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Amaro, R. E.

Publications and source records attributed to Amaro, R. E..

At least 19 recordsLinked to original sources

A Unified 3D Generative Model for Synthesizable Structure-Based Drug Design

Traditional screening-based drug discovery is inherently limited by the astronomical scale of the chemical space. Generative modelling offers a compelling alternative to the classical search paradigm and enables rational, bottom-up design of novel and target-specific small molecules. However, its impact has been hampered by challenges in synthetic accessibility of the designed compounds and lack of large-scale experimental validation. Here, we introduce LDDM (Large Drug Discovery Model), a generative framework that supports a range of drug discovery tasks, including constrained and unconstrained docking, fragment linking and growing, and de novo design. We further introduce a programmable design algorithm that enables accurate design of synthetically accessible compounds satisfying various fine-grained objectives. We experimentally validated the designed or optimised ligands for five therapeutically relevant protein targets. In all cases, LDDM achieved high success rates, allowing us to identify molecules with confirmed binding affinity while synthesizing only a small number of generated compounds. The best designs were structurally characterised through NMR spectroscopy and X-ray crystallography, demonstrating high prediction accuracy. Overall, LDDM provides a scalable and flexible platform for the rapid and tailored design of small molecules and non-natural peptides for therapeutic applications.

bioinformatics↗

Condensate-Like Organization in Respiratory Aerosols Modulates the Dynamics of an Airborne Virus

The molecular behavior of viruses within respiratory aerosols plays a critical role in airborne disease transmission yet remains largely inaccessible to experimental characterization. Here, we use a billion-atom all-atom molecular dynamics simulation of a virus-laden respiratory aerosol to uncover how respiratory proteins, lipids, ions, and water collectively assemble around SARS-CoV-2, giving rise to structured microenvironments that influence viral stability and spike dynamics. We find that respiratory components rapidly evolve into heterogeneous networks characterized by protein-rich aggregates, patchy lipid assemblies, and spatially structured ion and water dynamics. These features create distinct microenvironments that constrain molecular transport and stabilize regions surrounding the virion. Within this crowded aerosol context, we observe sustained and selective interactions between aerosol components and the viral spike protein, including preferential recruitment of surfactant lipids and persistent coordination by divalent cations. These interactions modulate spike conformational dynamics, enhancing domain breathing motions and flexibility at key hinge regions while preserving a stable membrane anchor. Together, these observations reveal a condensate-like physical regime in which multivalent aerosol components coalesce into a soft, heterogeneous matrix that selectively modulates viral protein dynamics under extreme crowding. By framing virus-laden respiratory aerosols within this physical context, this work establishes an in situ molecular framework for understanding how aerosols influence viral persistence and offers a platform for exploring mechanisms relevant to airborne disease transmission and mitigation strategies. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/721971v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@4d0f60org.highwire.dtl.DTLVardef@12c9d1forg.highwire.dtl.DTLVardef@1ff6c29org.highwire.dtl.DTLVardef@15feec_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisRespiratory aerosols exhibit condensate-like physical properties that govern the evolution of the particle and modulate the behavior of airborne SARS-CoV-2.

biophysics↗

Structural Dynamics and Allosteric Communication of a SARS-Like Bat Coronavirus Spike Glycoprotein

SARS-like bat coronaviruses (CoVs) pose ongoing public health risks due to their zoonotic potential, making it important to understand the molecular pathways driving their evolution. We recently showed that SHC014-CoV can infect human cell lines in an ACE2-dependent manner after acquiring two spike ectodomain mutations (F294L and A835D). However, how the wild-type (WT) SHC014 spike differs dynamically from these mutants remains unclear. Here, we built fully glycosylated ectodomain models of WT and three mutants (F294L, A835D, and the double mutant, DM) and performed triplicate 1-s all-atom molecular dynamics (MD) simulations for each variant. The two mutations exhibit epistasis, altering structural rearrangements relative to WT. Notably, the DM receptor binding domain (RBD) begins sampling the open conformation in our conventional MD. At the atomic level, the DM spike mitigates the dense negative packing introduced by A835D through a salt-bridge network, while F294L disrupts {pi}-mediated interactions, together enhancing RBD opening propensity--critical for viral entry. Increased flexibility of the subdomain-2 "620-loop" further modulates DM RBD openness. Dynamical network analysis identified three allosteric communication pathways. In WT and F294L, "Pathway 1" forms the baseline route linking the 620-loop to the RBD, whereas in A835D and DM it extends to the FPPR, reshaping long-range communication. "Pathway 2" is conserved across variants but is most prominent in WT and F294L. "Pathway 3" appears only in A835D and DM, compensating for reduced communication along Pathway 2. Overall, this work provides an atomistic perspective on SHC014 molecular adaptation during host-to-host transmission and highlights mechanistic features that may inform future therapeutic and pandemic-preparedness efforts. Statement of SignificanceBat coronaviruses are an important source of future pandemic threats, but we still know little about how small genetic changes help them infect humans. In this study, we used detailed computer simulations to watch how tiny mutations in a bat coronavirus spike protein change its motion and shape. We found that two specific mutations work together to make the spike more likely to open--a step required for the virus to enter human cells. By revealing how these molecular changes increase infection potential, our work helps improve understanding of coronavirus evolution and may guide strategies to prepare for future outbreaks.

