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.

A novel monovalent FGFR1 antagonist: Preclinical safety profiles in rodents and non-human primates.

Blocking Fibroblast Growth Factor Receptor 1 (FGFR1) is an attractive therapeutic option for treatment of cancer subtypes with amplification and over-expression of FGFR1. Selective targeting of FGFR1 can be achieved using an antibody-based approach, as small molecule inhibitors may not discriminate between FGFR1, 2, 3 and 4 due to their highly homologous kinase domain. However, development of classical bivalent FGFR1 directed antibodies has failed due to non-tolerated body weight decreases in preclinical species. M6123 is a novel IgG-like monovalent FGFR1 specific binder with enhanced Antibody-Dependent Cellular Cytotoxicity (ADCC) effector function and inhibits tumor growth significantly in FGFR1-dependent human xenograft models without reduced body weight in tumor-bearing mice. Toxicology studies reported here characterized the safety profile of M6123 in mouse, rat, and monkey. There were significant differences among animal species under similar M6123 exposure levels. Rats showed metastatic mineralization with an imbalance in serum phosphate at low doses, while mineralization was not found in mice or monkeys, even though hyperphosphatemia was detected in mice. Subtle differences in calcium/phosphate homoeostasis feedback loops may trigger the susceptibility to mineralization among animal species; nevertheless, the exact mechanism remains unknown. Monkeys showed marked, but reversible, decreases in peripheral blood NK cells and neutrophils. The latter was associated with considerably increased neutrophilic infiltrates in the liver sinusoids and red pulp of the spleen. These effects in monkeys are likely related to the enhanced ADCC activity of M6123. Overall, M6123 showed a superior safety profile in animals compared to bivalent FGFR1 antagonists or pan-FGFR inhibitors.

Biochemical, cellular and structural characterization of novel and selective ERK3 inhibitors.

Triazolo[4,5-d]pyrimidin-5-amines were identified from kinase selectivity screening as novel ERK3 inhibitors with sub-100 nanomolar potencies in a biochemical assay using MK5 as substrate and with an attractive kinase selectivity profile. ERK3 crystal structures clarified the inhibitor binding mode in the ATP pocket with impact on A-loop, GC-loop and αC-helix conformations suggesting a potential structural link towards MK5 interaction via the FHIEDE motif. The inhibitors also showed sub-100 nM potencies in a cellular ERK3 NanoBRET assay and with excellent correlation to the biochemical IC50s. This novel series provides valuable tool compounds to further investigate the biological function and activation mechanism of ERK3.

Peptide-Based Sandwich Immunoassay for the Quantification of the Membrane Transporter Multidrug Resistance Protein 1.

Multitransmembrane proteins are notoriously difficult to analyze. To date, rapid, and cost-efficient detection methods are lacking and only mass spectrometry-based systems allow reliable quantification of these proteins. Here, we present a novel type of sandwich immunoassay that is capable of sensitively detecting multidrug resistance protein 1 (MDR1), a prototypic 12-transmembrane-domains transporter. In a first assay step, complex samples are enzymatically fragmented into peptides as routinely done for mass spectrometry. A proteotypic peptide derived from MDR1 was chosen and antibodies targeting this peptide were used to build a sandwich immunoassay. Validation of the optimized assay showed good sensitivity, reproducibility and it allowed reliable quantification of MDR1; cross-validation by mass spectrometry demonstrated the applicability for routine analyses in clinical and pharmaceutical research. MDR1 was quantified in primary human renal cell carcinoma and corresponding normal tissue and down-regulation or expression loss was found in tumor tissue corroborating its importance in drug resistance and efficacy.

A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.

There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.

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 novel 7-azaindoles as PDK1 inhibitors.

A combined screening strategy using HTS together with focused kinase library and virtual screening led to the identification of diverse chemical series as PDK1 inhibitors. We focused our medicinal chemistry efforts on 7-azaindoles with low micromolar IC50s (e.g., 16: IC50=1.1µM) in the biochemical PDK1 assay. Our structure-guided optimization efforts considered also PDK1 X-ray structures with weaker binding fragments and resulted in 7-azaindoles with significantly improved biochemical PDK1 potency in the two-digit nanomolar range. However, the most potent analogues only showed moderate activities in a cellular mechanistic assay (42: IC50=2.3µM) together with either low microsomal stability or low permeability. The described structure-activity relationship together with PDK1 X-ray structures and early ADME data provided the basis for our subsequent hit-to-lead program.

