MK-8353: Discovery of an Orally Bioavailable Dual Mechanism ERK Inhibitor for Oncology.

The emergence and evolution of new immunological cancer therapies has sparked a rapidly growing interest in discovering novel pathways to treat cancer. Toward this aim, a novel series of pyrrolidine derivatives (compound 5) were identified as potent inhibitors of ERK1/2 with excellent kinase selectivity and dual mechanism of action but suffered from poor pharmacokinetics (PK). The challenge of PK was overcome by the discovery of a novel 3(S)-thiomethyl pyrrolidine analog 7. Lead optimization through focused structure-activity relationship led to the discovery of a clinical candidate MK-8353 suitable for twice daily oral dosing as a potential new cancer therapeutic.

Development of MK-8353, an orally administered ERK1/2 inhibitor, in patients with advanced solid tumors.

BACKGROUND: Constitutive activation of ERK1/2 occurs in various cancers, and its reactivation is a well-described resistance mechanism to MAPK inhibitors. ERK inhibitors may overcome the limitations of MAPK inhibitor blockade. The dual mechanism inhibitor SCH772984 has shown promising preclinical activity across various BRAFV600/RAS-mutant cancer cell lines and human cancer xenografts. METHODS: We have developed an orally bioavailable ERK inhibitor, MK-8353; conducted preclinical studies to demonstrate activity, pharmacodynamic endpoints, dosing, and schedule; completed a study in healthy volunteers (P07652); and subsequently performed a phase I clinical trial in patients with advanced solid tumors (MK-8353-001). In the P07652 study, MK-8353 was administered as a single dose in 10- to 400-mg dose cohorts, whereas in the MK-8353-001 study, MK-8353 was administered in 100- to 800-mg dose cohorts orally twice daily. Safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity were analyzed. RESULTS: MK-8353 exhibited comparable potency with SCH772984 across various preclinical cancer models. Forty-eight patients were enrolled in the P07652 study, and twenty-six patients were enrolled in the MK-8353-001 study. Adverse events included diarrhea (44%), fatigue (40%), nausea (32%), and rash (28%). Dose-limiting toxicity was observed in the 400-mg and 800-mg dose cohorts. Sufficient exposure to MK-8353 was noted that correlated with biological activity in preclinical data. Three of fifteen patients evaluable for treatment response in the MK-8353-001 study had partial response, all with BRAFV600-mutant melanomas. CONCLUSION: MK-8353 was well tolerated up to 400 mg twice daily and exhibited antitumor activity in patients with BRAFV600-mutant melanoma. However, antitumor activity was not particularly correlated with pharmacodynamic parameters. TRIAL REGISTRATION: ClinicalTrials.gov NCT01358331. FUNDING: Merck Sharp & Dohme Corp., a subsidiary of Merck & Co. Inc., and NIH (P01 CA168585 and R35 CA197633).

Structure-activity relationship study of 4-substituted piperidines at Leu26 moiety of novel p53-hDM2 inhibitors.

Led by the structural information of the screening hit with mDM2 protein, a structure modification of Leu26 moiety of the novel p53-hDM2 inhibitors was conducted. A structure-activity relationship study of 4-substituted piperidines revealed compound 20t with good potencies and excellent CYP450 profiles.

Discovery of Novel 3,3-Disubstituted Piperidines as Orally Bioavailable, Potent, and Efficacious HDM2-p53 Inhibitors.

A new subseries of substituted piperidines as p53-HDM2 inhibitors exemplified by 21 has been developed from the initial lead 1. Research focused on optimization of a crucial HDM2 Trp23-ligand interaction led to the identification of 2-(trifluoromethyl)thiophene as the preferred moiety. Further investigation of the Leu26 pocket resulted in potent, novel substituted piperidine inhibitors of the HDM2-p53 interaction that demonstrated tumor regression in several human cancer xenograft models in mice. The structure of HDM2 in complex with inhibitors 3, 10, and 21 is described.

Discovery of novel, dual mechanism ERK inhibitors by affinity selection screening of an inactive kinase.

An affinity-based mass spectrometry screening technology was used to identify novel binders to both nonphosphorylated and phosphorylated ERK2. Screening of inactive ERK2 identified a pyrrolidine analogue 1 that bound to both nonphosphorylated and phosphorylated ERK2 and inhibited ERK2 kinase activity. Chemical optimization identified compound 4 as a novel, potent, and highly selective ERK1,2 inhibitor which not only demonstrated inhibition of phosphorylation of ERK substrate p90RSK but also demonstrated inhibition of ERK1,2 phosphorylation on the activation loop. X-ray cocrystallography revealed that upon binding of compound 4 to ERK2, Tyr34 undergoes a rotation (flip) along with a shift in the poly-Gly rich loop to create a new binding pocket into which 4 can bind. This new binding mode represents a novel mechanism by which high affinity ATP-competitive compounds may achieve excellent kinase selectivity.