biophysics↗

pH-induced structural changes in SARS-CoV-2 spike variants

pH critically influences SARS-CoV-2 infectivity and stability by, for example, triggering pH-dependent conformational changes in the spike glycoprotein that can facilitate membrane fusion and viral entry into host cells. Using the emerging dynamical nonequilibrium molecular dynamics (D-NEMD) simulations approach, we investigated how biologically relevant pH shifts affect the functional dynamics of the fully glycosylated spike from the ancestral, Delta, and Omicron BA.1 variants of concern. For this, over 1100 nonequilibrium simulations were conducted to capture the pH-induced structural and dynamic changes that occur following transitions from physiological to acidic and alkaline conditions, with the former mimicking the low pH environment within endosomes, and the latter the high pH conditions accessible to nascent exhaled aerosols. D-NEMD reveals that pH changes trigger distinct, variant-specific conformational responses in key regions of the spike, including the receptor binding domain (RBD), fusion peptide proximal region (FPPR), and C-terminal domain (CTD). The ancestral spike shows broad pH sensitivity, characterised by directional motions and region-specific structural rearrangements that depend on the pH conditions. The spike of the Delta variant displays increased reactivity to alkaline pH, potentially explaining its reduced stability in alkaline aerosols. The spike of Omicron BA.1, in contrast, responds strongly to acidic conditions with spontaneous RBD opening and pronounced structural rearrangements in the FPPR and CTD. This behaviour aligns with this variants preference for endosomal entry and reduced reliance on TMPRSS2-mediated fusion. The Omicron BA.1 spike also shows increased resilience to alkaline pH, suggesting greater environmental stability. Our findings further emphasise the key role of glycans in spike activation, with glycan N234 stabilising the RBD "up" conformation during pH-induced transitions in Omicron under acidic conditions. These insights, together, highlight pH as a potential evolutionary pressure for SARS-CoV-2 and underscore the importance of glycosylation and environmental pH variability in shaping the behaviour of viral fusion proteins.

biochemistry↗

An unconventional HxD motif orchestrates coatomer-dependent coronavirus morphogenesis

Assembly of infectious coronaviruses requires spike (S) protein trafficking by host coatomer, typically via a dibasic signal in the S cytoplasmic tail. However, the human embecoviruses HKU1 and OC43, as well as the model virus MHV, lack this motif. Here we identify a conserved His-x-Asp (HxD) sequence that functions as an unconventional coatomer-binding signal. Structural and biochemical analyses show that the MHV HxD motif engages coatomer subunits through distinct conformations, while cellular imaging demonstrates its role in directing S to assembly sites with the viral M-protein. Disruption of HxD-coatomer interactions impairs S incorporation and provokes compensatory viral adaptations, including emergence of a canonical dibasic motif or mutations in M-protein. Electron microscopy further reveals profound alterations in virion surface architecture. These findings uncover HxD as a previously unrecognized coatomer-targeting motif, highlighting an unexpected flexibility in coronavirus assembly pathways and broadening understanding of the cellular machinery that shapes coronavirus morphogenesis.

