David Duhl

Mutation of a nuclear receptor gene, NR2E3 , causes enhanced S cone syndrome, a disorder of retinal cell fate

Hereditary human retinal degenerative diseases usually affect the mature photoreceptor topography by reducing the number of cells through apoptosis, resulting in loss of visual function. Only one inherited retinal disease, the enhanced S-cone syndrome (ESCS), manifests a gain in function of photoreceptors. ESCS is an autosomal recessive retinopathy in which patients have an increased sensitivity to blue light; perception of blue light is mediated by what is normally the least populous cone photoreceptor subtype, the S (short wavelength, blue) cones. People with ESCS also suffer visual loss, with night blindness occurring from early in life, varying degrees of L (long, red)- and M (middle, green)-cone vision, and retinal degeneration. The altered ratio of S- to L/M-cone photoreceptor sensitivity in ESCS may be due to abnormal cone cell fate determination during retinal development. In 94% of a cohort of ESCS probands we found mutations in NR2E3 (also known as PNR), which encodes a retinal nuclear receptor recently discovered to be a ligand-dependent transcription factor. Expression of NR2E3 was limited to the outer nuclear layer of the human retina. Our results suggest that NR2E3 has a role in determining photoreceptor phenotype during human retinogenesis.

Identification of the gene that, when mutated, causes the human obesity syndrome BBS4.

Bardet–Biedl syndrome (BBS, MIM 209900) is a heterogeneous autosomal recessive disorder characterized by obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation, and hypogenitalism. The disorder is also associated with diabetes mellitus, hypertension, and congenital heart disease. Six distinct BBS loci map to 11q13 (BBS1), 16q21 (BBS2), 3p13–p12 (BBS3), 15q22.3–q23 (BBS4), 2q31 (BBS5), and 20p12 (BBS6). Although BBS is rare in the general population (<1/100,000), there is considerable interest in identifying the genes causing BBS because components of the phenotype, such as obesity and diabetes, are common. We and others have demonstrated that BBS6 is caused by mutations in the gene MKKS (refs. 12,13), mutation of which also causes McKusick–Kaufman syndrome (hydrometrocolpos, post-axial polydactyly, and congenital heart defects). MKKS has sequence homology to the alpha subunit of a prokaryotic chaperonin in the thermosome Thermoplasma acidophilum. We recently identified a novel gene that causes BBS2. The BBS2 protein has no significant similarity to other chaperonins or known proteins. Here we report the positional cloning and identification of mutations in BBS patients in a novel gene designated BBS4.

The discovery of tetrahydro-β-carbolines as inhibitors of the kinesin Eg5

A series of tetrahydro-beta-carbolines were identified by HTS as inhibitors of the kinesin Eg5. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus of removing potential metabolic liabilities and improving cellular potency.

The Novel Oral Hsp90 Inhibitor NVP-HSP990 Exhibits Potent and Broad-spectrum Antitumor Activities In Vitro and In Vivo

A novel oral Hsp90 inhibitor, NVP-HSP990, has been developed and characterized in vitro and in vivo. In vitro, NVP-HSP990 exhibits single digit nanomolar IC50 values on three of the Hsp90 isoforms (Hsp90α, Hsp90β, and GRP94) and 320 nanomolar IC50 value on the fourth (TRAP-1), with selectivity against unrelated enzymes, receptors, and kinases. In c-Met amplified GTL-16 gastric tumor cells, NVP-HSP990 dissociated the Hsp90-p23 complex, depleted client protein c-Met, and induced Hsp70. NVP-HSP990 potently inhibited the growth of human cell lines and primary patient samples from a variety of tumor types. In vivo, NVP-HSP990 exhibits drug-like pharmaceutical and pharmacologic properties with high oral bioavailability. In the GTL-16 xenograft model, a single oral administration of 15 mg/kg of NVP-HSP990 induced sustained downregulation of c-Met and upregulation of Hsp70. In repeat dosing studies, NVP-HSP990 treatment resulted in tumor growth inhibition of GTL-16 and other human tumor xenograft models driven by well-defined oncogenic Hsp90 client proteins. On the basis of its pharmacologic profile and broad-spectrum antitumor activities, clinical trials have been initiated to evaluate NVP-HSP990 in advanced solid tumors. Mol Cancer Ther; 11(3); 730–9. ©2012 AACR.

