Biology subjects

Ruiz, F. X.

Publications and source records attributed to Ruiz, F. X..

7 recordsLinked to original sources

bioRxiv2026

Discovery of non-nucleoside inhibitors of the enterovirus D68 3D polymerase through crystallographic fragment and high-throughput biochemical screening

Enterovirus D68 (EV-D68) is a non-polio picornavirus that has caused increasing rates of severe respiratory illness and acute flaccid myelitis in children worldwide this century. There are no approved vaccines or antivirals for EV-D68. Thus, we conducted a crystallographic fragment screening (CFS) and a high-throughput screening (HTS) biochemical assay against the EV-D68 RNA-dependent RNA polymerase 3D (3Dpol) to identify ligandable sites and non-nucleoside compounds that can spearhead anti-enteroviral drug discovery. The CFS, involving 650 fragments, identified 68 hit compounds (~10% hit rate) distributed across 3Dpol, including the functionally relevant sites RNA template channel, Active site, and RNA primer channel, and the previously unknown "Thumb site II" and "Index-middle finger pocket". Inhibition assays confirmed that compounds binding to each site can inhibit EV-D68 3Dpol activity. The HTS, a fluorescence-based PicoGreen biochemical assay, permitted screening 50,000 compounds of the ChemBridge Premium Library (0.77% hit rate). After a second-round dose-response screening, we identified 5-aminoindazole as a promising scaffold that inhibits EV-D68 3Dpol, including hit-to-lead compound 727590, which displayed an IC50 value of 25 M and preliminary structure-activity relationships. These hits offer amenable starting points for discovery and development of non-nucleoside inhibitors and provide opportunities for structure-based drug design against enteroviruses. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/737532v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1a7cb78org.highwire.dtl.DTLVardef@11f4bb9org.highwire.dtl.DTLVardef@1a477e0org.highwire.dtl.DTLVardef@ba5391_HPS_FORMAT_FIGEXP M_FIG Created with biorender.com and PyMOL Molecular Graphics System, version 2.5.0. Schrodinger, LLC. C_FIG

biophysics↗

bioRxiv2025

Crystallographic fragment screening of the dengue virus polymerase reveals multiple binding sites for the development of non-nucleoside antiflavivirals

Dengue viruses (DENV) infect approximately 400 million people each year, and there are currently no effective therapeutics available. To explore potential starting points for antiviral drug development, we conducted a large-scale crystallographic fragment screen targeting the RNA-dependent RNA polymerase (RdRp) domain of the non-structural protein 5 (NS5) from DENV serotype 2. Our screening, which involved 1,108 fragments, identified 60 hit compounds across various known binding sites, including the active site, N pocket, and RNA tunnel. Additionally, we discovered a novel binding site and a fragment-binding hotspot in thumb site II. These structural findings open amenable avenues for developing non-nucleoside inhibitors and offer valuable insights for future structure-based drug design aimed at DENV and other flaviviral RdRps.

biophysics↗

bioRxiv2022

The Halo Library, a Tool for Rapid Identification of Ligand Binding Sites on Proteins Using Crystallographic Fragment Screening

X-ray crystallographic fragment screening (XCFS) uses fragment-sized molecules ([~]60 - 300 Da) to access binding sites on proteins that may be inaccessible to larger drug-like molecules (>300 Da). Previous studies from our lab and others have shown that fragments containing halogen atoms have a higher binding occurrence compared to non-halogenated fragments. Specifically, we showed that 4-halopyrazoles hold potential for predicting the likelihood of success of a XCFS campaign. Here, we designed the Halo Library containing 46 halogenated fragments (including the "universal fragment" 4-bromopyrazole). The basis of fragment selection was presence of (at least) one halogen atom, and binding to or inhibitory activity against (at least) two targets in literature. The library was screened against crystals of HIV-1 reverse transcriptase with drug rilpivirine, yielding an overall hit rate of 26%. Two new binding sites were discovered in addition to previously reported sites, and several hot spots were identified (i.e., sites with multiple fragment hits). This small library may thus provide a convenient tool for assessing feasibility of a target for XCFS, mapping hot spots and cryptic sites, as well as finding fragment binders that can be useful for developing drug leads.