biochemistry↗

Towards In Situ Dynamics of DNA-bound Full-Length p53 Tetramer

p53 is the most important tumor suppressor in humans as well as the most frequently mutated gene found in human cancers with [~]50% of all human tumors bearing p53 missense mutations that leave p53 inactive. Restoring the p53 activity proved to lead to tumor regression even in advanced tumors in mouse models-- and thus, is among the most attractive potential strategies for novel cancer therapy. Full-length p53 (fl-p53) consists of 393 residues and multiple domains; some folded and some disordered. Using crystal structures of folded domains and integrative molecular modelling techniques for disordered domains, we generated the first wild-type fl-p53 tetramer model bound to DNA. When solvated, the system size nears 500K atoms challenging extensive sampling. Using Anton2 supercomputer for microsecond-timescale simulations in explicit solvent and the rigorous Markov state model (MSM) framework, we elucidated the conformational landscape of wild-type p53 as well as two of the p53 hot-spot cancer mutants, Y220C and G245S, in a physiological DNA-bound, full-length tetramer context. In the simulated timescale, DNA-bound fl-p53 tetramer bent DNA and formed a compact complex with interactions between the N-terminal and DNA-binding domains (DBDs), and the C-terminal domains (CTDs) with DNA. WT fl-p53 tetramer also sampled a unique quaternary DBD organization not accessed by the cancer mutants. Free energy landscapes indicated differential dynamics for inner and outer p53 DBDs due to the dimer-dimer interface. The dynamics of the druggable L1/S3 pocket is also closely monitored. Ultimately the MSMs identified an underexplored loop 6 (L6) cryptic pocket and captured the effect of p53 tetramerization and cancer mutations. Significancep53, the most important tumor suppressor in humans, is found inactivated due to single point mutations in almost 50% of all human tumors. As restoring p53 activity is shown to achieve tumor regression even in advanced tumors in mice, there is a lot of interest in finding small molecules to reactivate p53 cancer mutants. p53 has 393 residues and is an intrinsically disordered protein with multiple unstructured and dynamic domains that are known to interact with and help the function of its folded domains. Adding another level of complexity, p53 forms a tetramer to bind DNA and start its transcriptional activity. Due to its high dynamicity, p53 evades full structural characterization leaving many open questions. We generated an integrative model weaving together structures of isolated p53 domains and explored it with molecular dynamics simulations at microsecond timescale. This allowed us to monitor various unresolved aspects with atomistic detail like the dynamics of druggable pockets, the interplay of p53 domains in the physiological complex and how they change in p53 cancer mutants compared to the wild-type p53. Our study provides insights into the structure and dynamics of a key tumor suppressor protein at its physiologically relevant state of full-length tetramer bound to DNA.

biophysics↗

seekrflow: Towards end-to-end automated simulation pipeline with machine-learned force fields for accelerated drug-target kinetic and ther-modynamic predictions

Accurate prediction of drug-target binding and unbinding kinetics and thermodynamics is essential for guiding drug discovery and lead optimization. However, traditional atomistic simulations are often too computationally expensive to capture rare events that govern ligand (un)binding. Several enhanced sampling methods exist to overcome these limitations, but they require extensive manual intervention and introduce variability and artifacts in free energy and kinetic estimates that limit high-throughput scalability. The present work introduces seekrflow, an automated multiscale milestoning simulation pipeline that streamlines the entire workflow from a single receptor-ligand input structure to kinetic and thermodynamic predictions in a single step. This integrated approach minimizes manual intervention, reduces computational overhead, and enhances the reproducibility and accuracy of kinetic and thermodynamic predictions. The accuracy and efficiency of the pipeline is demonstrated on multiple receptor-ligand complexes, including inhibitors of heat shock protein 90, threonine-tyrosine kinase, and the trypsin protein, with predicted kinetic parameters closely matching experimental estimates. seekrflow establishes a new benchmark for automated and high-throughput physics-based predictions of kinetics and thermodynamics. O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/669965v2_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@c4da23org.highwire.dtl.DTLVardef@1cd386eorg.highwire.dtl.DTLVardef@33f50corg.highwire.dtl.DTLVardef@11d1f5a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

N-Glycans Modulate HIV-1 Env Conformational Plasticity

Human immunodeficiency virus-1 (HIV-1) remains a global health crisis, with over 39 million people living with the virus and no effective vaccine available. Central to HIV infection and immune evasion is the envelope glycoprotein (Env), a heavily glycosylated class I fusion protein that mediates viral entry and is the sole immunogenic target. Despite the recent advancements provided by imaging techniques, the characterization of Envs structure and dynamics within its native membrane environment remains incomplete. Here, we present microsecond-long, all-atom molecular dynamics simulations of the full-length Env glycoprotein embedded in a biologically relevant lipid bilayer with a complete glycosylation profile. Our simulations reveal a pronounced tilting motion of Env relative to the membrane, with supporting evidence from cryo-electron tomography, which also captures Env tilting within the native membrane. Importantly, we identify a critical role for N-linked glycans at N88 and N611 in modulating the tilting transition. These findings highlight the dual role of Envs glycan shield as both a protective barrier against neutralizing antibodies and a structural modulator of conformational plasticity. While providing an atomically detailed view of Env in a native membrane environment and advancing the general understanding of its glycan shield and its vulnerabilities, this work also suggests a possible strategy to modulate Envs conformational plasticity.

