Discovery of an Orally Bioavailable and Selective PKMYT1 Inhibitor, RP-6306.

PKMYT1 is a regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with CCNE1 amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1. To address this need compound 1 was identified as a weak PKMYT1 inhibitor. Introduction of a dimethylphenol increased potency on PKMYT1. These dimethylphenol analogs were found to exist as atropisomers that could be separated and profiled as single enantiomers. Structure-based drug design enabled optimization of cell-based potency. Parallel optimization of ADME properties led to the identification of potent and selective inhibitors of PKMYT1. RP-6306 inhibits CCNE1-amplified tumor cell growth in several preclinical xenograft models. The first-in-class clinical candidate RP-6306 is currently being evaluated in Phase 1 clinical trials for treatment of various solid tumors.

CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition.

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

Synthesis and evaluation of NS5A inhibitors containing diverse heteroaromatic cores.

Inhibitors of the HCV NS5A nonstructural protein are showing promising clinical potential in the treatment of hepatitis C when used in combination with other direct-acting antiviral agents. Current NS5A clinical candidates such as daclatasvir, ledipasvir, and ombitasvir share a common pharmacophore that features a pair of (S)-methoxycarbonylvaline capped pyrrolidines linked to various cores by amides, imidazoles and/or benzimidazoles. In this Letter, we describe the evaluation of NS5A inhibitors which contain alternative heteroaromatic replacements for these amide mimetics. The SAR knowledge gleaned in the optimization of scaffolds containing benzoxazoles was parlayed toward the identification of potent NS5A inhibitors containing other heteroaromatic replacements such as indoles and imidazopyridines.

Benzimidazole-containing HCV NS5A inhibitors: effect of 4-substituted pyrrolidines in balancing genotype 1a and 1b potency.

The treatment of HCV with highly efficacious, well-tolerated, interferon-free regimens is a compelling clinical goal. Trials employing combinations of direct-acting antivirals that include NS5A inhibitors have shown significant promise in meeting this challenge. Herein, we describe our efforts to identify inhibitors of NS5A and report on the discovery of benzimidazole-containing analogs with subnanomolar potency against genotype 1a and 1b replicons. Our SAR exploration of 4-substituted pyrrolidines revealed that the subtle inclusion of a 4-methyl group could profoundly increase genotype 1a potency in multiple scaffold classes.

Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes.

Galectin-3 is unique among the galectin family of animal lectins in its biological activities and structure. Most members of the galectin family including galectin-1 possess apoptotic activities, whereas galectin-3 possesses anti-apoptotic activity. Galectin-3 is also the only chimera type galectin and consists of a nonlectin N-terminal domain and a C-terminal carbohydrate-binding domain. Recent sedimentation equilibrium and velocity studies show that murine galectin-3 is a monomer in the absence and presence of LacNAc, a monovalent sugar. However, quantitative precipitation studies in the present report indicate that galectin-3 precipitates as a pentamer with a series of divalent pentasaccharides with terminal LacNAc residues. Furthermore, the kinetics of precipitation are fast, on the order of seconds. This indicates that although the majority of galectin-3 in solution is a monomer, a rapid equilibrium exists between the monomer and a small percentage of pentamer. The latter, in turn, precipitates with the divalent oligosaccharides, resulting in rapid conversion of monomer to pentamer by mass action equilibria. Mixed quantitative precipitation experiments and electron microscopy suggest that galectin-3 forms heterogenous, disorganized cross-linking complexes with the multivalent carbohydrates. This contrasts with galectin-1 and many plant lectins that form homogeneous, organized cross-linked complexes. The results are discussed in terms of the biological properties of galectin-3.

First demonstration of differential inhibition of lectin binding by synthetic tri- and tetravalent glycoclusters from cross-coupling of rigidified 2-propynyl lactoside.

The interplay of mammalian lectins such as galectins with cellular glycoconjugates is intimately involved in crucial reaction pathways including tumor cell adhesion, migration or growth regulation. These clinically relevant functions explain the interest in designing glycoclusters with potent activity to interfere with lectin binding. In view of the perspective for medical applications the following objective arises: to correlate topological factors of ligand display most favorably to reactivity against endogenous lectins. To date, plant agglutinins have commonly been used as models. Properly addressing this issue we first prepared di- to tetravalent clusters from 2-propynyl lactoside under mild oxidative homocoupling conditions and using the Sonogashira palladium-catalyzed cross-coupling reaction with triiodobenzene or pentaerythritol cores. These products were tested for bioactivity in a competitive solid-phase assay using different labeled sugar receptors as probes, i,e. the beta-trefoil mistletoe lectin, the natural lactoside-binding immunoglobulin G fraction from human serum and three mammalian galectins from two subgroups. The lactose headgroups in the derivatives retained ligand properties. Differences in inhibitory capacity were marked between the galectins. In contrast to homodimeric proto-type galectins-1 and -7 significant inhibition of galectin-3 binding with a 7-fold increase in relative potency was observed for the trivalent compound. In comparison, the binding of the beta-trefoil mistletoe agglutinin was reduced best by tetravalent substances The result for galectin-3 was independently confirmed by haemagglutination and cytofluorometric cell binding assays. These data underline the feasibility of galectin-type target selectivity by compound design despite using an identical headgroup (lactose) in synthesis.

Efficient ruthenium carbenoid-catalyzed cross-metathesis of allyl halides with olefins.

[reaction: see text] Cross-metatheses of allyl halides and terminal olefins mediated by catalyst 2 are reported and showed good yield and excellent E/Z selectivity. The application of these compounds as alkylating reagents is also demonstrated.

Olefin self-metathesis as a new entry into xenotransplantation antagonists bearing the Galili antigen.

A hexameric disaccharide cluster bearing the terminal Gal alpha related xenotransplantation antigen was constructed using a sequence of ruthenium carbenoid catalyzed olefin self-metathesis of monoallylated tribenzyl pentaerythritol followed, after interconversion of benzyl ethers into para-iodobenzyl ethers, by a single step Sonogashira cross-coupling of six prop-2-ynyl glycosides onto a hexameric aryl iodide scaffold.