Development of small molecule inhibitors of natural killer group 2D receptor (NKG2D).

Natural killer group 2D (NKG2D) is a homodimeric activating immunoreceptor whose function is to detect and eliminate compromised cells upon binding to the NKG2D ligands (NKG2DL) major histocompatibility complex (MHC) molecules class I-related chain A (MICA) and B (MICB) and UL16 binding proteins (ULBP1-6). While typically present at low levels in healthy cells and tissue, NKG2DL expression can be induced by viral infection, cellular stress or transformation. Aberrant activity along the NKG2D/NKG2DL axis has been associated with autoimmune diseases due to the increased expression of NKG2D ligands in human disease tissue, making NKG2D inhibitors an attractive target for immunomodulation. Herein we describe the discovery and optimization of small molecule PPI (protein-protein interaction) inhibitors of NKG2D/NKG2DL. Rapid SAR was guided by structure-based drug design and accomplished by iterative singleton and parallel medicinal chemistry synthesis. These efforts resulted in the identification of several potent analogs (14, 21, 30, 45) with functional activity and improved LLE.

Discovery of a highly potent, selective, orally bioavailable inhibitor of KAT6A/B histone acetyltransferases with efficacy against KAT6A-high ER+ breast cancer.

KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.

Identification of small-molecule protein-protein interaction inhibitors for NKG2D.

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ+, and CD8+ T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein-protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure-activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

Design and Characterization of a Pyridone-Containing EZH2 Inhibitor Phosphate Prodrug.

A pyridone-derived phosphate prodrug of an enhancer of zeste homolog 2 (EZH2) inhibitor was designed and synthesized to improve the inhibitor's aqueous solubility. This prodrug (compound 5) was profiled in pharmacokinetic experiments to assess its ability to deliver the corresponding parent compound (compound 2) to animals in vivo following oral administration. Results from these studies showed that the prodrug was efficiently converted to its parent compound in vivo. In separate experiments, the prodrug demonstrated impressive in vivo tumor growth inhibition in a diffuse large B-cell lymphoma Karpas-422 cell line-derived xenograft model. The described prodrug strategy is expected to be generally applicable to poorly soluble pyridone-containing EZH2 inhibitors and provides a new option to enable such compounds to achieve sufficiently high exposures in vivo.

Harnessing Ionic Selectivity in Acetyltransferase Chemoproteomic Probes.

Chemical proteomics provides a powerful strategy for the high-throughput assignment of enzyme function or inhibitor selectivity. However, identifying optimized probes for an enzyme family member of interest and differentiating signal from the background remain persistent challenges in the field. To address this obstacle, here we report a physiochemical discernment strategy for optimizing chemical proteomics based on the coenzyme A (CoA) cofactor. First, we synthesize a pair of CoA-based sepharose pulldown resins differentiated by a single negatively charged residue and find this change alters their capture properties in gel-based profiling experiments. Next, we integrate these probes with quantitative proteomics and benchmark analysis of "probe selectivity" versus traditional "competitive chemical proteomics." This reveals that the former is well-suited for the identification of optimized pulldown probes for specific enzyme family members, while the latter may have advantages in discovery applications. Finally, we apply our anionic CoA pulldown probe to evaluate the selectivity of a recently reported small molecule N-terminal acetyltransferase inhibitor. These studies further validate the use of physical discriminant strategies in chemoproteomic hit identification and demonstrate how CoA-based chemoproteomic probes can be used to evaluate the selectivity of small molecule protein acetyltransferase inhibitors, an emerging class of preclinical therapeutic agents.

Characterization of Specific N-α-Acetyltransferase 50 (Naa50) Inhibitors Identified Using a DNA Encoded Library.

Two novel compounds were identified as Naa50 binders/inhibitors using DNA-encoded technology screening. Biophysical and biochemical data as well as cocrystal structures were obtained for both compounds (3a and 4a) to understand their mechanism of action. These data were also used to rationalize the binding affinity differences observed between the two compounds and a MLGP peptide-containing substrate. Cellular target engagement experiments further confirm the Naa50 binding of 4a and demonstrate its selectivity toward related enzymes (Naa10 and Naa60). Additional analogs of inhibitor 4a were also evaluated to study the binding mode observed in the cocrystal structures.

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.

Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance.

Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown. Here we report the crystal structures of the inhibitor-bound wild-type and Y641N PRC2. The structures illuminate an important role played by a stretch of 17 residues in the N-terminal region of EZH2, we call the activation loop, in the stimulation of the enzyme activity, inhibitor recognition and the potential development of the mutation-mediated drug resistance. The work presented here provides new avenues for the design and development of next-generation PRC2 inhibitors through establishment of a structure-based drug design platform.

SAH derived potent and selective EZH2 inhibitors.

