Jan E. Blanchard

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

Abstract 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 (IC50 = 0.5–7 μM) toward SARS-CoV 3CLpro.

Identification of Pharmacological Chaperones for Gaucher Disease and Characterization of Their Effects on β-Glucocerebrosidase by Hydrogen/Deuterium Exchange Mass Spectrometry

Point mutations in β‐glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lysosomes thereby causing Gaucher Disease (GD). Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs), increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), and allow increased lysosomal transport of the enzyme. A high‐throughput screen of the 50 000‐compound Maybridge library identified two, non‐carbohydrate‐based inhibitory molecules, a 2,4‐diamino‐5‐substituted quinazoline (IC50 5 μM) and a 5‐substituted pyridinyl‐2‐furamide (IC50 8 μM). They raised the levels of functional GCase 1.5–2.5‐fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium‐exchange mass spectrometry, identified a domain III active‐site loop (residues 243–249) as being significantly stabilized upon binding of isofagomine or either of these two new compounds; this suggests a common mechanism for PC enhancement of intracellular transport.

High-Throughput Screening Identifies Novel Inhibitors of the Acetyltransferase Activity of Escherichia coli GlmU

ABSTRACT The bifunctional GlmU protein catalyzes the formation of UDP-N-acetylglucosamine in a two-step reaction using the substrates glucosamine-1-phosphate, acetyl coenzyme A, and UTP. This metabolite is a common precursor to the synthesis of bacterial cell surface carbohydrate polymers, such as peptidoglycan, lipopolysaccharide, and wall teichoic acid that are involved in the maintenance of cell shape, permeability, and virulence. The C-terminal acetyltransferase domain of GlmU exhibits structural and mechanistic features unique to bacterial UDP-N-acetylglucosamine synthases, making it an excellent target for antibacterial design. In the work described here, we have developed an absorbance-based assay to screen diverse chemical libraries in high throughput for inhibitors to the acetyltransferase reaction of Escherichia coli GlmU. The primary screen of 50,000 drug-like small molecules identified 63 hits, 37 of which were specific to acetyltransferase activity of GlmU. Secondary screening and mode-of-inhibition studies identified potent inhibitors where compound binding within the acetyltransferase active site was requisite on the presence of glucosamine-1-phosphate and were competitive with the substrate acetyl coenzyme A. These molecules may represent novel chemical scaffolds for future antimicrobial drug discovery. In addition, this work outlines the utility of catalytic variants in targeting specific activities of bifunctional enzymes in high-throughput screens.

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.

A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia

Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery.

A High-Throughput Screen of the GTPase Activity of Escherichia coli EngA to Find an Inhibitor of Bacterial Ribosome Biogenesis

The synthesis of ribosomes is an essential process, which is aided by a variety of trans-acting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in a 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Furthermore, we describe the elimination of nonspecific inhibitors that were detergent sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counterscreens for nonspecificity, but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis.

Glossary of terms used in biomolecular screening (IUPAC Recommendations 2011)

Biomolecular screening is now a crucial component of the drug discovery process, and this glossary will be of use to practitioners in the field of screening and to those who interact with the screening community. The glossary contains definitions related to various aspects of the screening process such as assay types, data handling, and relevant technologies. Many of the terms used in this discipline are not covered by existing glossaries, and where they are, the definitions are often not appropriate for this field. Where appropriate, this document provides new or modified definitions to better reflect the new context. The field of biomolecular screening is multidisciplinary in nature, and this glossary, containing authoritative definitions, will be useful not only for regular practitioners, but also for those who make use of data generated during the screening process.

High-Throughput Screening of Model Bacteria

Small-molecule screening campaigns of model bacteria have been conducted extensively in biotechnology and pharmaceutical companies to search for novel compounds with antibacterial activity. Recently, there has been increasing interest in running such high-throughput screens within academic settings to answer questions in biology. In this respect, whole-cell screening has the particular advantage of identifying compounds with physical and chemical properties compatible with microbial cell permeation, thereby providing probes with which to study diverse aspects of microbial cell physiology and biochemistry. The focus of this chapter is to describe a general method of running a high-throughput screen against a model bacterium to identify small molecules with growth inhibitory activity. Once the primary bioactives have been identified, the determination of their dose-response relationships with the target microbe further characterizes their growth inhibitory effect.

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.

Pyrimethamine as a Potent and Selective Inhibitor of Acute Myeloid Leukemia Identified by High-throughput Drug Screening

Hematopoietic stem and progenitor cell differentiation are blocked in acute myeloid leukemia (AML) resulting in cytopenias and a high risk of death. Most patients with AML become resistant to treatment due to lack of effective cytotoxic and differentiation promoting compounds. High MN1 expression confers poor prognosis to AML patients and induces resistance to cytarabine and alltrans-retinoic acid (ATRA) induced differentiation. Using a high-throughput drug screening, we identified the dihydrofolate reductase (DHFR) antagonist pyrimethamine to be a potent inducer of apoptosis and differentiation in several murine and human leukemia cell lines. Oral pyrimethamine treatment was effective in two xenograft mouse models and specifically targeted leukemic cells in human AML cell lines and primary patient cells, while CD34+ cells from healthy donors were unaffected. The antileukemic effects of PMT could be partially rescued by excess folic acid, suggesting an oncogenic function of folate metabolism in AML. Thus, our study identifies pyrimethamine as a candidate drug that should be further evaluated in AML treatment.