Elucidation of novel TRAP1-Selective inhibitors that regulate mitochondrial processes.

Hsp90 isoform-selective inhibitors represent a new paradigm for novel anti-cancer drugs as each of the four isoforms have specific cellular localization, function, and client proteins. The mitochondrial isoform, TRAP1, is the least understood member of the Hsp90 family due to the lack of small molecule tools to study its biological function. Herein, we report novel TRAP1-selective inhibitors used to interrogate TRAP1's biological function along with co-crystal structures of such compounds bound to the N-terminus of TRAP1. Solution of the co-crystal structure allowed for a structure-based approach that resulted in compound 36, which is a 40 nM inhibitor with >250-fold TRAP1 selectivity over Grp94, the isoform with the highest structural similarity to TRAP1 within the N-terminal ATP binding site. Lead compounds 35 and 36 were found to selectively induce TRAP1 client protein degradation without inducing the heat shock response or disrupting Hsp90-cytosolic clients. They were also shown to inhibit OXPHOS, alter cellular metabolism towards glycolysis, disrupt TRAP1 tetramer stability, and disrupt the mitochondrial membrane potential.

Toehold-Mediated Shape Transition of Nucleic Acid Nanoparticles.

We introduce a toehold-mediated strand displacement strategy for regulated shape-switching of nucleic acid nanoparticles (NANPs) enabling their sequential transformation from triangular to hexagonal architectures at isothermal conditions. The successful shape transitions were confirmed by electrophoretic mobility shift assays, atomic force microscopy, and dynamic light scattering. Furthermore, implementation of split fluorogenic aptamers allowed for monitoring the individual transitions in real time. Three distinct RNA aptamers─malachite green (MG), broccoli, and mango─were embedded within NANPs as reporter domains to confirm shape transitions. While MG "lights up" within the square, pentagonal, and hexagonal constructs, the broccoli is activated only upon formation of pentagon and hexagon NANPs, and mango reports only the presence of hexagons. Moreover, the designed RNA fluorogenic platform can be employed to construct a logic gate that performs an AND operation with three single-stranded RNA inputs by implementing a non-sequential polygon transformation approach. Importantly, the polygonal scaffolds displayed promising potential as drug delivery agents and biosensors. All polygons exhibited effective cellular internalization followed by specific gene silencing when decorated with fluorophores and RNAi inducers. This work offers a new perspective for the design of toehold-mediated shape-switching nanodevices to activate different light-up aptamers for the development of biosensors, logic gates, and therapeutic devices in the nucleic acid nanotechnology.

Spike protein receptor-binding domains from SARS-CoV-2 variants of interest bind human ACE2 more tightly than the prototype spike protein.

Several SARS-CoV-2 variants of interest (VOI) have emerged since this virus was first identified as the etiologic agent responsible for COVID-19. Some of these variants have demonstrated differences in both virulence and transmissibility, as well as in evasion of immune responses in hosts vaccinated against the original strain of SARS-CoV-2. There remains a lack of definitive evidence that identifies the genetic elements that are responsible for the differences in transmissibility among these variants. One factor affecting transmissibility is the initial binding of the surface spike protein (SP) of SARS-CoV-2 to human angiotensin converting enzyme-2 (hACE2), the widely accepted receptor for SP. This step in the viral replication process is mediated by the receptor binding domain (RBD) of SP that is located on the surface of the virus. This current study was conducted with the aim of assessing potential differences in binding affinity between recombinant hACE2 and the RBDs of emergent SARS-CoV-2 WHO VOIs. Mutations that affect the binding affinity of SP play a dominant initial role in the infectivity of the virus.

Cell Entry and Unusual Replication of SARS-CoV-2.

BACKGROUND: SARS-CoV-2 is the causative virus for the CoVID-19 pandemic that has frequently mutated to continue to infect and resist available vaccines. Emerging new variants of the virus have complicated notions of immunity conferred by vaccines versus immunity that results from infection. While we continue to progress from epidemic to endemic as a result of this collective immunity, the pandemic remains a morbid and mortal problem. OBJECTIVE: The SARS-CoV-2 virus has a very complex manner of replication. The spike protein, one of the four structural proteins of the encapsulated virus, is central to the ability of the virus to penetrate cells to replicate. The objective of this review is to summarize these complex features of viral replication. METHODS: A review of the recent literature was performed on the biology of SARS-CoV-2 infection from published work from PubMed and works reported to preprint servers, e.g., bioRxiv and medRxiv. RESULTS AND CONCLUSION: The complex molecular and cellular biology involved in SARS-CoV-2 replication and the origination of >30 proteins from a single open reading frame (ORF) have been summarized, as well as the structural biology of spike protein, a critical factor in the cellular entry of the virus, which is a necessary feature for it to replicate and cause disease.