SAM-Competitive PRMT5 Inhibitor PF-06939999 Demonstrates Antitumor Activity in Splicing Dysregulated NSCLC with Decreased Liability of Drug Resistance.

Protein arginine methyltransferase 5 (PRMT5) overexpression in hematologic and solid tumors methylates arginine residues on cellular proteins involved in important cancer functions including cell-cycle regulation, mRNA splicing, cell differentiation, cell signaling, and apoptosis. PRMT5 methyltransferase function has been linked with high rates of tumor cell proliferation and decreased overall survival, and PRMT5 inhibitors are currently being explored as an approach for targeting cancer-specific dependencies due to PRMT5 catalytic function. Here, we describe the discovery of potent and selective S-adenosylmethionine (SAM) competitive PRMT5 inhibitors, with in vitro and in vivo characterization of clinical candidate PF-06939999. Acquired resistance mechanisms were explored through the development of drug resistant cell lines. Our data highlight compound-specific resistance mutations in the PRMT5 enzyme that demonstrate structural constraints in the cofactor binding site that prevent emergence of complete resistance to SAM site inhibitors. PRMT5 inhibition by PF-06939999 treatment reduced proliferation of non-small cell lung cancer (NSCLC) cells, with dose-dependent decreases in symmetric dimethyl arginine (SDMA) levels and changes in alternative splicing of numerous pre-mRNAs. Drug sensitivity to PF-06939999 in NSCLC cells associates with cancer pathways including MYC, cell cycle and spliceosome, and with mutations in splicing factors such as RBM10. Translation of efficacy in mouse tumor xenograft models with splicing mutations provides rationale for therapeutic use of PF-06939999 in the treatment of splicing dysregulated NSCLC.

Expanding control of the tumor cell cycle with a CDK2/4/6 inhibitor.

The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival in HR+/HER2- breast cancer when combined with anti-hormonals. We sought to discover PAL resistance mechanisms in preclinical models and through analysis of clinical transcriptome specimens, which coalesced on induction of MYC oncogene and Cyclin E/CDK2 activity. We propose that targeting the G1 kinases CDK2, CDK4, and CDK6 with a small-molecule overcomes resistance to CDK4/6 inhibition. We describe the pharmacodynamics and efficacy of PF-06873600 (PF3600), a pyridopyrimidine with potent inhibition of CDK2/4/6 activity and efficacy in multiple in vivo tumor models. Together with the clinical analysis, MYC activity predicts (PF3600) efficacy across multiple cell lineages. Finally, we find that CDK2/4/6 inhibition does not compromise tumor-specific immune checkpoint blockade responses in syngeneic models. We anticipate that (PF3600), currently in phase 1 clinical trials, offers a therapeutic option to cancer patients in whom CDK4/6 inhibition is insufficient to alter disease progression.

Discovery of PF-06873600, a CDK2/4/6 Inhibitor for the Treatment of Cancer.

Control of the cell cycle through selective pharmacological inhibition of CDK4/6 has proven beneficial in the treatment of breast cancer. Extending this level of control to additional cell cycle CDK isoforms represents an opportunity to expand to additional tumor types and potentially provide benefits to patients that develop tumors resistant to selective CDK4/6 inhibitors. However, broad-spectrum CDK inhibitors have a long history of failure due to safety concerns. In this approach, we describe the use of structure-based drug design and Free-Wilson analysis to optimize a series of CDK2/4/6 inhibitors. Further, we detail the use of molecular dynamics simulations to provide insights into the basis for selectivity against CDK9. Based on overall potency, selectivity, and ADME profile, PF-06873600 (22) was identified as a candidate for the treatment of cancer and advanced to phase 1 clinical trials.

Structure-Based Drug Design and Synthesis of PI3Kα-Selective Inhibitor (PF-06843195).

The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway is a frequently dysregulated pathway in human cancer, and PI3Kα is one of the most frequently mutated kinases in human cancer. A PI3Kα-selective inhibitor may provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family. Here, we describe our efforts to discover a PI3Kα-selective inhibitor by applying structure-based drug design (SBDD) and computational analysis. A novel series of compounds, exemplified by 2,2-difluoroethyl (3S)-3-{[2'-amino-5-fluoro-2-(morpholin-4-yl)-4,5'-bipyrimidin-6-yl]amino}-3-(hydroxymethyl)pyrrolidine-1-carboxylate (1) (PF-06843195), with high PI3Kα potency and unique PI3K isoform and mTOR selectivity were discovered. We describe here the details of the design and synthesis program that lead to the discovery of 1.

Optimization of Orally Bioavailable Enhancer of Zeste Homolog 2 (EZH2) Inhibitors Using Ligand and Property-Based Design Strategies: Identification of Development Candidate (R)-5,8-Dichloro-7-(methoxy(oxetan-3-yl)methyl)-2-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3,4-dihydroisoquinolin-1(2H)-one (PF-06821497).

A new series of lactam-derived EZH2 inhibitors was designed via ligand-based and physicochemical-property-based strategies to address metabolic stability and thermodynamic solubility issues associated with previous lead compound 1. The new inhibitors incorporated an sp3 hybridized carbon atom at the 7-position of the lactam moiety present in lead compound 1 as a replacement for a dimethylisoxazole group. This transformation enabled optimization of the physicochemical properties and potency compared to compound 1. Analysis of relationships between calculated log D (clogD) values and in vitro metabolic stability and permeability parameters identified a clogD range that afforded an increased probability of achieving favorable ADME data in a single molecule. Compound 23a exhibited the best overlap of potency and pharmaceutical properties as well as robust tumor growth inhibition in vivo and was therefore advanced as a development candidate (PF-06821497). A crystal structure of 23a in complex with the three-protein PRC2 complex enabled understanding of the key structural features required for optimal binding.

Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth.

Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K(d) = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC(50) = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC(50) = 4.7 +/- 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC(50) value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.