Evasion of apoptosis and treatment resistance in squamous cell carcinoma of the head and neck.

Combinations of surgery, radiotherapy and chemotherapy can eradicate tumors in patients with locally advanced squamous cell carcinoma of the head and neck (LA SCCHN), but a significant proportion of tumors progress, recur, or do not respond to therapy due to treatment resistance. The prognosis for these patients is poor, thus new approaches are needed to improve outcomes. Key resistance mechanisms to chemoradiotherapy (CRT) in patients with LA SCCHN are alterations to the pathways that mediate apoptosis, a form of programmed cell death. Targeting dysregulation of apoptotic pathways represents a rational therapeutic strategy in many types of cancer, with a number of proteins, including the pro-survival B-cell lymphoma 2 family and inhibitors of apoptosis proteins (IAPs), having been identified as druggable targets. This review discusses the mechanisms by which apoptosis occurs under physiological conditions, and how this process is abnormally restrained in LA SCCHN tumor cells, with treatment strategies aimed at re-enabling apoptosis in LA SCCHN also considered. In particular, the development of, and future opportunities for, IAP inhibitors in LA SCCHN are discussed, in light of recent encouraging proof-of-concept clinical trial data.

Fragment-based discovery of focal adhesion kinase inhibitors.

Chemically diverse fragment hits of focal adhesion kinase (FAK) were discovered by surface plasmon resonance (SPR) screening of our in-house fragment library. Site specific binding of the primary hits was confirmed in a competition setup using a high-affinity ATP-site inhibitor of FAK. Protein crystallography revealed the binding mode of 41 out of 48 selected fragment hits within the ATP-site. Structural comparison of the fragment binding modes with a DFG-out inhibitor of FAK initiated first synthetic follow-up optimization leading to improved binding affinity.

Inhibiting the inhibitors: Development of the IAP inhibitor xevinapant for the treatment of locally advanced squamous cell carcinoma of the head and neck.

Standard of care for patients with locally advanced squamous cell carcinoma of the head and neck (LA SCCHN) is surgery followed by chemoradiotherapy (CRT) or definitive CRT. However, approximately 50 % of patients with LA SCCHN develop disease recurrence or metastasis within 2 years of completing treatment, and the outcome for these patients is poor. Despite this, the current treatment landscape for LA SCCHN has remained relatively unchanged for more than 2 decades, and novel treatment options are urgently required. One of the key causes of disease recurrence is treatment resistance, which commonly occurs due to cancer cells' ability to evade apoptosis. Evasion of apoptosis has been in part attributed to the overexpression of inhibitor of apoptosis proteins (IAPs). IAPs, including X-linked IAP (XIAP) and cellular IAP 1 and 2 (cIAP1/2), are a class of proteins that regulate apoptosis induced by intrinsic and extrinsic apoptotic pathways. IAPs have been shown to be overexpressed in SCCHN, are associated with poor clinical outcomes, and are, therefore, a rational therapeutic target. To date, several IAP inhibitors have been investigated; however, only xevinapant, a potent, oral, small-molecule IAP inhibitor, has shown clinical proof of concept when combined with CRT. Specifically, xevinapant demonstrated superior efficacy in combination with CRT vs placebo + CRT in a randomized, double-blind, phase 2 trial in patients with unresected LA SCCHN. Here, we describe the current treatment landscape in LA SCCHN and provide the rationale for targeting IAPs and the clinical data reported for xevinapant.

Antitumor Efficacy of the Novel KIT Inhibitor IDRX-42 (Formerly M4205) in Patient- and Cell Line-Derived Xenograft Models of Gastrointestinal Stromal Tumor (GIST).

