Daniel Wettstein

Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.

Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.

Activation of Xenopus Genes Required for Lateral Inhibition and Neuronal Differentiation during Primary Neurogenesis

XNGN-1, a member of the neurogenin family of basic helix-loop-helix proteins, plays a critical role in promoting neuronal differentiation in Xenopus embryos. When ectopically expressed, XNGN-1 induces the expression of a set of genes required for neuronal differentiation such as XMyT1 and NeuroD. At the same time, however, XNGN-1 induces the expression of genes that antagonize neuronal differentiation by a process called lateral inhibition. Here, we present evidence that XNGN-1 activates the expression of genes required for differentiation and lateral inhibition by recruiting transcriptional coactivators p300/CBP (CREB-binding protein) or PCAF (p3OO/CBP-associated protein), both of which contain histone acetyltransferase (HAT) activity. Significantly, transcriptional activation of the genes in the lateral inhibitory pathway is less dependent on the HAT activity than is the activation of the genes that mediate differentiation. We propose that this difference enables the genes in the lateral inhibition pathway to be induced prior to the genes that promote differentiation, thus enabling lateral inhibition to establish a negative feedback loop and restrict the number of cells undergoing neuronal differentiation.

Characterization of the Cellular and Antitumor Effects of MPI-0479605, a Small-Molecule Inhibitor of the Mitotic Kinase Mps1

Mps1 is a dual specificity protein kinase that is essential for the bipolar attachment of chromosomes to the mitotic spindle and for maintaining the spindle assembly checkpoint until all chromosomes are properly attached. Mps1 is expressed at high levels during mitosis and is abundantly expressed in cancer cells. Disruption of Mps1 function induces aneuploidy and cell death. We report the identification of MPI-0479605, a potent and selective ATP competitive inhibitor of Mps1. Cells treated with MPI-0479605 undergo aberrant mitosis, resulting in aneuploidy and formation of micronuclei. In cells with wild-type p53, this promotes the induction of a postmitotic checkpoint characterized by the ATM- and RAD3-related-dependent activation of the p53–p21 pathway. In both wild-type and p53 mutant cells lines, there is a growth arrest and inhibition of DNA synthesis. Subsequently, cells undergo mitotic catastrophe and/or an apoptotic response. In xenograft models, MPI-0479605 inhibits tumor growth, suggesting that drugs targeting Mps1 may have utility as novel cancer therapeutics. Mol Cancer Ther; 10(12); 2267–75. ©2011 AACR.

Discovery of (2S)-1-[4-(2-{6-amino-8-[(6-bromo-1,3-benzodioxol-5-yl)sulfanyl]-9H-purin-9-yl}ethyl)piperidin-1-yl]-2-hydroxypropan-1-one (MPC-3100), a purine-based Hsp90 inhibitor.

Modulation of Hsp90 (heat shock protein 90) function has been recognized as an attractive approach for cancer treatment, since many cancer cells depend on Hsp90 to maintain cellular homeostasis. This has spurred the search for small-molecule Hsp90 inhibitors. Here we describe our lead optimization studies centered on the purine-based Hsp90 inhibitor 28a containing a piperidine moiety at the purine N9 position. In this study, key SAR was established for the piperidine N-substituent and for the congeners of the 1,3-benzodioxole at C8. These efforts led to the identification of orally bioavailable 28g that exhibits good in vitro profiles and a characteristic molecular biomarker signature of Hsp90 inhibition both in vitro and in vivo. Favorable pharmacokinetic properties along with significant antitumor effects in multiple human cancer xenograft models led to the selection of 28g (MPC-3100) as a clinical candidate.

Abstract 3237: Evaluation of the pharmacokinetics and efficacy of a novel pro-drug of the HSP90 inhibitor, MPC-3100, designed with improved solubility

MPC-3100 is a synthetic, orally bioavailable HSP90 inhibitor in clinical development. The low solubility of this compound requires a solubility enhancing agent to enable uniform oral bioavailability. A pro-drug of MPC-3100 with enhanced aqueous solubility was synthesized and evaluated for its physical-chemical and pharmacokinetic properties, and anti-tumor activity in mice. The alanine ester of MPC-3100 was synthesized by esterifying a hydroxyl group on the active compound, MPC-3100. Solubility and permeability were determined over the pH range of 5 – 7.5 using the PIon solubility analyzer and the double sink PAMPA method. To investigate esterase-mediated activation of the pro-drug, the pro-drug was incubated with biological matrices known to contain esterases (mouse and human plasma and liver microsomes). The pharmacokinetics of the active component of the pro-drug was determined at 360 mg/kg in female CD-1 mice after administration as an oral suspension in 2% carboxymethylcellulose (CMC). Plasma concentrations of the active compound, MPC-3100, were quantified by LC-ESI-MS/MS. Five million NCI-N87 human gastric carcinoma cells were implanted subcutaneously into athymic mice (nu/nu) to produce a mouse xenograft model. Efficacy was determined in this model using once daily oral dosing in 2% CMC for 21 days. The kinetic solubility of the alanine ester pro-drug of MPC-3100 at pH 6.5 was 536 µg/mL compared to 10.2 µg/mL for MPC-3100. The pro-drug had an apparent permeability of 2.7 × 10-6 cm/sec at pH 6.2; whereas, the apparent permeability of MPC-3100 was 420 × 10-6 cm/sec. The low permeability of the pro-drug suggests conversion to active would be required to occur in the lumen prior to MPC-3100 absorption. The pro-drug was converted to MPC-3100 (half-life The alanine ester pro-drug of MPC-3100 had a > 50-fold increase in aqueous solubility and was converted rapidly by mouse plasma and mouse and human liver microsomes into active MPC-3100 in vitro and in vivo. These improved properties resulted in pro-drug that was efficacious in a xenograft tumor model, when dosed without a solubility enhancing agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3237. doi:10.1158/1538-7445.AM2011-3237

