Jonathan D. Cechetto

High-throughput screening identifies inhibitors of the SARS coronavirus main proteinase.

n Abstractn n The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The main proteinase of SARS-CoV, 3CLpro, is an attractive target for therapeutics against SARS owing to its fundamental role in viral replication. We sought to identify novel inhibitors of 3CLpro to advance the development of appropriate therapies in the treatment of SARS. 3CLpro was cloned, expressed, and purified from the Tor2 isolate. A quenched fluorescence resonance energy transfer assay was developed for 3CLpro to screen the proteinase against 50,000 drug-like small molecules on a fully automated system. The primary screen identified 572 hits; through a series of virtual and experimental filters, this number was reduced to five novel small molecules that show potent inhibitory activity (IC50n = 0.5–7 μM) toward SARS-CoV 3CLpro.n n

Localization of Mitochondrial 60-kD Heat Shock Chaperonin Protein (Hsp60) in Pituitary Growth Hormone Secretory Granules and Pancreatic Zymogen Granules

We used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of Hsp60 in rat tissues. Western blot analysis, employing both monoclonal and polyclonal antibodies raised against mammalian Hsp60, shows that only a single 60-kD protein is reactive with the antibodies in brain, heart, kidney, liver, pancreas, pituitary, spleen, skeletal muscle, and adrenal gland. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows strong labeling of mitochondria in all tissues. However, in the anterior pitutary and in pancreatic acinar cells, Hsp60 also localizes in secretory granules. The labeled granules in the pituitary and pancreas were determined to be growth hormone granules and zymogen granules, respectively, using antibodies to growth hormone and carboxypeptidase A. Immunogold labeling of Hsp60 in all compartments was prevented by preadsorption of the antibodies with recombinant Hsp60. Biochemically purified zymogen granules free of mitochondrial contamination are shown by Western blot analysis to contain Hsp60, confirming the morphological localization results in pancreatic acinar cells. In kidney distal tubule cells, low Hsp60 reactivity is associated with infoldings of the basal plasma membrane. In comparison, the plasma membrane in kidney proximal tubule cells and in other tissues examined showed only background labeling. These findings raise interesting questions concerning translocation mechanisms and the cellular roles of Hsp60.

A mitochondrial ketogenic enzyme regulates its gene expression by association with the nuclear hormone receptor PPARα

Mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase (mHMG‐CoAS) is a key enzyme in ketogenesis, catalyzing the condensation of acetyl‐CoA and acetoacetyl‐CoA to generate HMG‐CoA, which is eventually converted to ketone bodies. Transcription of the nuclear‐encoded gene for mHMG‐CoAS is stimulated by peroxisome proliferator‐activated receptor (PPAR) α, a fatty acid‐activated nuclear hormone receptor. Here we show that the mHMG‐CoAS protein physically interacts with PPARα in vitro, and potentiates PPARα‐dependent transcriptional activation via the cognate PPAR response element of the mHMG‐CoAS gene in vivo. Immunofluorescence of transiently transfected cells demonstrated that in the presence of PPARα, mHMG‐CoAS is translocated into the nucleus. Binding to PPARα, stimulation of PPARα activity and nuclear penetration require the integrity of the sequence LXXLL in mHMG‐CoAS, a motif known to mediate the interaction between nuclear hormone receptors and coactivators. These findings reveal a novel mechanism of gene regulation whereby the product of a PPARα‐responsive gene, normally resident in the mitochondria, directly interacts with this nuclear hormone receptor to autoregulate its own nuclear transcription.

High throughput screening identifies novel inhibitors of Escherichia coli dihydrofolate reductase that are competitive with dihydrofolate

This communication describes the high-throughput screen of a diverse library of 50,000 small molecules against Escherichia coli dihydrofolate reductase to detect inhibitors. Sixty-two compounds were identified as having significant inhibitory activity against the enzyme. Secondary screening of these revealed twelve molecules that were competitive with dihydrofolate, nine of which have not been previously characterized as inhibitors of dihydrofolate reductase. These novel molecules ranged in potency (K(i)) from 26 nM to 11 microM and may represent fresh starting points for new small molecule therapeutics directed against dihydrofolate reductase.

Experimental Screening of Dihydrofolate Reductase Yields a “Test Set” of 50,000 Small Molecules for a Computational Data-Mining and Docking Competition

High-throughput screening (HTS) generates an abundance of data that are a valuable resource to be mined. Dockers and data miners can use “real-world” HTS data to test and further develop their tools. A screen of 50,000 diverse small molecules was carried out against Escherichia coli dihydrofolate reductase (DHFR) and compared with a previous screen of 50,000 compounds against the same target. Identical assays and conditions were maintained for both studies. Prior to the completion of the second screen, the original screening data were publicly released for use as a “training set,” and computational chemists and data analysts were challenged to predict the activity of compounds in this second “test set.” Upon completion, the primary screen of the test set generated no potent inhibitors of DHFR activity.

Anti-obesity Phenotypic Screening Looking to Increase OBR Cell Surface Expression

The leptin receptor, OBR, is involved in the regulation of whole-body energy homeostasis. Most obese people are resistant to leptin and do not respond to the hormone. The prevention and reversal of leptin resistance is one of the major current goals of obesity research. We showed previously that increased OBR cell surface expression concomitantly increases cellular leptin signaling and prevents obesity development in mice. Improvement of OBR cell surface expression can thus be considered as an interesting anti-obesity therapeutic strategy. To identify compounds that increase the surface expression of OBR, we developed a cell-based, phenotypic assay to perform a high-content screen (HCS) against a library of 50,000 chemical compounds. We identified 67 compounds that increased OBR cell surface expression with AC50 values in the low micromolar range and no effect on total OBR expression and cellular toxicity. Compounds were classified into 16 chemical clusters, of which 4 potentiated leptin-promoted signaling through the JAK2/STAT3 pathway. In conclusion, development of a robust phenotypic screening approach resulted in the discovery of four new scaffolds that demonstrate the desired biological activity and could constitute an original therapeutic solution against obesity and associated disorders.

High-Throughput Screening at McMaster University: Automation in Academe

In December 2001, the McMaster HTS Lab officially opened its doors. Since that time, we have made significant strides in demonstrating that university-based, high-throughput screening (HTS) is a viable proposition for academic scientists seeking to discover novel small molecule probes of biological function. Although the lab has been running screens for just over two years, the process of designing, building and maintaining the lab has been on-going for more than four years. As high-throughput screening technology moves from the industrial sector to the academic and small biotech sectors, strategies for setting up a successful, highly flexible HTS lab on a limited budget becomes very important. In the current communication, we outline some of the considerations in setting up the lab and some of our experiences to date with screening and automation in academe.