Chapter 20. Beyond kinases: Purine-binding enzymes as cancer targets

Publisher Summary This chapter discusses the role of purine-binding enzymes as cancer targets. It also provides an overview of kinesin spindle protein (KSP), a member of kinesin superfamily of MT motors, which convert the energy released from ATP hydrolysis into mechanical force for transport along microtubules in the cell. The ideal target for small molecule drug discovery is one that is essential for the disease state, yet nonessential for normal tissues. One recently emerged class of drugable targets is the protein kinases, exemplified by bcr-abl, which is the target of the highly successful new CML drug, Gleevec. The common drugable feature of the kinase class is the ability to bind ATP. The ATP purine moiety is bound in a hydrophobic environment of the active site and stabilized by key hydrogen bonds. Such a site favors binding of flat, aromatic heterocycles, a class of compound that has proven amenable to optimization and drug development, thus, making kinases eminently drugable. However, kinases are not the only class of enzyme that binds purines through key hydrogen bonds and hydrophobic stacking interactions; other such purine-binding enzymes include ATPases, GTPases, sulfotransferases, etc. Thus, these enzymes offer additional possibilities for drug discovery. To illustrate the potential of this emerging class of drug targets, this chapter focuses on the oncology and specifically on ATPases, GTPases and sulfotransferases.

Chapter 24. Obesity therapeutics: Prospects and perspectives

Publisher Summary This chapter presents an overview of obesity therapeutics. When measured by body mass index (BMI), a BMI of 30 is the threshold for obesity. Obesity is mischaracterized as a cosmetic or life style issue, when in reality it is a devastating disease with tremendous health and financial consequences. In the United States alone, it has been estimated that more than 300,000 deaths occur per year from obesity. This distressing effect on life expectancy is to a large extent related directly to the life threatening co-morbidities of obesity such as noninsulin-dependent diabetes, hypertension, coronary artery disease, and some forms of cancer. The less lethal comorbidities associated with obesity include gallstones, osteoarthritis, degenerative arthritis, and apnea. The chapter discusses the regulation of obesity through multiple integrated pathways and highlights historical approaches to obesity therapeutics. It also highlights current approaches to obesity therapeutics and discusses neuropeptide Y receptors, melanin-concentrating hormone receptor, and the role of protein tyrosine phosphatase 1B (PTP1B). The use of PTP1B inhibitors for the treatment of obesity and diabetes is an active area for anti-obesity research. The untapped potential of antiobesity drugs is also explored in the chapter.

Abstract A140: Evaluation of prediction of in vivo activity from in vitro combinations: Examples using a MEK1/2 inhibitor combined with docetaxel in NSCLC models.