A series of novel enhancer of zeste homolog 2 (EZH2) inhibitors was designed based on the chemical structure of the histone methyltransferase (HMT) inhibitor SAH (S-adenosyl-l-homocysteine). These nucleoside-based EZH2 inhibitors blocked the methylation of nucleosomes at H3K27 in biochemical assays employing both WT PRC2 complex as well as a Y641N mutant PRC2 complex. The most potent compound, 27, displayed IC50's against both complexes of 270 nM and 70 nM, respectively. To our knowledge, compound 27 is the most potent SAH-derived inhibitor of the EZH2 PRC2 complex yet identified. This compound also displayed improved potency, lipophilic efficiency (LipE), and selectivity profile against other lysine methyltransferases compared with SAH.

Chemogenetic evaluation of the mitotic kinesin CENP-E reveals a critical role in triple-negative breast cancer.

Breast cancer patients with tumors lacking the three diagnostic markers (ER, PR, and HER2) are classified as triple-negative (primarily basal-like) and have poor prognosis because there is no disease-specific therapy available. To address this unmet medical need, gene expression analyses using more than a thousand breast cancer samples were conducted, which identified elevated centromere protein E (CENP-E) expression in the basal-a molecular subtype relative to other subtypes. CENP-E, a mitotic kinesin component of the spindle assembly checkpoint, is shown to be induced in basal-a tumor cell lines by the mitotic spindle inhibitor drug docetaxel. CENP-E knockdown by inducible shRNA reduces basal-a breast cancer cell viability. A potent, selective CENP-E inhibitor (PF-2771) was used to define the contribution of CENP-E motor function to basal-like breast cancer. Mechanistic evaluation of PF-2771 in basal-a tumor cells links CENP-E-dependent molecular events (e.g., phosphorylation of histone H3 Ser-10; phospho-HH3-Ser10) to functional outcomes (e.g., chromosomal congression defects). Across a diverse panel of breast cell lines, CENP-E inhibition by PF-2771 selectively inhibits proliferation of basal breast cancer cell lines relative to premalignant ones and its response correlates with the degree of chromosomal instability. Pharmacokinetic-pharmacodynamic efficacy analysis in a basal-a xenograft tumor model shows that PF-2771 exposure is well correlated with increased phospho-HH3-Ser10 levels and tumor growth regression. Complete tumor regression is observed in a patient-derived, basal-a breast cancer xenograft tumor model treated with PF-2771. Tumor regression is also observed with PF-2771 in a taxane-resistant basal-a model. Taken together, CENP-E may be an effective therapeutic target for patients with triple-negative/basal-a breast cancer.

Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK).

Because of the critical roles of aberrant signaling in cancer, both c-MET and ALK receptor tyrosine kinases are attractive oncology targets for therapeutic intervention. The cocrystal structure of 3 (PHA-665752), bound to c-MET kinase domain, revealed a novel ATP site environment, which served as the target to guide parallel, multiattribute drug design. A novel 2-amino-5-aryl-3-benzyloxypyridine series was created to more effectively make the key interactions achieved with 3. In the novel series, the 2-aminopyridine core allowed a 3-benzyloxy group to reach into the same pocket as the 2,6-dichlorophenyl group of 3 via a more direct vector and thus with a better ligand efficiency (LE). Further optimization of the lead series generated the clinical candidate crizotinib (PF-02341066), which demonstrated potent in vitro and in vivo c-MET kinase and ALK inhibition, effective tumor growth inhibition, and good pharmaceutical properties.

Effective targeting of triple-negative breast cancer cells by PF-4942847, a novel oral inhibitor of Hsp 90.

PURPOSE: Triple-negative breast cancer (TNBC) patients have poor prognoses and survival outcomes such that the development of new targeted therapies is in strong demand. Mechanisms associated with high proliferation and aggressive tumor progression, such as PI3K/PTEN aberration, epidermal growth factor receptor (EGFR) overexpression, and cell-cycle upregulation, play important roles in TNBC. The molecular chaperone Hsp90 is required for the conformational maturation and stability of a variety of proteins in multiple pathways, such as EGFR, AKT, Raf, cdk4, etc. Therefore, an Hsp90 inhibitor may show therapeutic benefit in TNBC by targeting multiple pathways. EXPERIMENTAL DESIGN: The novel oral Hsp90 inhibitor PF-4942847 was characterized in multiple in vitro and in vivo assays to determine its antitumor activity in TNBC cell lines. In addition, the correlation of AKT degradation and Hsp70 induction in host peripheral blood lymphocytes (PBL) and xenograft tumors was determined. RESULTS: PF-4942847 induces degradation of multiple client proteins, cell-cycle block, apoptosis, and inhibits cell proliferation in TNBC lines, subsequently leading to tumor growth inhibition in mouse xenograft models. The correlation of AKT degradation and Hsp70 induction between PBLs and xenograft tumors reveals a differential modulation of Hsp90 activity between host and tumor tissues, and suggests that AKT degradation in PBLs may serve as a pharmacodynamic biomarker in future clinical development. CONCLUSIONS: The novel oral Hsp90 inhibitor, PF-4942847, is a candidate for clinical development in TNBC by collaboratively targeting multiple signaling pathways. In addition, AKT degradation in PBLs may serve as a biomarker in clinical development.

