High throughput screening for SARS-CoV-2 helicase inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for nearly 7 million deaths worldwide since its outbreak in late 2019. Even with the rapid development and production of vaccines and intensive research, there is still a huge need for specific anti-viral drugs that address the rapidly arising new variants. To address this concern, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) Centers, tasked with exploring approaches to target pathogens with pandemic potential, including SARS-CoV-2. In this study, we sought inhibitors of SARS-CoV2 non-structural protein 13 (nsP13) as potential antivirals, first developing a HTS-compatible assay to measure SARS-CoV2 nsP13 helicase activity. Here we present our effort in implementing the assay in a 1,536 well-plate format and in identifying nsP13 inhibitor hit compounds from a ∼650,000 compound library. The primary screen was robust (average Z' = 0.86 ± 0.05) and resulted in 7,009 primary hits. 1,763 of these compounds upon repeated retests were further confirmed, showing consistent inhibition. Following in-silico analysis, an additional orthogonal assay and titration assays, we identified 674 compounds with IC50 <10 µM. We confirmed activity of independent compound batches from de novo powders while also incorporating multiple counterscreen assays. Our study highlights the potential of this assay for use on HTS platforms to discover novel compounds inhibiting SARS-CoV2 nsP13, which merit further development as an effective SARS-CoV2 antiviral.

Light-controlled micron-scale molecular motion.

The micron-scale movement of biomolecules along supramolecular pathways, mastered by nature, is a remarkable system requiring strong yet reversible interactions between components under the action of a suitable stimulus. Responsive microscopic systems using a variety of stimuli have demonstrated impressive relative molecular motion. However, locating the position of a movable object that travels along self-assembled fibres under an irresistible force has yet to be achieved. Here, we describe a purely supramolecular system where a molecular 'traveller' moves along a 'path' over several microns when irradiated with visible light. Real-time imaging of the motion in the solvated state using total internal reflection fluorescence microscopy shows that anionic porphyrin molecules move along the fibres of a bis-imidazolium gel upon irradiation. Slight solvent changes mean movement and restructuring of the fibres giving microtoroids, indicating control of motion by fibre mechanics with solvent composition. The insight provided here may lead to the development of artificial travellers that can perform catalytic and other functions.

Discovery of Selective Inhibitors for In Vitro and In Vivo Interrogation of Skeletal Myosin II.

Myosin IIs, actin-based motors that utilize the chemical energy of adenosine 5'-triphosphate (ATP) to generate force, have potential as therapeutic targets. Their heavy chains differentiate the family into muscle (skeletal [SkMII], cardiac, smooth) and nonmuscle myosin IIs. Despite the therapeutic potential for muscle disorders, SkMII-specific inhibitors have not been reported and characterized. Here, we present the discovery, synthesis, and characterization of "skeletostatins," novel derivatives of the pan-myosin II inhibitor blebbistatin, with selectivity 40- to 170-fold for SkMII over all other myosin II family members. In addition, the skeletostatins bear improved potency, solubility, and photostability, without cytotoxicity. Based on its optimal in vitro profile, MT-134's in vivo tolerability, efficacy, and pharmacokinetics were determined. MT-134 was well-tolerated in mice, impaired motor performance, and had excellent exposure in muscles. Skeletostatins are useful probes for basic research and a strong starting point for drug development.