biophysics↗

Conformational dynamics and activation of membrane-associated human Group IVA cytosolic phospholipase A2 (cPLA2)

Cytosolic phospholipase A2 (cPLA2) associates with membranes where it hydrolyzes phospholipids containing arachidonic acid to initiate an inflammatory cascade. All-atom molecular dynamics simulations were employed to understand the activation process when cPLA2 associates with the endoplasmic reticulum (ER) membrane of macrophages where it acts. We found that membrane association causes the lid region of cPLA2 to undergo a closed-to-open state transition that is accompanied by the sideways movement of loop 495-540, allowing the exposure of a cluster of lysine residues (K488, K541, K543, and K544), which binds the allosteric activator PIP2 in the membrane. The active site of the open form of cPLA2, containing the catalytic dyad residues S228 and D549, exhibited a three-fold larger cavity than the closed form of cPLA2 in aqueous solution. These findings provide mechanistic insight as to how cPLA2 ER membrane association promotes major transitions between conformational states critical to allosteric activation and enzymatic phospholipid hydrolysis.

biophysics↗

D614G reshapes allosteric networks and opening mechanisms of SARS-CoV-2 spikes

The SARS-CoV-2 spike glycoprotein binds human epithelial cells and enables infection through a key conformational transition that exposes its receptor binding domain (RBD). Experimental evidence indicates that spike mutations, particularly the early D614G variant, alter the rate of this conformational shift, potentially increasing viral infectivity. To investigate how mutations reshape the conformational landscape, we conducted extensive weighted ensemble simulations of the Ancestral, Delta, and Omicron BA.1 spike strains along the RBD opening pathway. We observe that Ancestral, Delta, and Omicron BA.1 spike RBDs open differently, with Omicron BA.1 following a more direct opening profile until it reaches a "super-open" state wherein it begins to "peel", suggesting increased S1 flexibility. Via dynamical network analysis, we identified two allosteric communication networks uniting all S1 domains: the established N2R linker and a newly discovered anti-parallel R2N linker. In Delta and Omicron BA.1 variant spikes, RBD opening is facilitated by both linkers, while the Ancestral strain relies predominantly on the N2R linker. In the ancestral spike, the D614-K854 salt bridge impedes allosteric communication through the R2N linker, whereas the loss of this salt bridge in all subsequent VOCs alleviates local frustration and, we believe, accelerates RBD opening. Hydrogen-deuterium mass spectrometry experiments validate these altered dynamics in the D614 region across Ancestral, D614G, and Omicron BA.1 spikes. This study unveils a hidden allosteric network, connecting the NTD to the RBD via the 614-proximal region, and the D614G mutation reshapes the fitness landscape of these critical viral glycoproteins. Significance StatementOur work reveals how the D614G mutation in the SARS-CoV-2 spike protein reshapes its internal communication pathways and speeds up receptor binding domain (RBD) opening, providing mechanistic insight into the evolution and enhanced infectivity of SARS-CoV-2 variants of concern. We also describe differences in opening pathways and relative rates of opening for Delta and Omicron BA.1 spike RBDs relative to the original (Ancestral) coronavirus strain from Wuhan, China.

biophysics↗

SARS CoV-2 spike adopts distinct conformational ensembles in situ

Engineered recombinant Spike (S) has been invaluable for determining S structure and dynamics and is the basis for the design of most prevalent vaccines. While these vaccines have been highly efficacious for short-term protection from infection, protection waned with the emergence of variants (alpha through omicron). Here we report differences in conformational dynamics between native, membrane-embedded full-length S and recombinant S. Our virus-like particle (VLP) model mimics the native SARS CoV-2 virion by displaying S assembled with auxiliary E, M, and N proteins in a native membrane environment that captures the entirety of quaternary interactions mediated by S. Display of S on VLP obviates the requirement for stabilizing modifications that have been engineered into recombinant S for enhanced expression and solubility. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) reveals altered interprotomer contacts in VLP S trimers attributable to the presence of auxiliary proteins, membrane anchoring, and lack of engineered modifications. Our results reveal decreased dynamics in the S2 subunit and at sites spanning interprotomer contacts in VLP S with minimal differences in the N-terminal domain (NTD) and receptor binding domain (RBD). This carries implications for display of epitopes beyond NTD and RBD. In summary, despite affording efficient structural characterization, recombinant S distorts the intrinsic conformational ensemble of native S displayed on the virus surface.