Pharmacokinetic-pharmacodynamic modeling of biomarker response and tumor growth inhibition to an orally available heat shock protein 90 inhibitor in a human tumor xenograft mouse model.

PF04942847 [2-amino-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-methylphenyl}-N-(2,2-difluoropropyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide] was identified as an orally available, ATP-competitive, small-molecule inhibitor of heat shock protein 90 (HSP90). The objectives of the present study were: 1) to characterize the pharmacokinetic-pharmacodynamic relationship of the plasma concentrations of PF04942847 to the inhibition of HSP90-dependent protein kinase, AKT, as a biomarker and 2) to characterize the relationship of AKT degradation to tumor growth inhibition as a pharmacological response (antitumor efficacy). Athymic mice implanted with MDA-MB-231 human breast cancer cells were treated with PF04942847 once daily at doses selected to encompass ED(50) values. Plasma concentrations of PF04942847 were adequately described by a two-compartment pharmacokinetic model. A time delay (hysteresis) was observed between the plasma concentrations of PF04942847 and AKT degradation; therefore, a link model was used to account for the hysteresis. The model reasonably fit the time courses of AKT degradation with the estimated EC(50) of 18 ng/ml. For tumor growth inhibition, the signal transduction model reasonably fit the inhibition of individual tumor growth curves with the estimated EC(50) of 7.3 ng/ml. Thus, the EC(50) for AKT degradation approximately corresponded to the EC(50) to EC(80) for tumor growth inhibition, suggesting that 50% AKT degradation was required for significant antitumor efficacy (50-80%). The consistent relationship between AKT degradation and antitumor efficacy was also demonstrated by applying an integrated signal transduction model for linking AKT degradation to tumor growth inhibition. The present results will be helpful in determining the appropriate dosing regimen and guiding dose escalation to achieve efficacious systemic exposure in the clinic.

Design strategies to target crystallographic waters applied to the Hsp90 molecular chaperone.

A series of novel and potent small molecule Hsp90 inhibitors was optimized using X-ray crystal structures. These compounds bind in a deep pocket of the Hsp90 enzyme that is partially comprised by residues Asn51 and Ser52. Displacement of several water molecules observed crystallographically in this pocket using rule-based strategies led to significant improvements in inhibitor potency. An optimized inhibitor (compound 17) exhibited potent Hsp90 inhibition in ITC, biochemical, and cell-based assays (K(d)=1.3 nM, K(i)=15 nM, and cellular IC(50)=0.5 µM).

Optimization of potent, selective, and orally bioavailable pyrrolodinopyrimidine-containing inhibitors of heat shock protein 90. Identification of development candidate 2-amino-4-{4-chloro-2-[2-(4-fluoro-1H-pyrazol-1-yl)ethoxy]-6-methylphenyl}-N-(2,2-difluoropropyl)-5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidine-6-carboxamide.

A novel class of heat shock protein 90 (Hsp90) inhibitors was discovered by high-throughput screening and was subsequently optimized using a combination of structure-based design, parallel synthesis, and the application of medicinal chemistry principles. Through this process, the biochemical and cell-based potency of the original HTS lead were substantially improved along with the corresponding metabolic stability properties. These efforts culminated with the identification of a development candidate (compound 42) which displayed desired PK/PD relationships, significant efficacy in a melanoma A2058 xenograft tumor model, and attractive DMPK profiles.

RETRACTED: A novel class of specific Hsp90 small molecule inhibitors demonstrate in vitro and in vivo anti-tumor activity in human melanoma cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors. Following an investigation by Pfizer, Figures 2, 5B and 5C appear to be duplications and hence the conclusions in the manuscript cannot be verified. The Authors apologize for this inconvenience.

2-(6-Bromo-3-pyrid-yl)-8-methyl-imidazo[1,2-a]pyrazine.

The structure of the title compound, C(12)H(9)BrN(4), prepared by the reaction of 2-bromo-1-(6-bromo-3-pyrid-yl)ethanone with 2-amino-3-methyl-pyrazine indicates that the compound with the bromo-pyridyl substituent at position 2 of the imidazopyrazine fused-ring system represents the major product of this reaction. The plane of the pyridine ring forms a dihedral angle of 16.2 (2)° with the essentially planar (r.m.s. deviation = 0.006 Å) imidazopyrazine system. In the crystal, mol-ecules are linked by weak C-H⋯N inter-actions.