Emilio Diez

Identification of a Series of Tricyclic Natural Products as Potent Broad-Spectrum Inhibitors of Metallo-β-Lactamases

ABSTRACT This work describes the discovery and characterization of a novel series of tricyclic natural product-derived metallo-β-lactamase inhibitors. Natural product screening of the Bacillus cereus II enzyme identified an extract from a strain of Chaetomium funicola with inhibitory activity against metallo-β-lactamases. SB236050, SB238569, and SB236049 were successfully extracted and purified from this extract. The most active of these compounds was SB238569, which possessed Ki values of 79, 17, and 3.4 μM for the Bacillus cereus II, Pseudomonas aeruginosa IMP-1, and Bacteroides fragilis CfiA metallo-β-lactamases, respectively, yet none of the compounds exhibited any inhibitory activity against the Stenotrophomonas maltophilia L-1 metallo-β-lactamase (50% inhibitory concentration > 1,000 μM). The lack of activity against angiotensin-converting enzyme and serine β-lactamases demonstrated the selective nature of these compounds. The crystal structure of SB236050 complexed in the active site of CfiA has been obtained to a resolution of 2.5 Å. SB236050 exhibits key polar interactions with Lys184, Asn193, and His162 and a stacking interaction with the indole ring of Trp49 in the flap, which is in the closed conformation over the active site groove. SB236050 and SB238569 also demonstrate good antibacterial synergy with meropenem. Eight micrograms of SB236050 per ml gave rise to an eightfold drop in the MIC of meropenem for two clinical isolates of B. fragilis producing CfiA, making these strains sensitive to meropenem (MIC ≤ 4 μg/ml). Consequently, this series of metallo-β-lactamase inhibitors exhibit the most promising antibacterial synergy activity so far observed against organisms producing metallo-β-lactamases.

Process Validation and Screen Reproducibility in High-Throughput Screening

The use of large-scale compound screening has become a key component of drug discovery projects in both the pharmaceutical and the biotechnological industries. More recently, these activities have also been embraced by the academic community as a major tool for chemical genomic activities. High-throughput screening (HTS) activities constitute a major step in the initial drug discovery efforts and involve the use of large quantities of biological reagents, hundreds of thousands to millions of compounds, and the utilization of expensive equipment. All these factors make it very important to evaluate in advance of the HTS campaign any potential issues related to reproducibility of the experimentation and the quality of the results obtained at the end of these very costly activities. In this article, the authors describe how GlaxoSmithKline (GSK) has addressed the need of a true validation of the HTS process before embarking in full HTS campaigns. They present 2 different aspects of the so-called validation process: (1) optimization of the HTS workflow and its validation as a quality process and (2) the statistical evaluation of the HTS, focusing on the reproducibility of results and the ability to distinguish active from nonactive compounds in a vast collection of samples. The authors describe a variety of reproducibility indexes that are either innovative or have been adapted from generic medical diagnostic screening strategies. In addition, they exemplify how these validation tools have been implemented in a number of case studies at GSK. (Journal of Biomolecular Screening 2009:66-76)

Discovery of Enhancers of the Secretion of Leukemia Inhibitory Factor for the Treatment of Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease that involves activation of T cells, microglia, and astrocytes. There is a clear unmet medical need for MS, as current therapies reduce the relapse rate, but are unable to prevent the neurological deterioration. Leukemia inhibitory factor (LIF) is a proinflammatory cytokine that can also positively modulate the immune response, by inducing the inhibition of myelin-reactive TH17 differentiation, and by promoting oligodendrocyte-mediated myelination. The aim of this project was to find central nervous system (CNS)–permeable and orally available small molecules that upregulate production of endogenous LIF. We describe here the development of a phenotypic assay and screening of 1.7 million compounds to identify LIF enhancers using U87 MG cells. Five chemically tractable series of compounds and a few singletons were selected for further progression. Some of them were also active in a different LIF-expressing cell line and in primary rat astrocytes. Although further studies would be required to deconvolute the targets involved in LIF induction and to confirm activity of hits in more disease-relevant assays, our results have demonstrated the potential of the phenotypic approach to identify specific and chemically tractable small molecules that trigger the production of LIF in relevant cell lines.

Mining Natural-Products Screening Data for Target-Class Chemical Motifs

In this article, we describe two complementary data-mining approaches used to characterize the GlaxoSmithKline (GSK) natural-products set (NPS) based on information from the high-throughput screening (HTS) databases. Both methods rely on the aggregation and analysis of a large set of single-shot screening data for a number of biological assays, with the goal to reveal natural-product chemical motifs. One of them is an established method based on the data-driven clustering of compounds using a wide range of descriptors,1 whereas the other method partitions and hierarchically clusters the data to identify chemical cores.2,3 Both methods successfully find structural scaffolds that significantly hit different groups of discrete drug targets, compared with their relative frequency of demonstrating inhibitory activity in a large number of screens. We describe how these methods can be applied to unveil hidden information in large single-shot HTS data sets. Applied prospectively, this type of information could contribute to the design of new chemical templates for drug-target classes and guide synthetic efforts for lead optimization of tractable hits that are based on natural-product chemical motifs. Relevant findings for 7TM receptors (7TMRs), ion channels, class-7 transferases (protein kinases), hydrolases, and oxidoreductases will be discussed.

OP0233 Genome-Wide Pathway Analysis Reveals that VEGF Genetic Pathway Is Associated with Oral Ulcers in Systemic Lupus Erythematosus

Background Systemic lupus erythematosus (SLE) is a genetically complex rheumatic disease with heterogeneous clinical manifestations. Recent studies have suggested the existence of a genetic basis for the diverse SLE clinical phenotypes. Also, there is increasing evidence indicating that a substantial part of the genetic variation associated with complex diseases is explained by small-effect genes from common genetic pathways. Objectives The objective of the present study was to identify new genetic variation associated with SLE phenotypes using a genome-wide association study at the pathway level. Methods A total of 598,258 SNPs were genotyped in a discovery cohort of n=482 SLE patients of southern European ancestry using the Illumina platform Quad610. After quality control analysis, including ancestry estimation using principal-component analysis, genome-wide pathway analysis was performed. A total of 14 clinically relevant SLE phenotypes were tested for association with n>700 reference genetic pathways. Significantly associated pathways (corrected P-value <0.05) were subsequently tested for validation in an independent cohort of n=425 SLE patients from the same ancestry. Both discovery and validation cohort patients were Caucasian European and from Spanish origin, and were recruited by rheumatology departments from n=15 Spanish university hospitals. The validated genetic pathways were functionally characterized using in silico analysis on cell types of relevance in SLE pathogenesis. Results In the discovery stage, two genetic pathways were significantly associated with the presence of oral ulcers and antinuclear antibodies in SLE (PFDR<0.05). In the replication stage, we validated the association between oral ulcers and vascular endothelial growth factor (VEGF) genetic pathway (P=1.3e-2). Analyzing the transcriptional effect of the topical immunotherapies used for the treatment of oral ulcers in SLE, we found a significant differential expression of VEGF pathway genes (P<0.05). Conclusions In this work we have performed the first genome-wide association study for clinically relevant SLE phenotype using a pathway-based approach. With this new approach, we have identified and validated the association of VEGF genetic pathway with oral ulcers in SLE. These findings represent an important step towards the characterization of the genetic basis of phenotype heterogeneity in SLE. Disclosure of Interest None declared