In an attempt to combat the resistance of tumors to chemotherapy, we have already described at this year9s AACR, the systematic evaluation of over 11,000 different compound combinations using the human cancer cell line encyclopedia. Correlations of synergy with genetic features were identified and some novel synergies discovered. Here, we describe the efficacy, tolerability and PK-PD of 23 different in vivo combinations selected from the screens to determine the predictability of in vitro screening, including two examples from the combination of a MEK1/2-inhibitor (MEK162) with the taxane, docetaxel. In vitro screens were conducted as previously described using 3-day viability assays, and inhibition of proliferation determined relative to untreated samples, and the degree of synergy scored using different types of analyses: Gaddum, Bliss, Loewe. Only those combinations showing synergy over a wide range of concentrations were chosen for in vivo study. For in vivo studies, cells were injected s.c. in the flank of athymic nude mice, and once tumors reached a mean size of at least 100 mm 3 were treated for 2-4 weeks with the appropriate dose and schedule of the compounds either as monotherapy, or in combination. Efficacy and tolerability were determined at the endpoint using the T/C TVol and T/C BW respectively to derive a combination-index as previously described by Clarke (1997), where a negative-value (-CCI) indicated synergy. In most cases, PK-PD was also measured in plasma and tumour either at steady-state and/or the endpoint to study the mechanism of the interaction and to check for drug-drug interactions and their eventual impact on PD and efficacy. Thus far, we have studied in vivo 13 different molecular targets across 6 different histotypes to give 23 different combinations. No antagonism was seen in vivo (+CCI), and 19/23 were deemed synergistic (CCI ≤-0.1), of which 8 showed regression which was not seen with the individual monotherapies. Of the 4 combinations showing no interaction, 2 were predicted by the in vitro score and the other 2 showed negative drug-drug interactions. There were no significant correlations between the CCI and the different types of in vitro score (p>0.35), but perhaps more importantly, cut-offs could be identified suggesting synergy could be predicted (p≤0.02) although not the extent of the interaction. Several novel combinations were identified for clinical investigation, including MEK162 combined with docetaxel in KRAS-mutant NSCLC, which in two different models in vivo had a CCI≤ -0.1, with PD-analyses showing that cytotoxic doses of the taxane activated the MAPK-pathway which was blocked by the combination. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A140. Citation Format: Paul Martin J. Mcsheehy, Alex Cao, Giordi Caponigro, David Duhl, Brant Firestone, Tom Gesner, Daniel Guthy, Jocelyn Holash, Fred King, Joseph Lehar, Christopher Leroy, Manway Liu, Lilli Petruzzelli, Dale Porter, Daniel Menezes, Anupama Reddy, Johannes Roesel, Christian Schnell, Timothy Smith, Mark Stump, Markus Wartmann, Marion Wiesmann. Evaluation of prediction of in vivo activity from in vitro combinations: Examples using a MEK1/2 inhibitor combined with docetaxel in NSCLC models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A140.

Abstract 2058: Systematic evaluation of drug combinations in vitro and in vivo.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In recent years great advances have been made in developing targeted cancer therapeutics that produce dramatic responses in a subset of rationally selected patients. The initial breakthrough of the targeted design concept was established by the treatment of chronic myelogenous leukemia with Abl inhibitors and has been expanded to other cancer indications. As a consequence of this early success in CML, the identification and targeting of genetic lesions that confer cancer dependence has become an established strategy for drug discovery efforts. However, this approach has met with mixed degrees of success as confounding factors, such as tumor heterogeneity, have often resulted in partial responses and/or the emergence of resistance when targeted therapies were employed as single agents. To improve the therapeutic benefit in cancer, rationally-devised novel combinations of two or more agents are being explored clinically. To discover combinations that may be more effective therapies, an unbiased, systematic approach was used to identify drug combinations in vitro, using a panel of genetically diverse, and well characterized cell lines from the cancer cell line encyclopedia (CCLE: Barretina et al. Nature 2012). For three cancer indications, all pairwise combinations of 18 selected drugs (both novel inhibitors and standards of care) were tested as dose matrices in a proliferation assay. Synergistic interactions were scored using isobologram/Loewes excess inhibition and synergistic concentration ranges for each agent were identified. However, the clinical translation of positive combinations from in vitro matrix-based screens into clinically-relevant doses and schedules are challenging, due to host biology, tumor-stroma interactions, and the pharmacokinetic and pharmacodynamics of drug delivery. To explore this higher complexity, we evaluated the in vitro to in vivo translation of drug synergies in immune-compromised mouse tumor xenograft models. To recapitulate the pharmacological combination effects in vivo, mouse pharmacokinetic data and simulation was used to determine single agent doses that would result in the desired compound plasma concentration range and duration of action. Pharmakokinetics, pharmacodynamics, antitumor activity and tolerability of the combinations were then tested in tumor-bearing mice. Observed combination effects in vivo could in some cases be explained by either the expected biological pathway interactions or partially by physiological effects relating to drug-drug interactions. Citation Format: Marion Wiesmann, Mark Stump, Giordano Caponigro, David Duhl, Brant Firestone, Tom Gesner, Bjoern Gruenenfelder, Daniel Alexander Guthy, Jocelyn Holash, Fred King, Joseph Lehar, Christophe Leroy, Manway Liu, Lilli Petruzelli, Dale Porter, Paul McSheehy, Daniel Menezes, Anupama Reddy, Johannes Roesel, Christian Schnell, Timothy R. Smith, Markus Wartmann. Systematic evaluation of drug combinations in vitro and in vivo . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2013-2058