Michael R. Gehring

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.

Dihydroxylphenyl amides as inhibitors of the Hsp90 molecular chaperone.

Information from X-ray crystal structures were used to optimize the potency of a HTS hit in a Hsp90 competitive binding assay. A class of novel and potent small molecule Hsp90 inhibitors were thereby identified. Enantio-pure compounds 31 and 33 were potent in PGA-based competitive binding assay and inhibited proliferation of various human cancer cell lines in vitro, with IC(50) values averaging 20 nM.

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This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors.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.

Steady-state kinetic and inhibition studies of the mammalian target of rapamycin (mTOR) kinase domain and mTOR complexes.

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase and a major controller of cell growth. In cells, mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2. The mTORC1 complex can phosphorylate 4EBP1 and S6K1, two key regulators of translation initiation, whereas mTORC2 phosphorylates AKT1, an event required for AKT1 activation. Here, we expressed and purified human mTORC1 and mTORC2 from HEK-293 cells using FLAG-M2 affinity chromatography. Western blotting analysis using phospho-specific antibodies indicated that recombinant mTORC1 and mTORC2 exhibit distinct substrate preferences in vitro, consistent with their roles in cells. To improve our understanding of the enzymatic properties of mTOR alone and mTOR in its complex form, steady-state kinetic profiles of truncated mTOR containing the kinase domain (residues 1360-2549) and mTORC1 were determined. The results revealed that mTORC1 is catalytically less active than truncated mTOR, as evidenced by 4.7- and 3.1-fold decreases in catalytic efficiency, k(cat)/K(m), for ATP and 4EBP1, respectively. We also found that truncated mTOR undergoes autophosphorylation through an intramolecular mechanism. Mass spectrometric analysis identified two novel mTOR autophosphorylation sites, Ser2454 and either Thr2473 or Thr2474, in addition to the previously reported Ser2481 site. Truncated mTOR and mTORC1 were completely inhibited by ATP competitive inhibitors PI103 and BEZ235 and partially inhibited by rapamycin/FKBP12 in a noncompetitive fashion toward ATP. All inhibitors tested exhibited similar inhibitory potencies between mTORC1 and truncated mTOR containing the kinase domain. Our studies presented here provide the first detailed kinetic studies of a recombinant mTOR complex.

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.

Abstract 5779: The discovery of the potent and selective PI3K/mTOR dual inhibitor PF-04691502 through structure-based drug design

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays crucial roles in cell growth, proliferation and survival. Genomic aberrations in the PI3K pathway, such as mutational activation of PI3Kα or loss of function of tumor suppressor PTEN, have been closely linked to the development and progression of a wide range of cancers. Hence, inhibition of the key targets in the pathway, e.g. PI3K, AKT, mTOR, offers great potential for the treatment of cancer. In an effort to discover compounds that inhibit PI3Kα, a high throughput screen was carried out, and 4-methyl-pyrido-pyrimidine (MPP) derivatives were identified as potent and selective inhibitors of PI3Kα. For example, PF-00271897, 8-cyclopentyl-6-[3-(hydroxymethyl)phenyl]-4-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-7(8H)-one demonstrated PI3Kα Ki of 2.2 nM. Multiple crystal structures of inhibitors bound to PI3K gamma were determined to inform design and optimization of the ADMET properties of this lead series. Crystallographic studies with PI3K gamma protein indicated that the aminopyrimidine moiety forms two hydrogen bonds to the kinase backbone, and the aromatic moiety at the 6 position binds in a hydrophobic pocket. The X-ray structure suggested that the 4-methyl group on the MPP core structure conferred the excellent overall kinase selectivity to the series. The structure and SAR suggested optimization could come from keeping N-R group at 2 position very small and maintaining aromatic moiety at 6 position for hydrophobic interaction. Introduction of polar groups to the 8N side chains that are located in the ribose binding pocket increased both metabolic stability and solubility. Based on the overall properties, PF-04691502, 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one, was selected as a clinical candidate. PF-04691502 demonstrated Kis of 1.2-2.2 nM against PI3K α, β, γ and δ isoforms, and Ki of 9.1 nM against recombinant mTOR. PF-04691502 inhibited AKT phosphorylation at S473 in BT20 breast cancer line with IC50 of 12 nM. PF-04691502 is highly selective for inhibition of PI3K family kinases as shown by lack of activity against a panel of >75 protein kinases, including the class III PI3K hVps34. In the in vivo rat PK studies, PF-04691502 demonstrated the following properties: Clearance = 5.2 ml/min/kg, Vdss = 1.4 L/kg, T1/2 = 3.1 h, F% = 63%. The design, synthesis, in vitro potency SAR, selectivity, ADMET of the MPP derivatives will be discussed. The crystal structure of PF-04691502 in PI3K gamma will also be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5779.

Abstract 5043: Effects of a novel PI3 kinase/mTOR inhibitor on proliferation and pAKT signaling in canine lymphoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC PF-04691503 is a dual inhibitor of both PI3K and mTOR, inhibits PI3K signaling in cancer cell lines, and exhibits in vitro and in vivo anti-proliferative activity in PI3K-pathway driven cell lines. It is a nanomolar inhibitor of all 4 isoforms of the catalytic subunit of PI3K and of both TORC1 and TORC2. Anti-cancer activity of the inhibitor is hypothesized to be through inhibition of survival, proliferative, and anti-apoptotic processes. Phosphorylation of the protein kinase, Akt, is associated with activation of the phosphatidyl 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) signaling pathway, which plays a role in cell proliferation (Witzig and Kaufmann 2006). Recent studies by Gulmann et al (2005) showed increased pAKT in human lymphoma samples, and those of Rassidakis et al (2005) suggested that inhibition of Akt phosphorylation (pAKT) may be of value in the treatment of lymphoma. Using flow cytometry we report that cells obtained from 10 of 11 lymph node biopsies of dogs with lymphoma exhibit detectable pAKT using a phospho-Akt (Ser473) antibody, compared to an IgG isotype control or a competative phospho-Akt (Ser473) blocking peptide. Cells from normal canine lymph nodes do not exhibit detectable pAKT. The majority of the pAKT signal was generated from lymphoblasts present in the malignant, but not in the normal, lymph nodes. In separate studies, lymph node cells obtained from healthy dogs and dogs with lymphoma were stimulated in vitro with the mitogen, Con A. The novel PI3K/mTOR dual inhibitor, PF-04691503, produced dose dependent inhibition of proliferation as exemplified in a dog with T-cell lymphoma (EC50 = 18 nM)) and in a normal dog (EC50 = 53 nM)). No pAKT signal could be detected in peripheral blood mononuclear cells, from normal or lymphoma patients stimulated with hu-IGF-1 (the endogenous ligand for PI3), Con-A or LPS. These data suggest that PI-3 kinase and pAKT (1) are activated in canine lymphoma, (2) play a role in the lymphoproliferation associated with this disease, (3) represent legitimate targets for therapeutic intervention in lymphoma, and (4) can be studied ‘translationally’ in dogs as a model for humans. Gulmann C, Espina V, Petricoin E, et al. Proteomic analysis of apoptotic pathways reveals prognostic factors in follicular lymphoma. Clin Cancer Res 11:5847-5855, 2005. Rassidakis GZ, Feretzaki M, Atwell C, et al. Inhibition of Akt increases p27 Kip1 levels and induces cell cycle arrest in anaplastic large cell lymphoma. Blood 105:827-829, 2005. Witzig TE and Kaufmann SH. Inhibition of phosphatidyl 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway in hematologic malignancies. Current Treatment Options in Oncology 7:285-294, 2006. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5043.