Differential scanning fluorimetry as secondary screening platform for small molecule inhibitors of Bcl-XL.

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Small molecule inhibitors of Bcl-X(L) function have been discovered from diverse structure classes using rational drug design as well as high-throughput screening (HTS) approaches. However, most of the BH3 mimetics that have been identified via screening based on fluorescence polarization displayed an affinity for their presumed protein targets that is far lower than that of BH3-only proteins. Therefore, it is important to establish a simple and inexpensive secondary platform for hit validation which is pertinent to current efforts for developing compounds that mimic the action of BH3-only proteins as novel anticancer agents. These considerations prompted us to explore the differential scanning fluorimetry (DSF) method that is based on energetic coupling between ligand binding and protein unfolding. We have systematically tested known Bcl-X(L)/Bcl-2 inhibitors using DSF and have revealed distinct subsets of inhibitors. More importantly, we report that some of these inhibitors interacted selectively with glutathione S-transferase tagged Bcl-X(L), whereas certain inhibitors exhibited marked selectivity towards native untagged Bcl-X(L). Therefore, we propose that the affinity tag may cause a significant conformational switch in the Bcl-X(L), which results in the selectivity for certain subsets of small molecule inhibitors. This finding also implies that the previous screens involving tagged proteins need to be carefully reexamined while further investigations must ensure that the right conformation of protein is used in future screens.

N-Hydroxy-1,2-disubstituted-1H-benzimidazol-5-yl acrylamides as novel histone deacetylase inhibitors: design, synthesis, SAR studies, and in vivo antitumor activity.

A series of N-hydroxy-1,2-disubstituted-1H-benzimidazol-5-yl acrylamides were designed and synthesized as novel HDAC inhibitors. General SAR has been established for the substituents at positions 1 and 2, as well as the importance of the ethylene group and its attachment to position 5. Optimized compounds are much more potent than SAHA in both enzymatic and cellular assays. A representative compound, 23 (SB639), has demonstrated antitumor activity in a colon cancer xenograft model.

Overview of high-throughput screening.

High-throughput screening (HTS) is a key process used in drug discovery to identify hits from compound libraries that may become leads for medicinal chemistry optimization. This updated overview discusses the utilization of compound libraries, compounds derived from combinatorial and parallel synthesis campaigns and natural product sources; creation of mother and daughter plates; and compound storage, handling, and bar coding in HTS. The unit also presents an overview of established and emerging assay technologies (i.e., time-resolved fluorescence, fluorescence polarization, fluorescence-correlation spectroscopy, functional whole cell assays, and high-content assays) and their integration in automation hardware and IT systems. This revised unit provides updated descriptions of state-of-the-art instrumentation and technologies in this rapidly changing environment. The section on assay methodologies now also covers enzyme complementation assays and methods for high-throughput screening of ion channel activities. Finally, a section on criteria for assay robustness is included discussing the Z'-factor, which is now a widely accepted criterion for evaluation and validation of high throughput screening assays.

Genomics as knowledge enterprise: Implementing an electronic research habitat at the Biopolis Experimental Therapeutics Center.

The Experimental Therapeutics Center (ETC) has been established at Biopolis to advance translational research by bridging the gap between discovery science and commercialization. We describe the Electronic Research Habitat at ETC, a comprehensive hardware and software infrastructure designed to effectively manage terabyte data flows and storage, increase back office efficiency, enhance the scientific work experience, and satisfy rigorous regulatory and legal requirements. Our habitat design is secure, scalable and robust, and it strives to embody the core values of the knowledge-based workplace, thus contributing to the strategic goal of building a "knowledge economy" in the context of Singapore's on-going biotechnology initiative.

Function of the SIRT1 protein deacetylase in cancer.

