Cell-free assay technologies for the identification of active compounds

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The PCMBS collaborates closely with the MTP Assay Development and Screening Section (ADSS/Henrich) to automate, validate and complete high throughput screens (HTS), with the Chemical Diversity and Development Section (CDDS/Beutler) to access new sources of chemical diversity and with the Natural Products Chemistry Section (NPCS/Grkovic, Du) to prioritize extracts for natural product isolation and biochemically evaluate pure active compounds isolated by NPCS or CDDS. Examples of PCMBS efforts during this review period, include published papers on screens of natural products for selective inhibitors of the protease MALT the ubiquitin ligase CBLB, and the phosphodiesterase TDP1. Our studies on natural product inhibitors of TDP1 also resulted in a second paper describing a new class of cyclic peptides which are the first reported allosteric inhibitors of this phosphodiesterase. We have recently also completed a screen for inhibitors of the phosphodiesterase TDP2 with a manuscript in preparation. In addition, the PCMBS has continued to increase the implementation of biophysical screening methods for the identification of specific modulators of the thermal stability of both protein and RNA structures. Initially, this work resulted in the identification of a new small molecule scaffold that altered the stability of HIV-1 TAR RNA. This technology has now been expanded upon to allow for the facile screening of pure compounds and natural product samples in a high throughput format to identify modulators of RNA and protein stability. One recently published example was the identification of a natural product that selectively modulated the thermal stability of pre-miR-21. We have recently completed two new enzymatic screens, against the chimeric kinase DNAJB1-PRKACA (PKADJ) and inositol 1,3,4-triphosphate 5/6 kinase (ITPK1), as well as four biophysical screens; against the immunologically-pertinent metabolic enzyme Immune Responsive Gene 1 (IRG1), the diagnostic target for hepatocellular carcinoma glypican-3 (GPC-3) and the SARS-CoV-2 receptor binding domain (RBD), and DNA-reading protein BRD4. The PCMBS also evaluates compounds derived from MTP screens for their specific interactions against macromolecular targets. We have used both biophysical and biochemical techniques to evaluate inhibitors of pre-miR-21 RNA, TDP1, MALT1,CBLB, and SARS-CoV-2 viral entry.