Discovery and Selection of NVP-HSP990 as a Clinical Candidate

This chapter describes the discovery of NVP-Hsp990, a potent, orally bioavailable Hsp90 inhibitor currently in early clinical development. The program strategy for the discovery of Hsp990 is detailed from hit identification to in vivo pre-clinical evaluation. This approach relied heavily on the application of structure-based drug design to rapidly optimize biochemical potency, and identify opportunities for fine-tuning the in vitro and in vivo properties. A significant protein rearrangement is described, which enabled the identification of a highly potent inhibitor series. PK/PD/efficacy relationships are described that guided dose and schedule optimization for the clinical candidate.</jats:p

Directed evolution of D-2-keto-3-deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars

AbstractBackground: Exploitation and improvement of enzymes as catalysts for organic synthesis is of current interest in biocatalysis. A representative enzyme for investigation is the Escherichia coli D-2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes the highly specific reversible aldol reaction using the D-configurated KDPG as substrate.Results: Using in vitro evolution, the aldolase has been converted into aldolases with improved catalytic efficiency, altered substrate specificity and stereoselectivity. In particular, some evolved aldolases capable of accepting both D- and L- glyceraldehyde in the non-phosphorylated form as substrates for reversible aldol reaction have been obtained, providing a new direction to the enzymatic synthesis of both D- and L-sugars.Conclusions: This research has demonstrated the effectiveness of using in vitro evolution to rapidly alter the properties of an aldolase to improve its utility in asymmetric synthesis. The evolved aldolases, differing from the native enzyme which is highly phosphate- and D-sugar-dependent, catalyze the efficient synthesis of both D- and L-sugars from non-phosphorylated aldehydes and pyruvate. The principles and strategies described in this study should be applicable to other aldolases to further expand the scope of their synthetic utility

Abstract 2605: Identification and development of NVP-HSP990, a potent and orally active Hsp90 inhibitor for cancer treatment

Abstract Hsp90 is recognized as a potential target for the treatment of cancer, due to its chaperone functions that are required for maintaining the stability and/or active conformation of signalling molecules essential for tumorigenesis. The development of small molecule Hsp90 inhibitors has become an extremely competitive field of research. Here for the first time we present biological and pharmacological properties of NVP-HSP990, a potent oral Hsp90 inhibitor discovered by Novartis. In vitro, NVP-HSP990 potently inhibits all 4 isoforms of Hsp90 (Hsp90α, Hsp90β, TRAP-1 and GRP94) and has no major activities in a safety pharmacology panel of kinases, receptors and enzymes evaluated. NVP-HSP990 demonstrates broad-spectrum anti-tumor activities in tumor cell lines (IC 50s ranging from 4-35 nM) and primary patient samples (mean IC50 =49 nM). In vivo, NVP-HSP990 is highly bioavailable across species and a single oral administration of 15 mg/kg induces sustained target inhibition up to 7 days as indicated by downregulation of client protein (c-Met) and upregulation of Hsp70 in a cMet overexpressing gastric GTL16 xenograft tumor model. Weekly administration of NVP-HSP990 was efficacious in a broad range of mouse and rat human tumor xenograft models driven by well defined oncogenic Hsp90 client proteins. In summary, NVP-HSP990 represents a novel class of oral Hsp90 inhibitors with unique and superior pharmacological and drug-like properties. NVP-HSP990 is currently being evaluated in Phase I clinical trial in advanced solid tumors. In addition, we will present multiple secreted biomarkers for Hsp90 that were identified through a novel in-silico data mining approach. Several of these biomarkers have been validated both in vitro and in vivo, and have the potential to be utilized as clinical pharmaco-dynamic markers in the development of Hsp90 inhibitors. 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 2605. doi:10.1158/1538-7445.AM2011-2605</jats:p

One-Pot Synthesis of l-Fructose Using Coupled Multienzyme Systems Based on Rhamnulose-1-phosphate Aldolase

Two methods have been developed for the highly efficient enzymatic synthesis of l-fructose:  one is based on rhamnulose-1-phosphate aldolase and acid phosphatase using racemic glyceraldehyde and dihydroxyacetone phosphate as substrates; the other is to generate enantiomerically pure l-glyceraldehyde in situ from glycerol for the aldol reaction, using galactose oxidase catalyzed oxidation of glycerol in the presence of catalase. Using this four-enzyme system, enantiomerically pure l-fructose was obtained. Using the more expensive dihydroxyacetone phosphate, the yield was 55% after purification

Optimization and Mechanistic Characterization of Pyridopyrimidine Inhibitors of Bacterial Biotin Carboxylase

A major challenge for new antibiotic discovery is predicting the physicochemical properties that enable small molecules to permeate Gram-negative bacterial membranes. We have applied physicochemical lessons from previous work to redesign and improve the antibacterial potency of pyridopyrimidine inhibitors of biotin carboxylase (BC) by up to 64-fold and 16-fold against and , respectively. Antibacterial and enzyme potency assessments in the presence of an outer membrane-permeabilizing agent or in efflux-compromised strains indicate that penetration and efflux properties of many redesigned BC inhibitors could be improved to various extents. Spontaneous resistance to the improved pyridopyrimidine inhibitors in occurs at very low frequencies between 10 and 10. However, resistant isolates had alarmingly high minimum inhibitory concentration shifts (16- to \u3e128-fold) compared to the parent strain. Whole-genome sequencing of resistant isolates revealed that either BC target mutations or efflux pump overexpression can lead to the development of high-level resistance

THE NOVEL ORAL HSP90 INHIBITOR NVP-HSP990 EXHIBITS POTENT AND BROAD-SPECTRUM ANTI-TUMOR 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 nM IC50 values on three of the Hsp90 isoforms (Hsp90α, Hsp90β, and GRP94) and 320 nM IC50 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 pharmacological 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 pharmacological profile and broad spectrum anti-tumor activities, clinical trials have been initiated to evaluate NVP-HSP990 in advanced solid tumors