PURPOSE: The majority of gastrointestinal stromal tumors (GIST) are driven by constitutively activated KIT/PDGFRA kinases and are susceptible to treatment with tyrosine kinase inhibitors. During treatment, most of these tumors will develop secondary mutations in KIT or PDGFRA inducing drug resistance, so there is an unmet need for novel therapies. We tested the efficacy of IDRX-42, a novel selective KIT inhibitor with high activity toward the most relevant KIT mutations, in 4 GIST xenograft models. EXPERIMENTAL DESIGN: NMRI nu/nu mice were transplanted with patient-derived GIST xenograft models UZLX-GIST9 (KIT:p.P577del;W557LfsX5;D820G), UZLX-GIST2B (KIT:p.A502_Y503dup), UZLX-GIST25 (KIT:p.K642E), and the cell line-derived model GIST882 (KIT:p.K642E). Mice were treated daily with vehicle (control), imatinib (100 mg/kg), sunitinib (20 mg/kg), avapritinib (5 mg/kg), or IDRX-42 (10 mg/kg, 25 mg/kg). Efficacy was assessed by tumor volume evolution, histopathology, grading of histologic response, and IHC. The Kruskal-Wallis and Wilcoxon matched-pairs tests were used for statistical analysis, with P < 0.05 considered as significant. RESULTS: IDRX-42 (25 mg/kg) caused tumor volume shrinkage in UZLX-GIST25, GIST882, and UZLX-GIST2B, with a relative decrease to 45.6%, 57.3%, and 35.1% on the last day as compared with baseline, and tumor growth delay (160.9%) compared with control in UZLX-GIST9. Compared with controls, IDRX-42 (25 mg/kg) induced a significant decrease in mitosis. In UZLX-GIST25 and GIST882 grade 2-4 histologic response with myxoid degeneration was observed in all IDRX-42 (25 mg/kg)-treated tumors. CONCLUSIONS: IDRX-42 showed significant antitumor activity in patient- and cell line-derived GIST xenograft models. The novel kinase inhibitor induced volumetric responses, decreased mitotic activity, and had antiproliferative effects. In models with KIT exon 13 mutation IDRX-42 induced characteristic myxoid degeneration.

Identification of M4205─A Highly Selective Inhibitor of KIT Mutations for Treatment of Unresectable Metastatic or Recurrent Gastrointestinal Stromal Tumors.

The treatment of gastrointestinal stromal tumors (GISTs) driven by activating mutations in the KIT gene is a prime example of targeted therapy for treatment of cancer. The approval of the tyrosine kinase inhibitor imatinib has significantly improved patient survival, but emerging resistance under treatment and relapse is observed. Several additional KIT inhibitors have been approved; still, there is a high unmet need for KIT inhibitors with high selectivity and broad coverage of all clinically relevant KIT mutants. An imidazopyridine hit featuring excellent kinase selectivity was identified in a high-throughput screen (HTS) and optimized to the clinical candidate M4205 (IDRX-42). This molecule has a superior profile compared to approved drugs, suggesting a best-in-class potential for recurrent and metastatic GISTs driven by KIT mutations.

MSC-1186, a Highly Selective Pan-SRPK Inhibitor Based on an Exceptionally Decorated Benzimidazole-Pyrimidine Core.

The highly conserved catalytic sites in protein kinases make it difficult to identify ATP competitive inhibitors with kinome-wide selectivity. Serendipitously, during a dedicated fragment campaign for the focal adhesion kinase (FAK), a scaffold that had lost its initial FAK affinity showed remarkable potency and selectivity for serine-arginine-protein kinases 1-3 (SRPK1-3). Non-conserved interactions with the uniquely structured hinge region of the SRPK family were the key drivers of the exclusive selectivity of the discovered fragment hit. Structure-guided medicinal chemistry efforts led to the SRPK inhibitor MSC-1186, which fulfills all hallmarks of a reversible chemical probe, including nanomolar cellular potency and excellent kinome-wide selectivity. The combination of MSC-1186 with CDC2-like kinase (CLK) inhibitors showed additive attenuation of SR-protein phosphorylation compared to the single agents. MSC-1186 and negative control (MSC-5360) are chemical probes available via the Structural Genomics Consortium chemical probe program (https://www.sgc-ffm.uni-frankfurt.de/).