biophysics↗

Markovian State Models uncover Casein Kinase 1 dynamics that govern circadian period

Circadian rhythms in mammals are tightly regulated through phosphorylation of Period (PER) proteins by Casein Kinase 1 (CK1, subtypes {delta} and {varepsilon}). CK1 acts on at least two different regions of PER with opposing effects: phosphorylation of phosphodegron (pD) regions leads to PER degradation, while phosphorylation of the Familial Advanced Sleep Phase (FASP) region leads to PER stabilization. To investigate how substrate selectivity is encoded by the conformational dynamics of CK1, we performed a large set of independent molecular dynamics (MD) simulations of wildtype CK1 and the tau mutant (R178C) that biases kinase activity toward a pD. We used Markovian State Models (MSMs) to integrate the simulations into a single model of the conformational landscape of CK1 and used Gaussian accelerated molecular dynamics (GaMD) to build the first molecular model of CK1 and the unphosphorylated FASP motif. Together, these findings provide a mechanistic view of CK1, establishing how the activation loop acts as a key molecular switch to control substrate selectivity. We show that the tau mutant favors an alternative conformation of the activation loop and significantly accelerates the dynamics of CK1. This reshapes the binding cleft in a way that impairs FASP binding and would ultimately lead to PER destabilization and shorter circadian periods. Finally, we identified an allosteric pocket that could be targeted to bias this molecular switch. Our integrated approach offers a detailed model of CK1s conformational landscape and its relevance to normal, mutant, and druggable circadian timekeeping. Statement of SignificanceDisruption of circadian rhythms alters the temporal orchestration of vital cellular processes and increases the propensity for sleep disorders, metabolic disease, and cancer. Circadian rhythms are generated by a vast gene expression program controlled at the cellular level by a molecular clock comprised of dedicated clock proteins. Amongst the essential protein characters is Casein kinase 1 (CK1), which acts on multiple clock protein substrates. A delicate balance of CK1 activity on these substrates is crucial for proper circadian timekeeping, highlighting CK1 as a promising drug target to tune clock timing. This work aims to identify the conformational landscape of CK1 that underlies its substrate specificity and provide molecular insight for pharmacologic development that could modulate CK1 function for those suffering from clock-related syndromes.

biophysics↗

Regulatory interactions between APOBEC3B N- and C-terminal domains

APOBEC3B (A3B) is implicated in DNA mutations that facilitate tumor evolution. Although structures of its individual N- and C-terminal domains (NTD and CTD) have been resolved through X-ray crystallography, the full-length A3B (fl-A3B) structure remains elusive, limiting understanding of its dynamics and mechanisms. In particular, the APOBEC3B C-terminal domain (A3Bctd) active site is frequently closed in models and structures. In this study, we built several new models of fl-A3B using integrative structural biology methods and selected a top model for further dynamical investigation. We compared dynamics of the truncated (A3Bctd) to the fl-A3B via conventional and Gaussian accelerated molecular dynamics (MD) simulations. Subsequently, we employed weighted ensemble methods to explore the fl-A3B active site opening mechanism, finding that interactions at the NTD-CTD interface enhance the opening frequency of the fl-A3B active site. Our findings shed light on the structural dynamics of fl-A3B, which may offer new avenues for therapeutic intervention in cancer.

biophysics↗

An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4

Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the Endoplasmic Reticulum and is implicated in severe diseases such as various cancers and Alzheimers disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4s kinetics, catalytic parameters, substrate specificity, and binding modality we expressed and purified recombinant RHBDL4, and employed it in a Forster Resonance Energy Transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4s enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl -ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics (MD) simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4s active site that contributed to binding stability, offering valuable insights for optimizing the non-prime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4s catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.

