibition was demonstrated with this compound in colorectal xenograft models in vivo.

Structure-Based Optimization and Discovery of M3258, a Specific Inhibitor of the Immunoproteasome Subunit LMP7 (ß5i).

Proteasomes are broadly expressed key components of the ubiquitin-dependent protein degradation pathway containing catalytically active subunits (ß1, ß2, and ß5). LMP7 (ß5i) is a subunit of the immunoproteasome, an inducible isoform that is predominantly expressed in hematopoietic cells. Clinically effective pan-proteasome inhibitors for the treatment of multiple myeloma (MM) nonselectively target LMP7 and other subunits of the constitutive proteasome and immunoproteasome with comparable potency, which can limit the therapeutic applicability of these drugs. Here, we describe the discovery and structure-based hit optimization of novel amido boronic acids, which selectively inhibit LMP7 while sparing all other subunits. The exploitation of structural differences between the proteasome subunits culminated in the identification of the highly potent, exquisitely selective, and orally available LMP7 inhibitor 50 (M3258). Based on the strong antitumor activity observed with M3258 in MM models and a favorable preclinical data package, a phase I clinical trial was initiated in relapsed/refractory MM patients.

M3258 Is a Selective Inhibitor of the Immunoproteasome Subunit LMP7 (ß5i) Delivering Efficacy in Multiple Myeloma Models.

Large multifunctional peptidase 7 (LMP7/ß5i/PSMB8) is a proteolytic subunit of the immunoproteasome, which is predominantly expressed in normal and malignant hematolymphoid cells, including multiple myeloma, and contributes to the degradation of ubiquitinated proteins. Described herein for the first time is the preclinical profile of M3258; an orally bioavailable, potent, reversible and highly selective LMP7 inhibitor. M3258 demonstrated strong antitumor efficacy in multiple myeloma xenograft models, including a novel model of the human bone niche of multiple myeloma. M3258 treatment led to a significant and prolonged suppression of tumor LMP7 activity and ubiquitinated protein turnover and the induction of apoptosis in multiple myeloma cells both in vitro and in vivo Furthermore, M3258 showed superior antitumor efficacy in selected multiple myeloma and mantle cell lymphoma xenograft models compared with the approved nonselective proteasome inhibitors bortezomib and ixazomib. The differentiated preclinical profile of M3258 supported the initiation of a phase I study in patients with multiple myeloma (NCT04075721).

Identification of Methionine Aminopeptidase-2 (MetAP-2) Inhibitor M8891: A Clinical Compound for the Treatment of Cancer.

The recently disclosed next generation of reversible, selective, and potent MetAP-2 inhibitors introduced a cyclic tartronic diamide scaffold. However, the lead compound 1a suffered from enterohepatic circulation, preventing further development. Nevertheless, 1a served as a starting point for further optimization. Maintaining potent antiproliferation activity, while improving other compound properties, enabled the generation of an attractive array of new MetAP-2 inhibitors. The most promising derivatives were identified by a multiparameter analysis of the compound properties. Essential for the efficient selection of candidates with in vivo activity was the identification of molecules with a long residence time on the target protein, high permeability, and low efflux ratio not only in Caco-2 but also in the MDR-MDCK cell line. Orally bioavailable, potent, and reversible MetAP-2 inhibitors impede the growth of primary endothelial cells and demonstrated antitumoral activity in mouse models. This assessment led to the nomination of the clinical development compound M8891, which is currently in phase I clinical testing in oncology patients.

Discovery and Optimization of 2-Arylquinazolin-4-ones into a Potent and Selective Tankyrase Inhibitor Modulating Wnt Pathway Activity.

Tankyrases 1 and 2 (TNKS1/2) are promising pharmacological targets that recently gained interest for anticancer therapy in Wnt pathway dependent tumors. 2-Aryl-quinazolinones were identified and optimized into potent tankyrase inhibitors through SAR exploration around the quinazolinone core and the 4'-position of the phenyl residue. These efforts were supported by analysis of TNKS X-ray and WaterMap structures and resulted in compound 5k, a potent, selective tankyrase inhibitor with favorable pharmacokinetic properties. The X-ray structure of 5k in complex with TNKS1 was solved and confirmed the design hypothesis. Modulation of Wnt pathway activity was demonstrated with this compound in a colorectal xenograft model in vivo.

Discovery and Structure-Based Optimization of Next-Generation Reversible Methionine Aminopeptidase-2 (MetAP-2) Inhibitors.

