Biology subjects

Chavez, A.

Publications and source records attributed to Chavez, A..

28 records · Page 2Linked to original sources

bioRxiv2022

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful in reducing hospitalization or death due to COVID-191,2. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in increasing concentrations of nirmatrelvir using two independent approaches, including one on a large scale in 480 wells. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done at a larger scale with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones, each containing a unique mutation or a combination of mutations showed that the above precursor mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Structural explanations are discussed for some of the mutations that are proximal to the drug-binding site, as well as cross-resistance or lack thereof to ensitrelvir, another clinically important 3CL protease inhibitor. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next generation protease inhibitors.

microbiology↗

bioRxiv2022

The Functional Landscape of SARS-CoV-2 3CL Protease

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) as the etiologic agent of COVID-19 (coronavirus disease 2019) has drastically altered life globally. Numerous efforts have been placed on the development of therapeutics to treat SARS-CoV-2 infection. One particular target is the 3CL protease (3CLpro), which holds promise as it is essential to the virus and highly conserved among coronaviruses, suggesting that it may be possible to find broad inhibitors that treat not just SARS-CoV-2 but other coronavirus infections as well. While the 3CL protease has been studied by many groups for SARS-CoV-2 and other coronaviruses, our understanding of its tolerance to mutations is limited, knowledge which is particularly important as 3CL protease inhibitors become utilized clinically. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the SARS-CoV-2 3CLpro, and validate our results both in yeast and in authentic viruses. We reveal that the 3CLpro is highly malleable and is capable of tolerating mutations throughout the protein, including within the substrate binding pocket. Yet, we also identify specific residues that appear immutable for function of the protease, suggesting that these interactions may be novel targets for the design of future 3CLpro inhibitors. Finally, we utilize our screening results as a basis to identify E166V as a resistance-conferring mutation against the therapeutic 3CLpro inhibitor, nirmatrelvir, in clinical use. Collectively, the functional map presented herein may serve as a guide for further understanding of the biological properties of the 3CL protease and for drug development for current and future coronavirus pandemics.

microbiology↗

bioRxiv2021

Isolation and comparative analysis of antibodies that broadly neutralize sarbecoviruses

The devastation caused by SARS-CoV-2 has made clear the importance of pandemic preparedness. To address future zoonotic outbreaks due to related viruses in the sarbecovirus subgenus, we identified a human monoclonal antibody, 10-40, that neutralized or bound all sarbecoviruses tested in vitro and protected against SARS-CoV-2 and SARS-CoV in vivo. Comparative studies with other receptor-binding domain (RBD)-directed antibodies showed 10-40 to have the greatest breadth against sarbecoviruses and thus its promise as an agent for pandemic preparedness. Moreover, structural analyses on 10-40 and similar antibodies not only defined an epitope cluster in the inner face of the RBD that is well conserved among sarbecoviruses, but also uncovered a new antibody class with a common CDRH3 motif. Our analyses also suggested that elicitation of this class of antibodies may not be overly difficult, an observation that bodes well for the development of a pan-sarbecovirus vaccine. One sentence summaryA monoclonal antibody that neutralizes or binds all sarbecoviruses tested and represents a reproducible antibody class.

microbiology↗

bioRxiv2021

Interrogation of genome-wide, experimentally dissected gene regulatory networks reveals mechanisms underlying dynamic cellular state control

Pooled CRISPRi-mediated silencing of >1,000 transcriptional regulators expressed in single colorectal adenocarcinoma cells, followed by single-cell RNA-seq profiling at two timepoints, 1 day and 4 days, allowed reverse engineering the underlying tumor context-specific, causal regulatory network. Furthermore, the availability of experimentally derived, highly multiplexed gene reporter assays for each regulator, as identified by this analysis, allowed accurate assessment of differential protein activity following silencing of each regulator, thus providing proof-of-concept for generating comprehensive, tissue-specific networks of transcriptional and post-translational interactions. Analysis of this causal network allowed elucidation of complex autoregulatory mechanisms that have eluded previous computational approaches and supported systematic elucidation of cooperative mechanisms, where one regulatory protein can modulate the activity of another regulatory protein, as well as transcriptional mimicry, where one regulatory protein can phenocopy others.