Overcoming MET-mediated resistance in oncogene-driven NSCLC.

This study evaluates the efficacy of combining targeted therapies with MET or SHP2 inhibitors to overcome MET-mediated resistance in different NSCLC subtypes. A prevalence study was conducted for MET amplification and overexpression in samples from patients with NSCLC who relapsed on ALK, ROS1, or RET tyrosine kinase inhibitors. MET-mediated resistance was detected in 37.5% of tissue biopsies, which allow the detection of MET overexpression, compared to 7.4% of liquid biopsies. The development of drug resistance by MET overexpression was confirmed in EGFRex19del-, KRASG12C-, HER2ex20ins-, and TPM3-NTRK1-mutant cell lines. The combination of targeted therapy with MET or SHP2 inhibitors was found to overcome MET-mediated resistance in both in vitro and in vivo assays. This study highlights the importance of considering MET overexpression as a resistance driver to NSCLC targeted therapies to better identify patients who could potentially benefit from combination approaches with MET or SHP2 inhibitors.

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).

FGFR1 overexpression in non-small cell lung cancer is mediated by genetic and epigenetic mechanisms and is a determinant of FGFR1 inhibitor response.

Amplification of fibroblast growth factor receptor 1 (FGFR1) in non-small cell lung cancer (NSCLC) has been considered as an actionable drug target. However, pan-FGFR tyrosine kinase inhibitors did not demonstrate convincing clinical efficacy in FGFR1-amplified NSCLC patients. This study aimed to characterise the molecular context of FGFR1 expression and to define biomarkers predictive of FGFR1 inhibitor response. In this study, 635 NSCLC samples were characterised for FGFR1 protein expression by immunohistochemistry and copy number gain (CNG) by in situ hybridisation (n = 298) or DNA microarray (n = 189). FGFR1 gene expression (n = 369) and immune cell profiles (n = 309) were also examined. Furthermore, gene expression, methylation and microRNA data from The Cancer Genome Atlas (TCGA) were compared. A panel of FGFR1-amplified NSCLC patient-derived xenograft (PDX) models were tested for response to the selective FGFR1 antagonist M6123. A minority of patients demonstrated FGFR1 CNG (10.5%) or increased FGFR1 mRNA (8.7%) and protein expression (4.4%). FGFR1 CNG correlated weakly with FGFR1 gene and protein expression. Tumours overexpressing FGFR1 protein were typically devoid of driver alterations (e.g. EGFR, KRAS) and showed reduced infiltration of T-lymphocytes and lower PD-L1 expression. Promoter methylation and microRNA were identified as regulators of FGFR1 expression in NSCLC and other cancers. Finally, NSCLC PDX models demonstrating FGFR1 amplification and FGFR1 protein overexpression were sensitive to M6123. The unique molecular and immune features of tumours with high FGFR1 expression provide a rationale to stratify patients in future clinical trials of FGFR1 pathway-targeting agents.

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.

SHP2 Inhibition Influences Therapeutic Response to Tepotinib in Tumors with MET Alterations.

Tepotinib is an oral MET inhibitor approved for metastatic non-small cell lung cancer (NSCLC) harboring MET exon 14 (METex14) skipping mutations. Examining treatment-naive or tepotinib-resistant cells with MET amplification or METex14 skipping mutations identifies other receptor tyrosine kinases (RTKs) that co-exist in cells prior to tepotinib exposure and become more prominent upon tepotinib resistance. In a small cohort of patients with lung cancer with MET genetic alterations treated with tepotinib, gene copy number gains of other RTKs were found at baseline and affected treatment outcome. An Src homology 2 domain-containing phosphatase 2 (SHP2) inhibitor delayed the emergence of tepotinib resistance and synergized with tepotinib in treatment-naive and tepotinib-resistant cells as well as in xenograft models. Alternative signaling pathways potentially diminish the effect of tepotinib monotherapy, and the combination of tepotinib with an SHP2 inhibitor enables the control of tumor growth in cells with MET genetic alterations.