Pei-Pei Kung

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.

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.

Dihydroxyphenylisoindoline amides as orally bioavailable inhibitors of the heat shock protein 90 (hsp90) molecular chaperone.

The discovery and optimization of potency and metabolic stability of a novel class of dihyroxyphenylisoindoline amides as Hsp90 inhibitors are presented. Optimization of a screening hit using structure-based design and modification of log D and chemical structural features led to the identification of a class of orally bioavailable non-quinone-containing Hsp90 inhibitors. This class is exemplified by 14 and 15, which possess improved cell potency and pharmacokinetic profiles compared with the original screening hit.

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.

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. Clin Cancer Res; 17(16); 5432–42. ©2011 AACR.

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. Mol Cancer Ther; 13(8); 2104–15. ©2014 AACR.

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.

Solution-phase parallel synthesis of Hsp90 inhibitors.

As part of an oncology chemistry program directed toward discovery of orally bioavailable inhibitors of the 90 kDa heat shock protein (Hsp90), several solution-phase libraries were designed and prepared. A 2 x 89 library of racemic resorcinol amides was prepared affording 131 purified compounds. After evaluation in a binding assay, followed by an AKT-Luminex cellular assay, three potent analogs had functional activity between 0.1 and 0.3 microM. Resolution by preparative chiral SFC chromatography led to (+)-15, (+)-16, and (+)-17 having functional IC(50) = 27, 43, and 190 nM, respectively. (+)-15 exhibited high clearance in human hepatocytes driven primarily by glucuronidation as confirmed by metabolite identification. A second 8 x 14 exploratory library was designed to investigate heterocyclic replacements of the resorcinol ring. The second library highlights the use of the (-)-sparteine-mediated enantioselective Pd-catalyzed alpha-arylation of N-Boc-pyrrolidine to prepare chiral 2-arylpyrrolidines in parallel.

Methyl 3-[3-(ethoxy­carbon­yl)thio­ureido]-1H-pyrazole-5-carboxyl­ate

The title compound, C9H12N4O4S, was proven to be the product of the reaction of methyl 5-amino-1H-pyrazole-3-carboxylate with ethyl isothiocyanatocarbonate. All non-H atoms of the molecule are planar, the mean deviation from the least squares plane being 0.048 Å. The intramolecular N—H⋯O bond involving the NH-group, which links the thiourea and pyrazole fragments, closes a six-membered pseudo-heterocyclic ring, and two more hydrogen bonds (N—H⋯O with the participation of the pyrazole NH group and N—H⋯S involving the second thiourea NH group) link the molecules into infinite chains running along [10].

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

The structure of the title compound, C12H9BrN4, prepared by the reaction of 2-bromo-1-(6-bromo-3-pyridyl)ethanone with 2-amino-3-methylpyrazine indicates that the compound with the bromopyridyl 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, molecules are linked by weak C—H⋯N interactions.