Category: Assay Development and Screening
Fragment Based Drug Discovery (FBDD) has become a well-established strategy for developing new lead compounds. It allows fast, simple and robust screening, with the potential to become the tool of choice for many drug discovery pipelines against new and demanding targets. This approach has been applied against aspartate semialdehyde dehydrogenase (ASADH), an enzyme that functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. ASADH catalyzes the NADPH-dependent reductive dephosphorylation of β-aspartyl phosphate to produce the key intermediate aspartate semialdehyde. Here, we present the current state of antifungal drug development against ASADH using a coupled enzymatic assay (Z’ = 0.82) to measure ASADH activity, where phenazine methosulfate (PMS), an electron carrier from NADPH, will reduce iodonitrotetrazolium (INT), an internal dye to a wine red colored compound formazan which absorbs at 500 nm. This assay was developed to avoid UV background absorbance from many of the library compounds. As an initial study ~1,000 compounds from our customized fragment libraries were screened against ASADH, followed by determination of the inhibition constant (Ki) values of the most potent hits. Encouragingly, these results showed that most of the hits obtained already have Ki values in the lower micromolar range. Several of these initial hits were selected for structural-activity relationship (SAR) evaluation, leading to optimized inhibitors against fungal ASADH with inhibition constants in the low micromolar/high nanomolar range while still retaining high ligand efficiency values. The efficacy of these hits was further validated using a sensitive cell-based assay against the growth of Candida cells. The most potent hits in this cell-based assay have MIC values approaching ~2 μg/ml. Cheminformatic analysis of our fragment libraries indicate that the majority of the compounds are in accordance with the "Rule of Three"; ideal fragments for FBDD. Further screening of ~1000 drug-like compounds from an NIH clinical collection library revealed that many of these hits have core scaffolds that are representative of those obtained from the fragment library hits, confirming that fragments can serve as hit predictors and as a guide for optimized inhibitor selection. Native PAGE studies and small angle x-ray scattering (SAXS) analysis showed that the fungal ASADHs exist as a functional tetramer, in contrast to the bacterial ASADHs that exist as dimers. Some of these newly identified inhibitors showed a non-competitive mode of inhibition, functioning to bind in the tetrameric interface and causing dissociation into inactive dimers. These compounds will be used as starting points for designing protein-protein interaction inhibitors with a unique mode of action against our fungal enzyme target. Our long-term goal is to develop drugs that selectively target these fungal enzymes and inhibit the growth of these pathogenic fungal species.
Gopal Dahal– Graduate Teaching Assistant, University of Toledo, Toledo, OH
Graduate Teaching Assistant
University of Toledo
I am a 5th year graduate student at The Department of Chemistry and Biochemistry at The University of Toledo, Ohio. I completed my Bachelor of Pharmacy from Kathmandu University, Nepal in 2011. My graduate research involves the development of novel antifungal inhibitors targeting enzymes involved in amino acid biosynthetic pathway by using fragment library screening and high throughput screening complemented by structure based drug design. Besides this, my research also focuses on enzymatic assay development using UV/Vis spectrophotometer, HPLC, UPLC, GC, ESI-MS and analyzing protein-ligand and protein-protein interactions using biophysical techniques like X-ray Crystallography, Isothermal Titration Calorimetry (ITC), Surface Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR). Also, I have experience in cell based assay development against pathogenic microorganisms and study of their biofilm inhibition.