The NAD(+)-dependent protein deacetylase SIRT1 is linked to cellular survival pathways by virtue of keeping the tumor suppressor gene p53 and members of the forkhead transcription factor family deacetylated. To validate SIRT1 as a therapeutic anti-cancer target, we performed immunohistochemistry experiments to study the in vivo expression of SIRT1 in cancer specimens. We show that human SIRT1 is highly expressed in cancer cell lines as well as in tissue samples from colon carcinoma patients. Interestingly, there is a strong cytosolic component in the SIRT1 expression pattern. We further characterized SIRT1 in p53-wild-type and -mutant cell lines and show that SIRT1 mRNA-knockdown leads to a p53-independent decrease of cell proliferation and induction of apoptosis. In addition, SIRT1 expression has been found to be inducible upon DNA damage. A previously discovered small molecule SIRT1 inhibitor with nanomolar in vitro activity has been tested in cancer relevant assays. The SIRT1 inhibitory compound showed no potent anti-proliferative activity despite hitting its molecular target within tumor cells. From these studies we conclude that it may not be sufficient to block the catalytic function of SIRT1, and that its survival effects may be mainly brought about by means other then the deacetylase function. The increased cytosolic expression of SIRT1 in cancer cells could be an indicator of such novel functions.

SIRT1 modulating compounds from high-throughput screening as anti-inflammatory and insulin-sensitizing agents.

The nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase SIRT1 has been linked to fatty acid metabolism via suppression of peroxysome proliferator-activated receptor gamma (PPAR-gamma) and to inflammatory processes by deacetylating the transcription factor NF-kappaB. First, modulation of SIRT1 activity affects lipid accumulation in adipocytes, which has an impact on the etiology of a variety of human metabolic diseases such as obesity and insulin-resistant diabetes. Second, activation of SIRT1 suppresses inflammation via regulation of cytokine expression. Using high-throughput screening, the authors identified compounds with SIRT1 activating and inhibiting potential. The biological activity of these SIRT1-modulating compounds was confirmed in cell-based assays using mouse adipocytes, as well as human THP-1 monocytes. SIRT1 activators were found to be potent lipolytic agents, reducing the overall lipid content of fully differentiated NIH L1 adipocytes. In addition, the same compounds have anti-inflammatory properties, as became evident by the reduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). In contrast, a SIRT1 inhibitory compound showed a stimulatory activity on the differentiation of adipocytes, a feature often linked to insulin sensitization.

Development of human calcitonin gene-related peptide (CGRP) receptor antagonists. 1. Potent and selective small molecule CGRP antagonists. 1-[N2-[3,5-dibromo-N-[[4-(3,4-dihydro-2(1H)-oxoquinazolin-3-yl)-1-piperidinyl]carbonyl]-D-tyrosyl]-l-lysyl]-4-(4-pyridinyl)piperazine: the first CGRP antagonist for clinical trials in acute migraine.

Although the triptans have greatly improved the acute treatment of migraine headache, there are yet many shortcomings. Therefore, new strategies for the treatment of migraine are needed which offer advantages over current therapy, e.g. triptans. Our novel approach was based on the hypothesis that the release of calcitonin gene-related peptide (CGRP) could play a causative role in migraine headache. Thus we initiated a program aimed at the design and synthesis of small molecule CGRP receptor antagonists. High throughput screening led to the identification of (R)-Tyr-(S)-Lys dipeptide-like compounds that showed weak but unequivocal binding to the human CGRP receptor. Lead optimization afforded highly potent CGRP antagonists, the prototype being compound 19 (BIBN4096). This compound exhibiting a favorable biological profile was selected for initial clinical trials. A proof of concept study indicated that intravenous application of 19 was effective in the treatment of acute migraine headache. This finding strongly supports our initial working hypothesis that CGRP plays an important role in the pathophysiology of migraine.

Emerging trends in high-throughput screening.

Novel technologies are emerging for high-throughput screening, driven by the needs and fine-tuning of established drug discovery activities, as well as by the emergence of novel target classes resulting from the deciphering of the human genome. Disciplines other than biology have now entered the screening scene, as bioinformatics, micro-technology and analytics provide powerful methodologies and applications that were not previously suitable for high-throughput screening. Many of these will move high-throughput screening from a numbers game to a content- and information-based approach to identify leads for novel disease targets.