Ronald Frank

Identification of myxobacteria-derived HIV inhibitors by a high-throughput two-step infectivity assay

BackgroundDrug-resistance and therapy failure due to drug-drug interactions are the main challenges in current treatment against Human Immunodeficiency Virus (HIV) infection. As such, there is a continuous need for the development of new and more potent anti-HIV drugs. Here we established a high-throughput screen based on the highly permissive TZM-bl cell line to identify novel HIV inhibitors. The assay allows discriminating compounds acting on early and/or late steps of the HIV replication cycle.ResultsThe platform was used to screen a unique library of secondary metabolites derived from myxobacteria. Several hits with good anti-HIV profiles were identified. Five of the initial hits were tested for their antiviral potency. Four myxobacterial compounds, sulfangolid C, soraphen F, epothilon D and spirangien B, showed EC50 values in the nM range with SIu2009>u200915. Interestingly, we found a high amount of overlapping hits compared with a previous screen for Hepatitis C Virus (HCV) using the same library.ConclusionThe unique structures and mode-of-actions of these natural compounds make myxobacteria an attractive source of chemicals for the development of broad-spectrum antivirals. Further biological and structural studies of our initial hits might help recognize smaller drug-like derivatives that in turn could be synthesized and further optimized.

Myxobacteria: natural pharmaceutical factories

Myxobacteria are amongst the top producers of natural products. The diversity and unique structural properties of their secondary metabolites is what make these social microbes highly attractive for drug discovery. Screening of products derived from these bacteria has revealed a puzzling amount of hits against infectious and non-infectious human diseases. Preying mainly on other bacteria and fungi, why would these ancient hunters manufacture compounds beneficial for us? The answer may be the targeting of shared processes and structural features conserved throughout evolution.

The myxobacterial metabolite ratjadone A inhibits HIV infection by blocking the Rev/CRM1-mediated nuclear export pathway

BackgroundThe nuclear export of unspliced and partially spliced HIV-1 mRNA is mediated by the recognition of a leucine-rich nuclear export signal (NES) in the HIV Rev protein by the host protein CRM1/Exportin1. This makes the CRM1-Rev complex an attractive target for the development of new antiviral drugs. Here we tested the anti-HIV efficacy of ratjadone A, a CRM1 inhibitor derived from myxobacteria.ResultsRatjadone A inhibits HIV infection in vitro in a dose-dependent manner with EC50 values at the nanomolar range. The inhibitory effect of ratjadone A occurs around 12 hours post-infection and is specific for the Rev/CRM1-mediated nuclear export pathway. By using a drug affinity responsive target stability (DARTS) assay we could demonstrate that ratjadone A interferes with the formation of the CRM1-Rev-NES complex by binding to CRM1 but not to Rev.ConclusionRatjadone A exhibits strong anti-HIV activity but low selectivity due to toxic effects. Although this limits its potential use as a therapeutic drug, further studies with derivatives of ratjadones might help to overcome these difficulties in the future.

Design of a general-purpose European compound screening library for EU-OPENSCREEN

This work describes a collaborative effort to define and apply a protocol for the rational selection of a general‐purpose screening library, to be used by the screening platforms affiliated with the EU‐OPENSCREEN initiative. It is designed as a standard source of compounds for primary screening against novel biological targets, at the request of research partners. Given the general nature of the potential applications of this compound collection, the focus of the selection strategy lies on ensuring chemical stability, absence of reactive compounds, screening‐compliant physicochemical properties, loose compliance to drug‐likeness criteria (as drug design is a major, but not exclusive application), and maximal diversity/coverage of chemical space, aimed at providing hits for a wide spectrum of drugable targets. Finally, practical availability/cost issues cannot be avoided. The main goal of this publication is to inform potential future users of this library about its conception, sources, and characteristics. The outline of the selection procedure, notably of the filtering rules designed by a large committee of European medicinal chemists and chemoinformaticians, may be of general methodological interest for the screening/medicinal chemistry community. The selection task of 200K molecules out of a pre‐filtered set of 1.4M candidates was shared by five independent European research groups, each picking a subset of 40K compounds according to their own in‐house methodology and expertise. An in‐depth analysis of chemical space coverage of the library serves not only to characterize the collection, but also to compare the various chemoinformatics‐driven selection procedures of maximal diversity sets. Compound selections contributed by various participating groups were mapped onto general‐purpose self‐organizing maps (SOMs) built on the basis of marketed drugs and bioactive reference molecules. In this way, the occupancy of chemical space by the EU‐OPENSCREEN library could be directly compared with distributions of known bioactives of various classes. This mapping highlights the relevance of the selection and shows how the consensus reached by merging the five different 40K selections contributes to achieve this relevance. The approach also allows one to readily identify subsets of target‐ or target‐class‐oriented compounds from the EU‐OPENSCREEN library to suit the needs of the diverse range of potential users. The final EU‐OPENSCREEN library, assembled by merging five independent selections of 40K compounds from various expert groups, represents an excellent example of a Europe‐wide collaborative effort toward the common objective of building best‐in‐class European open screening platforms.

Screening of small molecules affecting mammalian P-body assembly uncovers links with diverse intracellular processes and organelle physiology

Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. The mechanism of P-body assembly has been typically addressed by depleting P-body components. Here we apply a complementary approach and establish an automated cell-based assay platform to screen for molecules affecting P-body assembly. From a unique library of compounds derived from myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA), a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also inhibited, under stress conditions, phosphorylation of eIF2α and stress granule formation. Other hits uncovered interesting novel links between P-body assembly, lipid metabolism, and internal organelle physiology. The obtained results provide a chemical toolbox to manipulate P-body assembly and function.

Synthetic Genes as a Powerful Tool for Protein Structure-Function Analysis

Proteins effect their versatile biological functions by folding into defined tertiary structures. A folded protein chain assembles precisely the relevant atomic groups that participate in substrate binding and catalysis, and provides the necessary stability and flexibility of the active conformation. The principles for building such complex structures from selected amino acid sequences are still poorly understood. Advanced techniques such as X-ray crystallography and other spectro-scopic methods (NMR, IR, CD) have brought a deeper insight into the three-dimensional architecture and physicochemical properties of proteins. However, conclusions and theories derived from these studies require experimental verification.