Pivotal Role of an Aliphatic Side Chain in the Development of an HDM2 Inhibitor.

Introduction of an aliphatic side chain to a key position of a novel piperidine-based HDM2 inhibitor scaffold resulted in significant potency gains, enabling further series progression.

Core modification of substituted piperidines as novel inhibitors of HDM2-p53 protein-protein interaction.

The discovery of 3,3-disubstituted piperidine 1 as novel p53-HDM2 inhibitors prompted us to implement subsequent SAR follow up directed towards piperidine core modifications. Conformational restrictions and further functionalization of the piperidine core were investigated as a strategy to gain additional interactions with HDM2. Substitutions at positions 4, 5 and 6 of the piperidine ring were explored. Although some substitutions were tolerated, no significant improvement in potency was observed compared to 1. Incorporation of an allyl side chain at position 2 provided a drastic improvement in binding potency.

Substituted piperidines as HDM2 inhibitors.

Novel small molecule HDM2 inhibitor, substituted piperidine, was identified. Initial SAR study indicated potential for several position optimizations. Additional potency enhancement was achieved by introducing a sidechain off the aromatic ring. DMPK study of one of the active compounds has shown a moderate oral PK and reasonable bioavailability.

Modulating the interaction between CDK2 and cyclin A with a quinoline-based inhibitor.

A new class of quinoline-based kinase inhibitors has been discovered that both disrupt cyclin dependent 2 (CDK2) interaction with its cyclin A subunit and act as ATP competitive inhibitors. The key strategy for discovering this class of protein-protein disrupter compounds was to screen the monomer CDK2 in an affinity-selection/mass spectrometry-based technique and to perform secondary assays that identified compounds that bound only to the inactive CDK2 monomer and not the active CDK2/cyclin A heterodimer. Through a series of chemical modifications the affinity (Kd) of the original hit improved from 1 to 0.005µM.

Structure-based design and optimization of 2-aminothiazole-4-carboxamide as a new class of CHK1 inhibitors.

Drug design efforts in the emerging 2-aminothiazole-4-carboxamide class of CHK1 inhibitors have uncovered specific combinations of key substructures within the molecule; resulting in significant improvements in cell-based activity while retaining a greater than one hundred-fold selectivity against CDK2. The X-ray crystal structure of a complex between compound 39 and the CHK1 protein detailing a 'U-shaped' topology and key interactions with the protein surface at the ATP site is also reported.

Discovery of liver-targeted inhibitors of stearoyl-CoA desaturase (SCD1).

Inhibitors based on a benzo-fused spirocyclic oxazepine scaffold were discovered for stearoyl-coenzyme A (CoA) desaturase 1 (SCD1) and subsequently optimized to potent compounds with favorable pharmacokinetic profiles and in vivo efficacy in reducing the desaturation index in a mouse model. Initial optimization revealed potency preferences for the oxazepine core and benzylic positions, while substituents on the piperidine portions were more tolerant and allowed for tuning of potency and PK properties. After preparation and testing of a range of functional groups on the piperidine nitrogen, three classes of analogs were identified with single digit nanomolar potency: glycine amides, heterocycle-linked amides, and thiazoles. Responding to concerns about target localization and potential mechanism-based side effects, an initial effort was also made to improve liver concentration in an available rat PK model. An advanced compound 17m with a 5-carboxy-2-thiazole substructure appended to the spirocyclic piperidine scaffold was developed which satisfied the in vitro and in vivo requirements for more detailed studies.

Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4.

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.

Discovery of a Novel Series of CHK1 Kinase Inhibitors with a Distinctive Hinge Binding Mode.

A novel series of CHK1 inhibitors with a distinctive hinge binding mode, exemplified by 2-aryl-N-(2-(piperazin-1-yl)phenyl)thiazole-4-carboxamide, was discovered through high-throughput screening using the affinity selection-mass spectrometry (AS-MS)-based Automated Ligand Identification System (ALIS) platform. Structure-based ligand design and optimization led to significant improvements in potency to the single digit nanomolar range and hundred-fold selectivity against CDK2.

Discovery of novel imidazo[1,2-a]pyrazin-8-amines as Brk/PTK6 inhibitors.

A series of substituted imidazo[1,2-a]pyrazin-8-amines were discovered as novel breast tumor kinase (Brk)/protein tyrosine kinase 6 (PTK6) inhibitors. Tool compounds with low-nanomolar Brk inhibition activity, high selectivity towards other kinases and desirable DMPK properties were achieved to enable the exploration of Brk as an oncology target.

Small molecule inhibition of the TNF family cytokine CD40 ligand through a subunit fracture mechanism.

BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, aka CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging. Therefore, to understand how BIO8898 achieves this feat, we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC(50) = 25 µM and inhibited CD40L-dependent apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the protein's 3-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.

