The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has resulted in a global health and economic crisis of unprecedented scale. The high transmissibility of SARS-CoV-2, combined with a lack of population immunity and prevalence of severe clinical outcomes, urges the rapid development of effective therapeutic countermeasures. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2. In an expeditious process taking only twelve working days, sybodies were selected entirely in vitro from three large combinatorial libraries, using ribosome and phage display. We obtained six strongly enriched sybody pools against the isolated RBD and identified 63 unique anti-RBD sybodies which also interact in the context of the full-length SARS-CoV-2 spike protein. It is anticipated that compact binders such as these sybodies could feasibly be developed into an inhalable drug that can be used as a convenient prophylaxis against COVID-19. Moreover, generation of polyvalent antivirals, via fusion of anti-RBD sybodies to additional small binders recognizing secondary epitopes, could enhance the therapeutic potential and guard against escape mutants. We present full sequence information and detailed protocols for the identified sybodies, as a freely accessible resource. This report will be updated as we further characterize the identified sybodies, in terms of affinities, scaled-up purification yields, and their potential to neutralize SARS-CoV-2 infections.
Summary The major facilitator superfamily transporter Rv1410 and the lipoprotein LprG (Rv1411) are encoded by a conserved two‐gene operon and contribute to virulence in Mycobacterium tuberculosis. Rv1410 was originally postulated to function as a drug efflux pump, but recent studies suggested that Rv1410 and LprG work in concert to insert triacylglycerides and lipoarabinomannans into the outer membrane. Here, we conducted microscopic analyses of Mycobacterium smegmatis lacking the operon and observed a cell separation defect, while surface rigidity measured by atomic force microscopy was found to be increased. Whereas Rv1410 expressed in Lactococcus lactis did not confer drug resistance, deletion of the operon in Mycobacterium abscessus and M. smegmatis resulted in increased susceptibility toward vancomycin, novobiocin and rifampicin. A homology model of Rv1410 revealed a periplasmic loop as well as a highly conserved aspartate, which were found to be essential for the operon’s function. Interestingly, influx of the fluorescent dyes BCECF‐AM and calcein‐AM in de‐energized M. smegmatis cells was faster in the deletion mutant. Our results unambiguously show that elevated drug susceptibility in the deletion mutant is caused by increased drug influx through a defective mycobacterial cell envelope and not by drug efflux mediated by Rv1410.
Mycobacterium tuberculosis (Mtb) can withstand months of antibiotic treatment. An important goal of tuberculosis research is shortening the treatment to reduce the burden on patients, increase adherence to the drug regimen and thereby slow the spread of drug resistance. Inhibition of drug efflux pumps by small molecules has been advocated as a promising strategy to attack persistent Mtb and shorten therapy. Although mycobacterial drug efflux pumps have been broadly investigated, mechanistic studies are scarce. In this critical review, we shed light on drug efflux in its larger mechanistic context by considering the intricate interplay between membrane transporters annotated as drug efflux pumps, membrane energetics, efflux inhibitors and cell wall biosynthesis processes. We conclude that a great wealth of data on mycobacterial transporters is insufficient to distinguish by what mechanism they contribute to drug resistance. Recent studies suggest that some drug efflux pumps transport structural lipids of the mycobacterial cell wall and that the action of certain drug efflux inhibitors involves dissipation of the proton-motive force, thereby draining the energy source of all active membrane transporters. We propose recommendations on the generation and interpretation of drug efflux data to reduce ambiguities and promote assigning novel roles to mycobacterial membrane transporters.
The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.
Molecular research on mycobacteria relies on a multitude of tools for the genetic manipulation of these clinically important bacteria. However, a uniform set of vectors allowing for standardized cloning procedures is not available. Here, we developed a versatile series of mycobacterial vectors for gene deletion, complementation and protein production and purification. The vectors are compatible with fragment exchange (FX) cloning, a recently developed high-throughput cloning principle taking advantage of the type IIS restriction enzyme SapI and its capacity to generate sticky trinucleotide ends outside of its recognition sequence. FX cloning allows for the efficient cloning into an entry vector and the facile transfer of the sequenced insert into a variety of destination vectors. We generated a set of mycobacterial expression vectors spanning a wide range of expression strengths, tagging variants and selection markers to rapidly screen for the optimal expression construct in order to purify membrane proteins from the model organism Mycobacterium smegmatis. Further, we generated a series of suicide vectors containing two counterselection markers and used them to delete twenty genes encoding for potential drug efflux pumps in M. smegmatis. The vectors will further facilitate genetic and biochemical research on various mycobacterial species.
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