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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.

PDF - 98K, Effect of PF-2771 on breast cell line proliferation and CIN index analysis.

PDF - 68K, Broad kinase selectivity analysis of PF-2771.

PDF - 76K, Cell cycle analysis of PF-2771 treatment of MDA-MB-468 tumor cells.

PDF - 165K, Comparison of the effect of PF-2771 and a Eg5/KSP inhibitor on the microtubule network.

PDF - 238K, Supplementary Materials and Methods

PDF - 219K, PK/PD/Efficacy model analysis.

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.

PDf - 114K, Analysis of mouse body weight in the tumor growth models.

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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.

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Supplementary Tables 1-2, Figures 1-3 from An Orally Available Small-Molecule Inhibitor of c-Met, PF-2341066, Exhibits Cytoreductive Antitumor Efficacy through Antiproliferative and Antiangiogenic Mechanisms

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been implicated in the progression of several human cancers and are attractive therapeutic targets. PF-2341066 was identified as a potent, orally bioavailable, ATP-competitive small-molecule inhibitor of the catalytic activity of c-Met kinase. PF-2341066 was selective for c-Met (and anaplastic lymphoma kinase) compared with a panel of >120 diverse tyrosine and serine-threonine kinases. PF-2341066 potently inhibited c-Met phosphorylation and c-Met–dependent proliferation, migration, or invasion of human tumor cells in vitro (IC50 values, 5–20 nmol/L). In addition, PF-2341066 potently inhibited HGF-stimulated endothelial cell survival or invasion and serum-stimulated tubulogenesis in vitro, suggesting that this agent also exhibits antiangiogenic properties. PF-2341066 showed efficacy at well-tolerated doses, including marked cytoreductive antitumor activity, in several tumor models that expressed activated c-Met. The antitumor efficacy of PF-2341066 was dose dependent and showed a strong correlation to inhibition of c-Met phosphorylation in vivo. Near-maximal inhibition of c-Met activity for the full dosing interval was necessary to maximize the efficacy of PF-2341066. Additional mechanism-of-action studies showed dose-dependent inhibition of c-Met–dependent signal transduction, tumor cell proliferation (Ki67), induction of apoptosis (caspase-3), and reduction of microvessel density (CD31). These results indicated that the antitumor activity of PF-2341066 may be mediated by direct effects on tumor cell growth or survival as well as antiangiogenic mechanisms. Collectively, these results show the therapeutic potential of targeting c-Met with selective small-molecule inhibitors for the treatment of human cancers. [Cancer Res 2007;67(9):4408–17]

[Image: see text] 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.

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.

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-pr...

KAT6A is a lysine histone acetyltransferase (HAT) of the MYST family of HATs. KAT6A, and its paralog KAT6B, have been shown to acetylate histone H3K23Ac and regulate diverse biological processes, including transcription, cell-cycle progression, stem cell maintenance and development. Molecular dysregulation of KAT6A has been observed in several cancers, including amplifications in breast, lung, ovarian cancer along with oncogenic fusions in AML. In breast cancer, KAT6A is amplified as part of the 8p11 amplicon in 10-15% of the patient population, which correlates with a worse clinical outcome in the estrogen receptor+ (ER+) subtype. Here we present identification of a first-in-class potent KAT6A/KAT6B tool inhibitor CTx-648 (PF-9363), that possesses high selectivity versus other MYST family members (KAT7, KAT5, KAT8) and other KATs, demonstrating anti-tumor activity in breast cancer. Using genetic and pharmacological approaches, we have demonstrated several ER+ breast cancer cell lines including KAT6A amplified and over-expressing models, are dependent on KAT6A enzymatic function. Epigenomic profiling studies using bulk and nascent RNA-seq combined with ATAC-seq revealed CTx-648 leads to downregulation of a specific set of genes involved in ESR1 pathway, cell cycle and stem cell pathways. In vivo target validation studies showed strong anti-tumor activity of CTx-648 in several ER+ breast cancer cell line and patient-derived xenograft models, including models harboring endocrine therapy resistance ESR1 mutations, highlighting promise for this novel therapy in ER+ breast cancer population. Based on the strength of the pre-clinical data, a selective KAT6 inhibitor (PF-07248144) is now commencing a Phase 1 clinical study in Advanced or Metastatic Solid Tumors. Citation Format: Shikhar Sharma, Jay Chung, Sean Uryu, Amanda Rickard, Natalie Nady, Showkhin Khan, Zhenxiong Wang, Yong Zhang, Haikuo Zhang, Pei-Pei Kung, Eric Greenwald, Karen Maegley, Patrick Bingham, Hieu Lam, Ylva E. Bozikis, Hendrik Falk, Elizabeth Allan, Vicky M. Avery, Miriam S. Butler, Michelle A. Camerino, Catalina Carrasco-Pozo, Susan A. Charman, Melissa J. Davis, Mark A. Dawson, Dawson Sarah-Jane, Melanie de Silva, Matthew L. Dennis, Olan Dolezal, Rachel Lagiakos, Geoffrey J. Lindeman, Laura MacPherson, Stewart Nuttall, Thomas S. Peat, Bin Ren, Alexandra E. Stupple, Elliot Surgenor, Chin Wee Tan, Tim Thomas, Jane E. Visvader, Anne K. Voss, Francois Vaillant, Karen L. White, James Whittle, Yuqing Yang, Soroor Hediyeh-Zadeh, Paul A. Stupple, Ian P. Street, Brendon J. Monahan, Thomas Paul. First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1130.

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., Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Here, the authors present crystal structures of the inhibitor-bound wild-type and a mutant form of PRC2.

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.

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 sp

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.

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.

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.