systems biology↗

bioRxiv2020

A simplified cell-based assay to identify coronavirus 3CL protease inhibitors

We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease (3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the utility of our approach by performing a curated compound screen along with structure-activity profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these studies, we observed concordance between data emerging from this assay and from live virus assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that will inevitably arise.

microbiology↗

bioRxiv2020

Lead compounds for the development of SARS-CoV-2 3CL protease inhibitors

We report the identification of three structurally diverse compounds - compound 4, GC376, and MAC-5576 - as inhibitors of the SARS-CoV-2 3CL protease. Structures of each of these compounds in complex with the protease revealed strategies for further development, as well as general principles for designing SARS-CoV-2 3CL protease inhibitors. These compounds may therefore serve as leads for the basis of building effective SARS-CoV-2 3CL protease inhibitors.

microbiology↗

bioRxiv2018

Rational design of a compact CRISPR-Cas9 activator for AAV- mediated delivery

Akin to Zinc Finger and Transcription Activator Like Effector based transcriptional modulators, nuclease-null CRISPR-Cas9 provides a groundbreaking programmable DNA binding platform, begetting an arsenal of targetable regulators for transcriptional and epigenetic perturbation, by either directly tethering, or recruiting, transcription enhancing effectors to either component of the Cas9/guide RNA complex. Application of these programmable regulators is now gaining traction for the modulation of disease-causing genes or activation of therapeutic genes, in vivo. Adeno-Associated Virus (AAV) is an optimal delivery vehicle for in vivo delivery of such regulators to adult somatic tissue, due to the efficacy of viral delivery with minimal concerns about immunogenicity or integration. However, present Cas9 activator systems are notably beyond the packaging capacity of a single AAV delivery vector capsid. Here, we engineer a compact CRISPR-Cas9 activator for convenient AAV-mediated delivery. We validate efficacy of the CRISPR-Cas9 transcriptional activation using AAV delivery in several cell lines.

synthetic biology↗

bioRxiv2018

Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks.

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double strand break (DSB), Cas9 remains stably bound to it. Here we show persistent Cas9 binding blocks access to DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches one direction towards the Cas9-DSB complex. By exploiting these RNA polymerase-Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of PAM sequences and a simple method of improving selection of highly active sgRNA for genome editing.

molecular biology↗

bioRxiv2018

Accurate analysis of genuine CRISPR editing events with ampliCan

We present ampliCan, an analysis tool for genome editing that unites highly precise quantification and visualization of genuine genome editing events. ampliCan features nuclease-optimized alignments, filtering of experimental artifacts, event-specific normalization, off-target read detection and quantifies insertions, deletions, HDR repair as well as targeted base editing. It is scalable to thousands of amplicon sequencing-based experiments from any genome editing experiment, including CRISPR. It enables automated integration of controls and accounts for biases at every step of the analysis. We benchmarked ampliCan on both real and simulated datasets against other leading tools, demonstrating that it outperformed all in the face of common confounding factors.

bioinformatics↗

bioRxiv2017

High-throughput creation and functional profiling of eukaryotic DNA sequence variant libraries using CRISPR/Cas9

Construction of genetic variant libraries with phenotypic measurement is central to advancing todays functional genomics, and remains a grand challenge. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions and insertions), along with methods for tracking their fitness en masse. We demonstrate the utility of our approach in performing focused analysis of hundreds of mutants of a single protein and in investigating the biological function of an entire family of poorly characterized genetic elements. Our platform allows fundamental biology questions to be investigated in a quick, easy and affordable manner.

bioengineering↗