biochemistry↗

Cryo-EM visualizes multiple steps of dynein activation pathway

Cytoplasmic dynein-1 (dynein) is an essential molecular motor controlled in part by autoinhibition. We recently identified a structure of partially autoinhibited dynein bound to Lis1, a key dynein regulator mutated in the neurodevelopmental disease lissencephaly. This structure provides an intermediate state in dyneins activation pathway; however, other structural information is needed to fully explain Lis1 function in dynein activation. Here, we used cryo-EM and samples incubated with ATP for different times to reveal novel conformations that we propose represent intermediate states in the dyneins activation pathway. We solved sixteen high-resolution structures, including seven distinct dynein and dynein-Lis1 structures from the same sample. Our data also support a model in which Lis1 relieves dynein autoinhibition by increasing its basal ATP hydrolysis rate and promoting conformations compatible with complex assembly and motility. Together, this analysis advances our understanding of dynein activation and the contribution of Lis1 to this process.

biophysics↗

Prediction of Threonine-Tyrosine Kinase Receptor-LigandUnbinding Kinetics with Multiscale Milestoning andMetadynamics

Accurately describing protein-ligand binding and unbinding kinetics remains challenging. Computational calculations are difficult and costly, while experimental measurements often lack molecular detail and can be unobtainable. Here we extend our multiscale milestoning method, Simulation-Enabled Estimation of Kinetics Rates (SEEKR), with metadynamics molecular dynamics simulations to yield accurate small molecule drug residence times. Using the pharmaceutically relevant threonine-tyrosine kinase (TTK) and eight long-residence-time (tens of seconds to hours) inhibitors, we demonstrate accurate prediction of absolute and rank-ordered ligand residence times and free energies of binding. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/606239v1_ufig1.gif" ALT="Figure 1"> View larger version (99K): org.highwire.dtl.DTLVardef@59d2dborg.highwire.dtl.DTLVardef@12244c4org.highwire.dtl.DTLVardef@d0a8a5org.highwire.dtl.DTLVardef@f01a4b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

Epistasis between N-terminal and receptor-binding domains drives cell entry in a bat coronavirus spike

Understanding the zoonotic risks posed by bat coronaviruses (CoVs) is critical for pandemic preparedness. Herein, we generated recombinant vesicular stomatitis viruses (rVSVs) bearing spikes from divergent bat CoVs to investigate their cell entry mechanisms. Unexpectedly, the successful recovery of rVSVs bearing the spike from SHC014, a SARS-like bat CoV, was associated with the acquisition of a novel substitution in the S2 fusion peptide-proximal region (FPPR). This substitution enhanced viral entry in both VSV and coronavirus contexts by increasing the availability of the spike receptor-binding domain to recognize its cellular receptor, ACE2. A second substitution in the spike N-terminal domain, uncovered through forward-genetic selection, interacted epistatically with the FPPR substitution to synergistically enhance spike:ACE2 interaction and viral entry. Our findings identify genetic pathways for adaptation by bat CoVs during spillover and host-to-host transmission, fitness trade-offs inherent to these pathways, and potential Achilles heels that could be targeted with countermeasures.

microbiology↗

Development of Allosteric Small Molecule APOBEC3B Inhibitors from In Silico Screening

The APOBEC3 (A3) family of enzymes are zinc metalloenzymes that catalyze the conversion of 2'- deoxycytidine to 2'-deoxyuridine in single stranded DNA. APOBEC3B (A3B), a member of the A3 family, has emerged as a key driver of genomic instability in many cancer types by mutating host DNA to drive tumorigenesis and therapy resistance. Small molecule inhibitors of A3B would extend the durability of current therapies by limiting mutations that promote tumor escape and therapy resistance. Consequently, a computer aided drug discovery (CADD) campaign was employed to identify inhibitors of A3B. Through molecular dynamics (MD) simulations and computational solvent mapping analysis, we identified a novel putative allosteric pocket on the c-terminal domain of A3B and virtually screened the ChemBridge Diversity Set ([~]110,000 small molecules) against both the active and predicted allosteric sites. High scoring compounds were selected for in vitro testing and triage, resulting in 13 candidate inhibitors. Using cysteine reactive probes, one of the original hit compounds was mapped to the computationally identified allosteric pocket. However, after resynthesis of representative chemotypes and further investigation using in vitro assays, none of the chemotypes retained inhibitory activity. Further analysis revealed that most of the inhibition observed in the primary assay was due to metal contamination in the screening sample, which resulted in the removal of the catalytic zinc from the enzyme active site. Although validated A3B inhibitors were not discovered in this study, we report a ligandable allosteric site on A3B and several cautionary insights for researchers developing small molecule inhibitors of zinc metalloenzymes. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/591187v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b7e577org.highwire.dtl.DTLVardef@13a1a43org.highwire.dtl.DTLVardef@192309eorg.highwire.dtl.DTLVardef@b60a31_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