Discovery of an l-alanine ester prodrug of the Hsp90 inhibitor, MPC-3100

Various types of Hsp90 inhibitors have been and continue to undergo clinical investigation. One development candidate is the purine-based, synthetic Hsp90 inhibitor 1 (MPC-3100), which successfully completed a phase I clinical study. However, further clinical development of 1 was hindered by poor solubility and consequent formulation issues and promoted development of a more water soluble prodrug. Towards this end, numerous pro-moieties were explored in vitro and in vivo. These studies resulted in identification of L-alanine ester mesylate, 2i (MPC-0767), which exhibited improved aqueous solubility, adequate chemical stability, and rapid bioconversion without the need for solubilizing excipients. Based on improved physical characteristics and favorable PK and PD profiles, 2i mesylate was selected for further development. A convergent, scalable, chromatography-free synthesis for 2i mesylate was developed to support further clinical evaluation.

Abstract 3233: Comparative in vitro and in vivo metabolism of MPC-3100, an oral HSP90 inhibitor, in rat, dog, monkey and human

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL MPC-3100, an 8, 9-disubstituted purine, is an orally bioavailable HSP90 inhibitor currently in Phase 1 clinical development. The objectives of these studies were to compare the metabolism of MPC-3100 in preclinical species to select the species most appropriate for toxicological testing and to identify the major phase I and II metabolites formed both in vitro and in vivo in rats, dogs, monkeys and humans. MPC-3100 was incubated with liver microsomes from rats, dogs, monkeys, and humans. In addition, urine, feces, and bile were collected from rats dosed with MPC-3100 intravenously (5 mg/kg) or orally (50 mg/kg), and urine was collected from dogs (2 mg/kg) and cynomolgus monkeys (2.5 mg/kg) dosed intravenously. Metabolites were identified by liquid chromatography electrospray-ionization mass spectrometry. Quantitative analysis was performed with an AB Sciex 4000 Q-trap and qualitative analysis was conducted on a high resolution Agilent Q-TOF 6520 mass spectrometer. Six authentic standards were synthesized and used to confirm structural identity. In human liver microsomes, four distinct peaks were observed following chromatographic analysis. Three of these were conclusively identified using synthetic standards, accurate mass, and chromatographic retention time. The most abundant metabolite in all species was the catechol. In human, monkey, and dog liver microsomes, the next most abundant metabolite was formed by oxidation of the 2-hydroxypropan-1-one moiety to propane-1, 2-dione. A third metabolite present in all incubations was the de-amidated product of MPC-3100. It was formed in microsomes in the absence of NADPH suggesting that its formation was due to pH-mediated hydrolysis. A fourth metabolite formed by the addition of oxygen (+16 Da) within the methylenedioxyphenyl ring was assigned based solely upon its product ion spectrum. Following intravenous administration of MPC-3100 to rats, fourteen metabolites were observed in the feces; whereas, only 6 metabolites were observed in urine. No glucuronides were found in either the urine or feces. Less than 1% of the dose was recovered in rat urine; whereas, up to 40% of the dose was recovered as MPC-3100 and metabolites in feces over a 24 hour period. As many as 25 different metabolites were observed in the bile based upon differences in their retention time and molecular weight. Most of the metabolites in the bile resulted from either glucuronidation or sulfation, some of which were conclusively identified with authentic standards. Rat, dog, and monkey liver microsomes all produced the four major metabolites formed in human liver microsomes. Three of these metabolites formed in human microsomes were conclusively identified by comparison to authentic synthetic standards. Although MPC-3100 and several metabolites were found in rat, dog and monkey urine, the primary route of elimination of MPC-3100 was through biliary excretion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3233. doi:10.1158/1538-7445.AM2011-3233

Abstract 2628: Antitumor activity of MPC-3100, a synthetic Hsp90 inhibitor, in combination with erlotinib and sorafenib

