Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma.

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2- breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Å2), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpKa = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse Kp,uu = 0.20-0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.

A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells.

EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkin's lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.

Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas.

EZH2, the catalytic subunit of the PRC2 complex, catalyzes the mono- through trimethylation of lysine 27 on histone H3 (H3K27). Histone H3K27 trimethylation is a mechanism for suppressing transcription of specific genes that are proximal to the site of histone modification. Point mutations of the EZH2 gene (Tyr641) have been reported to be linked to subsets of human B-cell lymphoma. The mutant allele is always found associated with a wild-type allele (heterozygous) in disease cells, and the mutations were reported to ablate the enzymatic activity of the PRC2 complex for methylating an unmodified peptide substrate. Here we demonstrate that the WT enzyme displays greatest catalytic efficiency (k(cat)/K) for the zero to monomethylation reaction of H3K27 and diminished efficiency for subsequent (mono- to di- and di- to trimethylation) reactions. In stark contrast, the disease-associated Y641 mutations display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to the WT enzyme. These results imply that the malignant phenotype of disease requires the combined activities of a H3K27 monomethylating enzyme (PRC2 containing WT EZH2 or EZH1) together with the mutant PRC2s for augmented conversion of H3K27 to the trimethylated form. To our knowledge, this is the first example of a human disease that is dependent on the coordinated activities of normal and disease-associated mutant enzymatic function.

Structure-Based Design of Potent, Selective, and Orally Bioavailable VPS34 Kinase Inhibitors.

VPS34 is a class III phosphoinositide 3-kinase involved in endosomal trafficking and autophagosome formation. Inhibitors of VPS34 were believed to have value as anticancer agents, but genetic and pharmacological data suggest that sustained inhibition of VPS34 kinase activity may not be well tolerated. Here we disclose the identification of a novel series of dihydropyrazolopyrazinone compounds represented by compound 5 as potent, selective, and orally bioavailable VPS34 inhibitors through a structure-based design strategy. A water-interacting hydrogen bond acceptor within an appropriate distance to a hinge-binding element was found to afford significant VPS34 potency across chemical scaffolds. The selectivity of compound 5 over PIK family kinases arises from interactions between the hinge-binding element and the pseudo-gatekeeper residue Met682. As recent in vivo pharmacology data suggests that sustained inhibition of VPS34 kinase activity may not be tolerated, structure-activity relationships leading to VPS34 inhibition may be helpful for avoiding this target in other ATP-competitive kinase programs.

Discovery of Spiro-azaindoline Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1).

Hematopoietic progenitor kinase 1 (HPK1) is implicated as a negative regulator of T-cell receptor-induced T-cell activation. Studies using HPK1 kinase-dead knock-in animals have demonstrated the loss of HPK1 kinase activity resulted in an increase in T-cell function and tumor growth inhibition in glioma models. Herein, we describe the discovery of a series of small molecule inhibitors of HPK1. Using a structure-based drug design approach, the kinase selectivity of the molecules was significantly improved by inducing and stabilizing an unusual P-loop folded binding mode. The metabolic liabilities of the initial 7-azaindole high-throughput screening hit were mitigated by addressing a key metabolic soft spot along with physicochemical property-based optimization. The resulting spiro-azaindoline HPK1 inhibitors demonstrated improved in vitro ADME properties and the ability to induce cytokine production in primary human T-cells.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

Hematopoietic Progenitor Kinase-1 Structure in a Domain-Swapped Dimer.

Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator of T cell receptor signaling, which dampens T cell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented T cell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context.

Chemogenetic analysis of human protein methyltransferases.

A survey of the human genome was performed to understand the constituency of protein methyltransferases (both protein arginine and lysine methyltransferases) and the relatedness of their catalytic domains. We identified 51 protein lysine methyltransferase proteins based on similarity to the canonical Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain. Disruptor of telomeric silencing-1-like, a known protein lysine methyltransferase, did not fit within the protein lysine methyltransferase family, but did group with the protein arginine methyltransferases, along with 44 other proteins, including the METTL and NOP2/Sun domain family proteins. We show that a representative METTL, METTL11A, demonstrates catalytic activity as a histone methyltransferase. We also solved the co-crystal structures of disruptor of telomeric silencing-1-like with S-adenosylmethionine and S-adenosylhomocysteine bound in its active site. The conformation of both ligands is virtually identical to that found in known protein arginine methyltransferases, METTL and NOP2/Sun domain family proteins and is distinct from that seen in the Drosophila Su(var)3-9, enhancer of zeste (E(z)), and trithorax (trx) domain protein lysine methyltransferases. We have developed biochemical assays for 11 members of the protein methyltransferase target class and have profiled the affinity of three ligands for these enzymes: the common methyl-donating substrate S-adenosylmethionine; the common reaction product S-adenosylhomocysteine; and the natural product sinefungin. The affinity of each of these ligands is mapped onto the family trees of the protein lysine methyltransferases and protein arginine methyltransferases to reveal patterns of ligand recognition by these enzymes.

Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor.

Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.