Co- and post-translational processing are crucial maturation steps to generate functional proteins. MetAP-2 plays an important role in this process, and inhibition of its proteolytic activity has been shown to be important for angiogenesis and tumor growth, suggesting that small-molecule inhibitors of MetAP-2 may be promising options for the treatment of cancer. This work describes the discovery and structure-based hit optimization of a novel MetAP-2 inhibitory scaffold. Of critical importance, a cyclic tartronic diamide coordinates the MetAP-2 metal ion in the active site while additional side chains of the molecule were designed to occupy the lipophilic methionine side chain recognition pocket as well as the shallow cavity at the opening of the active site. The racemic screening hit from HTS campaign 11a was discovered with an enzymatic IC50 of 150 nM. The resynthesized eutomer confirmed this activity and inhibited HUVEC proliferation with an IC50 of 1.9 µM. Its structural analysis revealed a sophisticated interaction pattern of polar and lipophilic contacts that were used to improve cellular potency to an IC50 of 15 nM. In parallel, the molecular properties were optimized on plasma exposure and antitumor efficacy which led to the identification of advanced lead 21.

A novel tankyrase inhibitor, MSC2504877, enhances the effects of clinical CDK4/6 inhibitors.

Inhibition of the PARP superfamily tankyrase enzymes suppresses Wnt/ß-catenin signalling in tumour cells. Here, we describe here a novel, drug-like small molecule inhibitor of tankyrase MSC2504877 that inhibits the growth of APC mutant colorectal tumour cells. Parallel siRNA and drug sensitivity screens showed that the clinical CDK4/6 inhibitor palbociclib, causes enhanced sensitivity to MSC2504877. This tankyrase inhibitor-CDK4/6 inhibitor combinatorial effect is not limited to palbociclib and MSC2504877 and is elicited with other CDK4/6 inhibitors and toolbox tankyrase inhibitors. The addition of MSC2504877 to palbociclib enhances G1 cell cycle arrest and cellular senescence in tumour cells. MSC2504877 exposure suppresses the upregulation of Cyclin D2 and Cyclin E2 caused by palbociclib and enhances the suppression of phospho-Rb, providing a mechanistic explanation for these effects. The combination of MSC2504877 and palbociclib was also effective in suppressing the cellular hyperproliferative phenotype seen in Apc defective intestinal stem cells in vivo. However, the presence of an oncogenic Kras p.G12D mutation in mice reversed the effects of the MSC2504877/palbociclib combination, suggesting one molecular route that could lead to drug resistance.

Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates.

The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC50: 1µM) led to the most potent compound 27 (IC50: 0.038µM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.

Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases.

Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors.

Structure-Based Optimization of Potent, Selective, and Orally Bioavailable CDK8 Inhibitors Discovered by High-Throughput Screening.

The mediator complex-associated cyclin dependent kinase CDK8 regulates ß-catenin-dependent transcription following activation of WNT signaling. Multiple lines of evidence suggest CDK8 may act as an oncogene in the development of colorectal cancer. Here we describe the successful optimization of an imidazo-thiadiazole series of CDK8 inhibitors that was identified in a high-throughput screening campaign and further progressed by structure-based design. In several optimization cycles, we improved the microsomal stability, potency, and kinase selectivity. The initial imidazo-thiadiazole scaffold was replaced by a 3-methyl-1H-pyrazolo[3,4-b]-pyridine which resulted in compound 25 (MSC2530818) that displayed excellent kinase selectivity, biochemical and cellular potency, microsomal stability, and is orally bioavailable. Furthermore, we demonstrated modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8 activity, and tumor growth inhibition in an APC mutant SW620 human colorectal carcinoma xenograft model after oral administration. Compound 25 demonstrated suitable potency and selectivity to progress into preclinical in vivo efficacy and safety studies.

2,8-Disubstituted-1,6-Naphthyridines and 4,6-Disubstituted-Isoquinolines with Potent, Selective Affinity for CDK8/19.

We demonstrate a designed scaffold-hop approach to the discovery of 2,8-disubstituted-1,6-naphthyridine- and 4,6-disubstituted-isoquinoline-based dual CDK8/19 ligands. Optimized compounds in both series exhibited rapid aldehyde oxidase-mediated metabolism, which could be abrogated by introduction of an amino substituent at C5 of the 1,6-naphthyridine scaffold or at C1 of the isoquinoline scaffold. Compounds 51 and 59 were progressed to in vivo pharmacokinetic studies, and 51 also demonstrated sustained inhibition of STAT1(SER727) phosphorylation, a biomarker of CDK8 inhibition, in an SW620 colorectal carcinoma human tumor xenograft model following oral dosing.

Discovery of potent and selective CDK8 inhibitors from an HSP90 pharmacophore.

Here we describe the discovery and optimization of 3-benzylindazoles as potent and selective inhibitors of CDK8, also modulating CDK19, discovered from a high-throughput screening (HTS) campaign sampling the Merck compound collection. The primary hits with strong HSP90 affinity were subsequently optimized to potent and selective CDK8 inhibitors which demonstrate inhibition of WNT pathway activity in cell-based assays. X-ray crystallographic data demonstrated that 3-benzylindazoles occupy the ATP binding site of CDK8 and adopt a Type I binding mode. Medicinal chemistry optimization successfully led to improved potency, physicochemical properties and oral pharmacokinetics. Modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8, was demonstrated in an APC-mutant SW620 human colorectal carcinoma xenograft model following oral administration.

Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19.

The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer.