GS-441524-Diphosphate-Ribose Derivatives as Nanomolar Binders and Fluorescence Polarization Tracers for SARS-CoV-2 and Other Viral Macrodomains.

Viral macrodomains that can bind to or hydrolyze protein adenosine diphosphate ribosylation (ADP-ribosylation) have emerged as promising targets for antiviral drug development. Many inhibitor development efforts have been directed against the severe acute respiratory syndrome coronavirus 2 macrodomain 1 (SARS-CoV-2 Mac1). However, potent inhibitors for viral macrodomains are still lacking, with the best inhibitors still in the micromolar range. Based on GS-441524, a remdesivir precursor, and our previous studies, we have designed and synthesized potent binders of SARS-CoV-2 Mac1 and other viral macrodomains including those of Middle East respiratory syndrome coronavirus (MERS-CoV), Venezuelan equine encephalitis virus (VEEV), and Chikungunya virus (CHIKV). We show that the 1'-CN group of GS-441524 promotes binding to all four viral macrodomains tested while capping the 1″-OH of GS-441524-diphosphate-ribose with a simple phenyl ring further contributes to binding. Incorporating these two structural features, the best binders show 20- to 6000-fold increases in binding affinity over ADP-ribose for SARS-CoV-2, MERS-CoV, VEEV, and CHIKV macrodomains. Moreover, building on these potent binders, we have developed two highly sensitive fluorescence polarization tracers that only require nanomolar proteins and can effectively resolve the binding affinities of nanomolar inhibitors. Our findings and probes described here will facilitate future development of more potent viral macrodomain inhibitors.

A Fluorescence Polarization Assay for Macrodomains Facilitates the Identification of Potent Inhibitors of the SARS-CoV-2 Macrodomain.

Viral macrodomains, which can bind to and/or hydrolyze adenine diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested to counteract host immune response and be viable targets for the development of antiviral drugs. Therefore, developing high-throughput screening (HTS) techniques for macrodomain inhibitors is of great interest. Herein, using a novel tracer TAMRA-ADPr, an ADP-ribose compound conjugated with tetramethylrhodamine, we developed a robust fluorescence polarization assay for various viral and human macrodomains including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1, MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC50 6.4 µM) and GS441524 (IC50 15.2 µM), two literature-reported small-molecule inhibitors of SARS-CoV-2 Macro1. Our data suggest that GS441524 is highly selective for SARS-CoV-2 Macro1 over other human and viral macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p-nitrophenol, IC50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl umbelliferone, IC50 590 nM) as the most potent SARS-CoV-2 Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2 Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes to the binding affinity. Our data demonstrate that this fluorescence polarization assay is a useful addition to the HTS methods for the identification of macrodomain inhibitors.

Fibrinogen-related protein, FGL2, of hamster cauda epididymal fluid: Purification, kinetic analysis of its prothrombinase activity, and its role in segregation of nonviable spermatozoa.

Although the epididymal environment promotes the maturation and survival of spermatozoa, not all spermatozoa remain viable during passage through the epididymis. Does the epididymis has a protective mechanism(s) to segregate the viable sperm from defective spermatozoa? Previously, we identified 260/280 kDa oligomers (termed eFGL-Epididymal Fibrinogen-Like oligomer) are composed of two disulfide-linked subunits: a 64 kDa polypeptide identified as fibrinogen-like protein-2 (FGL2) and a 33 kDa polypeptide identified as fibrinogen-like protein-1 (FGL1). Our morphological studies demonstrated that the eFGL, secreted from the principal cells of the cauda epididymis, is polymerized into a death cocoon-like complex (DCF), masking defective luminal spermatozoa but, not the viable sperm population. In the present study, we purified FGL2 from hamster cauda epididymal fluid toward homogeneity and its prothrombinase catalytic activity was examined. Time-course conversion studies revealed that all prothrombin was converted to thrombin by purified hamster FGL2. Our biochemical studies demonstrate that FGL2 is a lipid-activated serine protease and functions as a lectin by binding specific carbohydrate residues. Co-immunoprecipitation analysis demonstrated that FGL2 of cauda epididymal fluid is ubiquitinated but not the FGL1. We propose that FGL2/FGL1 oligomers represent a novel and unique mechanism to shield the viable sperm population from degenerating spermatozoa contained within the tubule lumen.