Hsp90 is required for conformational maturation and stability of numerous key signaling proteins (clients) involved in cell proliferation and transformation. Here we describe two novel Hsp90 inhibitors, PF-4470296 and PF-3823863, demonstrate a differential sensitivity in B-Raf mutant (V600E) versus wild-type protein degradation. These two inhibitors inhibit proliferation and anchorage-independent growth, and abolish in vivo xenograft tumor growth in melanoma cells regardless of B-Raf mutation status. Mutant B-Raf protein and other Hsp90 clients, such as cMet, ErbB2, C-Raf, and AKT, are degraded in cells and xeno- graft tumors. Our results indicate that Hsp90 inhibitors induce anti-tumor activity in mel- anoma cells and are likely to show therapeutic benefit in melanoma patients by collaboratively targeting multiple pathways.

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.

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.

A robust, regioselective synthesis of 3-amino-1,2,4-triazoles is described. This reaction employs a key intermediate 2, which is coupled to carboxylic acids in good yields to afford intermediates 3a–d. These entities, in turn, react with a variety of hydrazines or hydrazine hydrochlorides to provide proposed intermediates 4a–j, which under microwave conditions cyclize to the desired 3-amino-1,2,4-triazoles (compounds 5a–j). This approach permits the rapid synthesis of regioselective N1-substituted 3-amino-1,2,4-triazoles, and is shown to afford a variety of such compounds in 34–70% isolated yields.

A series of quinoline-containing c-Met inhibitors were prepared and studied. Chemistry was developed to introduce a pyridyl moiety onto the 2-aryl ring present in a lead molecule which mitigated the potential for quinone formation relative to the original compound. The study also assessed the importance of an acylthiourea moiety present in the lead structure for effective binding to the c-Met protein ATP site.

Synthesis of a RACEMIC 4′-ethynyl-2′-deoxyribose intermediate using stereoselective chelation control is presented. This intermediate is further transformed to 4′-ethynyl-2′-deoxy- and 4′-ethynyl-2′,3′-dideoxy-2′,3′-didehydronucleoside analogues.

Epigenetic alterations are an important hallmark of cancer, and the enzymes that modify histone tails have emerged as attractive drug targets. The histone methyltransferase EZH2 is the catalytic subunit of PRC2, a highly conserved protein complex that regulates gene expression by methylating lysine 27 on histone H3. EZH2 is frequently overexpressed in cancer, and oncogenic gain-of-function mutations have been identified in both hematological malignancies and solid tumors. In cancer cells, the aberrant activity of the enzyme contributes to tumorigenesis by altering cell fate decisions and regulating pathways involved in proliferation, differentiation, and cell migration. Early validation efforts relied on the use of RNAi technology and non-specific small molecule inhibitors to down-regulate EZH2 and destabilize the PRC2 complex. The discovery of catalytic inhibitors of EZH2 has provided an invaluable tool for further elucidating the role of this enzyme in cancer, and preclinical studies in EZH2-mutant non-Hodgkin lymphoma have driven the clinical development of these agents. This review focuses on the use of catalytic small molecule inhibitors to identify solid tumor indications that are dependent on aberrant EZH2 methyltransferase activity. The emerging data suggests that EZH2 inhibitors will have therapeutic potential that extends beyond hematological malignancies to the solid tumor setting.

The structure-based design, chemical synthesis, and biological evaluation of novel MTAP substrates are described. These compounds incorporate various C5′-moieties and are shown to have different k cat / K m values compared with the natural MTAP substrate (MTA).

An efficient and flexible synthesis of cyclopentane and hydroxylated cyclopentane phosphonic acid analogues is described. The key step involves the opening of an epoxide with either a nucleoside base or a selenyl anion toaccess the target molecules.

A series of novel benzimidazoles were efficiently synthesized using both solution- and solid-phase chemistry. These compounds were found to bind to the bacterial 16S ribosomal RNA A-site with micromolar affinities using unique mass spectrometry-based assays.

A novel route for the synthesis of tetrahydropyrazine-2-ones on solid support through intramolecular Mitsunobu reactions is reported. The syntheses employ reductive amination of commercially available amino alcohols to attach them to ArgoGel-MB-CHO resin. Amino acids are then coupled to the derivatized solid support for construction of the ring systems and a Mitsunobu reaction between the activated amino acids and amino alcohols forms the derivatized tetrahydropyrazine-2-one rings.

Capillary electrophoresis has been applied to monitor model reactions in solution-phase combinatorial chemistry. In particular, the simultaneous alkylation reactions of secondary amines with a series of benzyl halides has been investigated. Reactant and product concentrations were monitored using capillary electrophoresis in a non-aqueous buffer system. The simplified sample preparation was a key feature making this an attractive method of analysis. The results demonstrate that capillary electrophoresis is a useful tool for monitoring reactions to determine initial rates, rate constants, and extinction correlation coefficients for quantitative analysis in combinatorial chemistry, and is a broadly applicable technique for the analysis of a variety of organic and bioorganic transformations. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:169–177, 1998/1999.

Use of solution phase combinatorial library synthesis led to the discovery of several oxyamine-containing antibacterial compounds. Solution-phase simultaneous addition of meta-substituted benzyl bromides and “fix-last” combinatorial strategies were used to prepare libraries. Additional structure−activity relationship studies were conducted by reductive cleavage of the oxyamine moiety and led to loss of antibacterial activity. Several single compounds were designed and synthesized on the basis of library screening results and were shown to have antibacterial activity.

Several piperazine libraries were prepared using solution phase simultaneous addition of functionalities methodology as well as the "library from library" concept. The resulting piperazine libraries displayed antimicrobial activity against both gram-positive and gram-negative bacteria.