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: MPC-3100 is a fully synthetic, orally bioavailable, Hsp90 inhibitor in clinical development. It is active as a single agent in xenograft models with many cancer types including colon, gastric, ovary, prostate, breast, lung and myeloid leukemia. We evaluate here the activity of MPC-3100 in combination with erlotinib or sorafenib in xenograft models. Methods: Three to five million cells, depending on cell-type, were implanted subcutaneously into athymic mice (nu/nu) for tumor studies. Dosing was initiated when median tumor volume was >100 mm3. All compounds were dosed orally, once daily. MPC-3100 (100 mg/kg) and erlotinib (100 mg/kg) were dosed on Days 1-21 and sorafenib (60 mg/kg) was dosed on Days 1-9. Results: Anti-tumor activity for the combination of MPC-3100 and erlotinib was compared to that of the single agents in a lung cancer (A549) xenograft model sensitive to EGFR inhibition. Administration of MPC-3100 or erlotinib resulted in 52% and 64% tumor growth inhibition (TGI), respectively relative to vehicle by the end of dosing on Day 19. By contrast, the combination of MPC-3100 and erlotinib resulted in 30% tumor regression over the same period. Thus the combination of MPC-3100 and erlotinib was more effective at inhibiting tumor growth than either agent alone (p<0.05). The combined anti-tumor activity of MPC-3100 and sorafenib was compared to administration of the single agents in a xenograft model with the melanoma cell line A375 that harbors the activating B-raf mutation, V600E. The median tumor volume (MTV) of the cohorts dosed with vehicle, MPC-3100 or sorafenib as single agents was comparable and increased by 4.8-, 6.4- and 8.2-fold, respectively by the end of dosing on study Day 20, whereas MTV of the cohort dosed with a combination of MPC-3100 and sorafenib increased by only 1.6-fold. The combination of MPC-3100 and sorafenib resulted in 66% tumor growth inhibition relative to vehicle on Day 20 and was more effective at inhibiting tumor growth than single agent (p<0.05). Conclusions: The combination of MPC-3100 with erlotinib or sorafenib shows greater anti-tumor activity than either agent alone. Thus, in addition to its broad activity in xenograft models as a single agent, MPC-3100 has the potential to be combined with other targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2628. doi:10.1158/1538-7445.AM2011-2628

Abstract 2617: MPC-3100, a synthetic Hsp90 inhibitor, induces biomarker changes in vitro and in vivo

Introduction: MPC-3100 is a fully synthetic, orally bioavailable, Hsp90 inhibitor in clinical development. It is broadly active in xenograft models with anti-tumor activity ranging from tumor regression to tumor growth inhibition in many cancer types including colon, gastric, ovarian, prostate, breast, lung and myeloid leukemia. Here we evaluate the effect of MPC-3100 on stability of client proteins in cells and xenograft tumors. We also determine the effect on Hsp70 protein levels, a biomarker of Hsp90 inhibition, in peripheral blood mononuclear cells (PBMCs) from cancer patients receiving MPC-3100. Methods: Her2, Akt, Cdk4, c-Raf and Hsp70 client protein levels were monitored in cell culture with protein immunoblots. Formalin-fixed, paraffin-embedded sections of xenograft tumors from mice dosed orally with MPC-3100 were analyzed by immunohistochemistry (IHC) to monitor changes in Her2, Akt and Hsp70 protein levels. To monitor changes in Hsp70 in cancer patients treated with MPC-3100, PBMCs were collected prior to drug administration, 8 and 24 hours post-dose on Day 1 and 24 hours post-dose on Days 7 and 21 of the first treatment cycle. Hsp70 protein levels were determined by ELISA. Results: Exposure of HCT-116, NCI-N87 and DU-145 cells to MPC-3100 in vitro resulted in a time-dependent reduction in client protein levels with maximal reduction by 24 hours. The IC 50 values for client protein reduction ranged from 0.1 μM to 0.5 μM, comparable to the cellular cytotoxicity values of MPC-3100 at 72 hours for the various cell lines. IHC revealed reduction in Her2 and Akt protein in N87 xenografts in mice given a single oral dose of 200 mg/kg MPC-3100 relative to tumors from animals dosed with vehicle. Healthy volunteer PBMCs exposed to 1 μM MPC-3100 for 24 hours ex vivo revealed a reduction in Akt, c-Raf and Cdk4 protein levels ranging from 50% to 90%. PBMCs from cancer patients receiving MPC-3100 showed an increase of 28 to 589 ng of Hsp70 protein per mg total protein over baseline by Day 8. The increase in Hsp70 expression was seen as early as 8 hours after the first dose and sustained through at least Day 22. Conclusions: The changes in client proteins and biomarkers observed in cells and tumor xenografts exposed to MPC-3100 confirm that the cellular cytotoxic activity and anti-tumor activity in xenografts are a result of Hsp90 inhibition. The consistent increase in Hsp70 expression in PBMCs from cancer patients receiving MPC-3100 indicates that Hsp90 function is inhibited in patients at doses that have been well tolerated in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2617. doi:10.1158/1538-7445.AM2011-2617