Small-molecule inhibitors of FABP4/5 ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity.

Fatty acid binding protein-4 (FABP4) and FABP5 are two closely related FA binding proteins expressed primarily in adipose tissue and/or macrophages. The small-molecule FABP4 inhibitor BMS309403 was previously reported to improve insulin sensitivity in leptin-deficient Lep(ob)/Lep(ob) (ob/ob) mice. However, this compound was not extensively characterized in the more physiologically relevant animal model of mice with diet-induced obesity (DIO). Here, we report the discovery and characterization of a novel series of FABP4/5 dual inhibitors represented by Compounds 1-3. Compared with BMS309403, the compounds had significant in vitro potency toward both FABP4 and FABP5. In cell-based assays, Compounds 2 and 3 were more potent than BMS309403 to inhibit lipolysis in 3T3-L1 adipocytes and in primary human adipocytes. They also inhibited MCP-1 release from THP-1 macrophages as well as from primary human macrophages. When chronically administered to DIO mice, BMS309403 and Compound 3 reduced plasma triglyceride and free FA levels. Compound 3 reduced plasma free FAs at a lower dose level than BMS309403. However, no significant change was observed in insulin, glucose, or glucose tolerance. Our results indicate that the FABP4/5 inhibitors ameliorate dyslipidemia but not insulin resistance in DIO mice.

Discovery and Hit-to-Lead Optimization of Non-ATP Competitive MK2 (MAPKAPK2) Inhibitors.

A novel series of non-ATP-competitive MK2 inhibitors based on a furan-2-carboxyamide scaffold was discovered through high-throughput screening using the affinity selection-mass spectrometry-based Automated Ligand Identification System platform. Medicinal chemistry efforts optimized the initial screening hit to leadlike compounds with significant improvements in biochemical and cellular potencies, while maintaining excellent kinase selectivity and in vitro pharmacokinetic properties. Biophysical and biochemical studies confirmed the unique non-ATP-competitive binding mode of this series and suggested that highly selective inhibitors of MK2 should be feasible by targeting the outside ATP pocket.

Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle protein motor domain.

Affinity selection-mass spectrometry (AS-MS) screening of kinesin spindle protein (KSP) followed by enzyme inhibition studies and temperature-dependent circular dichroism (TdCD) characterization was utilized to identify a series of benzimidazole compounds. This series also binds in the presence of Ispinesib, a known anticancer KSP inhibitor in phase I/II clinical trials for breast cancer. TdCD and AS-MS analyses support simultaneous binding implying existence of a novel non-Ispinesib binding pocket within KSP. Additional TdCD analyses demonstrate direct binding of these compounds to Ispinesib-resistant mutants (D130V, A133D, and A133D + D130V double mutant), further strengthening the hypothesis that the compounds bind to a distinct binding pocket. Also importantly, binding to this pocket causes uncompetitive inhibition of KSP ATPase activity. The uncompetitive inhibition with respect to ATP is also confirmed by the requirement of nucleotide for binding of the compounds. After preliminary affinity optimization, the benzimidazole series exhibited distinctive antimitotic activity as evidenced by blockade of bipolar spindle formation and appearance of monoasters. Cancer cell growth inhibition was also demonstrated either as a single agent or in combination with Ispinesib. The combination was additive as predicted by the binding studies using TdCD and AS-MS analyses. The available data support the existence of a KSP inhibitory site hitherto unknown in the literature. The data also suggest that targeting this novel site could be a productive strategy for eluding Ispinesib-resistant tumors. Finally, AS-MS and TdCD techniques are general in scope and may enable screening other targets in the presence of known drugs, clinical candidates, or tool compounds that bind to the protein of interest in an effort to identify potency-enhancing small molecules that increase efficacy and impede resistance in combination therapy.

Structure and activity relationships of tartrate-based TACE inhibitors.

The syntheses and structure-activity relationships of the tartrate-based TACE inhibitors are discussed. The optimization of both the prime and non-prime sites led to compounds with picomolar activity. Several analogs demonstrated good rat pharmacokinetics.

Inhibitors of hepatitis C virus polymerase: synthesis and characterization of novel 2-oxy-6-fluoro-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-benzamides.

SAR exploration from an initial hit, (S)-N-(2-cyclohexenylethyl)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)benzamide (1), identified using our proprietary automated ligand identification system (ALIS),(1) has led to a novel series of selective hepatitis C virus (HCV) NS5B polymerase inhibitors with improved in vitro potency as exemplified by (S)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)-N-isopentyl-N-methylbenzamidecarboxamide (41) (IC(50)=0.5 microM). The crystal structure of an analogue (44) was solved and provided rationalization of the SAR of this series, which binds in a distinct manner in the palm domain of NS5B, consistent with biochemical analysis using enzyme mutant variants. These data warrant further lead optimization efforts on this novel series of non-nucleoside inhibitors targeting the HCV polymerase.