biochemistry↗

bioRxiv2022

Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro

SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its [~]30 kb RNA genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of cleavages as: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering yielded three-dimensional models of the nsp7-10/11 polyprotein. Our data suggest that the nsp7- 10/11 structure in complex with Mpro strongly resembles the unbound polyprotein, and that both polyprotein conformation and junction accessibility determine the preference and order of cleavages. Finally, we used limited proteolysis assays to characterize the effect of a series of inhibitors/binders on Mpro processing of nsp7-11 and Mpro inhibition using a polyprotein substrate. TeaserWe elucidated the structural basis of order of cleavage of SARS-CoV-2 nsp7-11 polyprotein, with implications for Mpro inhibition.

biochemistry↗

bioRxiv2021

Cryo-EM Structure of the Pol Polyprotein Provides Insights into HIV Maturation

Many retroviral proteins are initially translated from unspliced full-length RNA as polyprotein precursors that are subsequently processed by the viral protease (PR) to yield the mature forms. In HIV-1, the enzymes, PR, reverse transcriptase (RT), and integrase (IN), are produced as part of the Gag-Pol polyprotein. While structures of the mature proteins have aided our understanding of catalytic mechanisms and the design of antiretroviral drugs, knowledge of the architecture and functional implications of the immature forms prior to PR-mediated cleavage is limited. We developed a system to produce and purify the HIV-1 Pol polyprotein intermediate precursor and determined its high-resolution cryo-EM structure. The RT portion of the polyprotein has an architecture similar to the mature RT p66/p51 heterodimer, and dimerization of the RT portion draws together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate the assembly and maturation of the polyprotein precursors.

biophysics↗

bioRxiv2021

Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

Coronavirus (CoV) non-structural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase (RdRp) and other nsps. To date, no structure of full-length SARS-CoV-2 nsp7:nsp8 complex has been published. Current understanding of this complex is based on structures from truncated constructs or with missing electron densities and complexes from related CoV species with which SARS-CoV-2 nsp7 and nsp8 share upwards of 90% sequence identity. Despite available structures being solved using crystallography and cryo-EM representing detailed snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the complex and how it assembles to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex. The results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without altering nsp7-nsp8 interactions.

biophysics↗

bioRxiv2020

Crystal structure of prototype foamy virus (PFV) protease-reverse transcriptase fusion (PR-RT) reveals conformational plasticity: implications for function

In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs), express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 [A] resolution crystal structure of the mature PR-RT from prototype FV (PFV) that carries out both proteolytic processing and reverse transcription. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins. IMPORTANCERetroviruses such as HIV and other human and animal pathogens have evolved to maximize the protein-coding capacity of their genomes. Retroviruses synthesize large polyprotein precursors that are subsequently cleaved to produce mature functional enzymes and structural proteins from the same mRNA template. These polyproteins play an important role in the viral life cycle. We report the monomeric structure of the prototype foamy virus protease-reverse transcriptase fusion (PR-RT) which serves as both a protease and reverse transcriptase. The monomeric PR domain is folded in a similar way as a single subunit of dimeric HIV-1 PR. PR is anchored close to the fingers subdomain of the RT by a long C-terminal extension (CTE) that folds into two helices that pack against the palm subdomain of the RT. The RT is folded similarly to the p51 subunit of HIV-1 RT with the RNase H subdomain packed against the thumb and opposite the connection and palm subdomains. This structure offers insight into the structural arrangement of the domains and subdomains of immature retroviral polyproteins and provides support for the concept of developing drugs that target interfaces between immature enzymes that are present only in retroviral polyprotein precursors.

biophysics↗