Development and Comprehensive Benchmark of a High-Quality AMBER-Consistent Small Molecule Force Field with Broad Chemical Space Coverage for Molecular Modeling and Free Energy Calculation.

Biomolecular simulations have become an essential tool in contemporary drug discovery, and molecular mechanics force fields (FFs) constitute its cornerstone. Developing a high quality and broad coverage general FF is a significant undertaking that requires substantial expert knowledge and computing resources, which is beyond the scope of general practitioners. Existing FFs originate from only a limited number of groups and organizations, and they either suffer from limited numbers of training sets, lower than desired quality because of oversimplified representations, or are costly for the molecular modeling community to access. To address these issues, in this work, we developed an AMBER-consistent small molecule FF with extensive chemical space coverage, and we provide Open Access parameters for the entire modeling community. To validate our FF, we carried out benchmarks of quantum mechanics (QM)/molecular mechanics conformer comparison and free energy perturbation calculations on several benchmark data sets. Our FF achieves a higher level of performance at reproducing QM energies and geometries than two popular open-source FFs, OpenFF2 and GAFF2. In relative binding free energy calculations for 31 protein-ligand data sets, comprising 1079 pairs of ligands, the new FF achieves an overall root-mean-square error of 1.19 kcal/mol for ΔΔG and 0.92 kcal/mol for ΔG on a subset of 463 ligands without bespoke fitting to the data sets. The results are on par with those of the leading commercial series of OPLS FFs.

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.

Selective, Small-Molecule Co-Factor Binding Site Inhibition of a Su(var)3-9, Enhancer of Zeste, Trithorax Domain Containing Lysine Methyltransferase.

The first chemical probe to primarily occupy the co-factor binding site of a Su(var)3-9, enhancer of a zeste, trithorax (SET) domain containing protein lysine methyltransferase (PKMT) is reported. Protein methyltransferases require S-adenosylmethionine (SAM) as a co-factor (methyl donor) for enzymatic activity. However, SAM itself represents a poor medicinal chemistry starting point for a selective, cell-active inhibitor given its extreme physicochemical properties and its role in multiple cellular processes. A previously untested medicinal chemistry strategy of deliberate file enrichment around molecules bearing the hallmarks of SAM, but with improved lead-like properties from the outset, yielded viable hits against SET and MYND domain-containing protein 2 (SMYD2) that were shown to bind in the co-factor site. These leads were optimized to identify a highly biochemically potent, PKMT-selective, and cell-active chemical probe. While substrate-based inhibitors of PKMTs are known, this represents a novel, co-factor-derived strategy for the inhibition of SMYD2 which may also prove applicable to lysine methyltransferase family members previously thought of as intractable.

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.

Design and Synthesis of Pyridone-Containing 3,4-Dihydroisoquinoline-1(2H)-ones as a Novel Class of Enhancer of Zeste Homolog 2 (EZH2) Inhibitors.

A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound 18. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound 31. Compound 31 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.

Design, synthesis, and evaluation of 3,4-dihydro-2H-[1,4]diazepino[6,7,1-hi]indol-1-ones as inhibitors of poly(ADP-ribose) polymerase.

The design, synthesis, and biological evaluation of potent inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) are reported. A novel series of 3,4-dihydro-2H-[1,4]diazepino[6,7,1-hi]indol-1-ones were designed using a combination of protein structure-based drug design, molecular modeling, and structure-activity relationships (SAR). These novel submicromolar inhibitors possess a tricyclic ring system conformationally restricting the benzamide in the preferred cis orientation. The compounds were designed to optimize space-filling and atomic interactions within the NAD+ binding site of PARP-1. Previously described and newly adapted methods were applied to syntheses of these tricyclic inhibitors. Various modifications were made to the diazepinoindolones at the 6- and 7-positions in order to study this region of the active site and optimize noncovalent interactions. The electron density of derivative 28 bound to chicken PARP-1 revealed that the oxime makes a tight hydrogen bond with the catalytic gamma-carboxylate of glutamic acid (Glu) 988 in accordance with our original designs and models. Most of the compounds have been evaluated for inhibition of human PARP-1. Selected inhibitors were also tested for the ability to potentiate the cytotoxic effect of the DNA-damaging agent Topotecan.

Novel tricyclic poly(ADP-ribose) polymerase-1 inhibitors with potent anticancer chemopotentiating activity: design, synthesis, and X-ray cocrystal structure.

A series of novel compounds have been designed that are potent inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1), and the activity and physical properties have been characterized. The new structural classes, 3,4,5,6-tetrahydro-1H-azepino[5,4,3-cd]indol-6-ones and 3,4-dihydropyrrolo[4,3,2-de]isoquinolin-5-(1H)-ones, have conformationally locked benzamide cores that specifically interact with the PARP-1 protein. The compounds have been evaluated with in vitro cellular assays that measure the ability of the PARP-1 inhibitors to enhance the effect of cytotoxic agents against cancer cell lines.

Anticancer chemosensitization and radiosensitization by the novel poly(ADP-ribose) polymerase-1 inhibitor AG14361.

BACKGROUND: Poly(ADP-ribose) polymerase-1 (PARP-1) facilitates the repair of DNA strand breaks. Inhibiting PARP-1 increases the cytotoxicity of DNA-damaging chemotherapy and radiation therapy in vitro. Because classical PARP-1 inhibitors have limited clinical utility, we investigated whether AG14361, a novel potent PARP-1 inhibitor (inhibition constant <5 nM), enhances the effects of chemotherapy and radiation therapy in human cancer cell cultures and xenografts. METHODS: The effect of AG14361 on the antitumor activity of the DNA alkylating agent temozolomide, topoisomerase I poisons topotecan or irinotecan, or x-irradiation or gamma-radiation was investigated in human cancer cell lines A549, LoVo, and SW620 by proliferation and survival assays and in xenografts in mice by tumor volume determination. The specificity of AG14361 for PARP-1 was investigated by microarray analysis and by antiproliferation and acute toxicity assays in PARP-1-/- and PARP-1+/+ cells and mice. After intraperitoneal administration, the concentration of AG14361 was determined in mouse plasma and tissues, and its effect on PARP-1 activity was determined in tumor homogenates. All statistical tests were two-sided. RESULTS: AG14361 at 0.4 micro M did not affect cancer cell gene expression or growth, but it did increase the antiproliferative activity of temozolomide (e.g., in LoVo cells by 5.5-fold, 95% confidence interval [CI] = 4.9-fold to 5.9-fold; P =.004) and topotecan (e.g., in LoVo cells by 1.6-fold, 95% CI = 1.3-fold to 1.9-fold; P =.002) and inhibited recovery from potentially lethal gamma-radiation damage in LoVo cells by 73% (95% CI = 48% to 98%). In vivo, nontoxic doses of AG14361 increased the delay of LoVo xenograft growth induced by irinotecan, x-irradiation, or temozolomide by two- to threefold. The combination of AG14361 and temozolomide caused complete regression of SW620 xenograft tumors. AG14361 was retained in xenografts in which PARP-1 activity was inhibited by more than 75% for at least 4 hours. CONCLUSION: AG14361 is, to our knowledge, the first high-potency PARP-1 inhibitor with the specificity and in vivo activity to enhance chemotherapy and radiation therapy of human cancer.