Biology subjects

Kirby, K. A.

Publications and source records attributed to Kirby, K. A..

12 recordsLinked to original sources

bioRxiv2026

Structural and Mechanistic Basis of F227C-Mediated Hypersusceptibility to Islatravir in HIV-1 Reverse Transcriptase

Islatravir (ISL; 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA)), a first-in-class nucleoside reverse transcriptase (RT) translocation inhibitor (NRTTI), was recently approved by the FDA for the treatment of HIV-1 infection in combination with the non-nucleoside RT inhibitor (NNRTI), doravirine (DOR). Notably, the RT mutation F227C, which confers clinical resistance to multiple NNRTIs, including DOR, unexpectedly increases susceptibility to ISL. To elucidate the mechanistic basis of this hypersusceptibility, we determined a 1.8 angstrom crystal structure of F227C RT in complex with a ddGMP-terminated primer/template and ISL-triphosphate. The structure reveals conformational rearrangements that propagate into a cleft, thereby affecting ATP-mediated unblocking of chain-terminating antivirals. Complementary biochemical assays showed that although F227C does not significantly affect ISL incorporation, it alters RT translocation and impairs ATP-dependent phosphorolytic excision of ISL-terminated primers, thereby enhancing ISL susceptibility. These findings establish direct structural and mechanistic links between NNRTI resistance and ISL hypersusceptibility, providing a structural foundation for rationally designed, resistance-informed combination regimens that exploit this unique collateral sensitivity.

biochemistry↗

bioRxiv2026

Unraveling the Mechanism of HIV-1 Hypersusceptibility to Tenofovir Imparted by Islatravir Resistance Mutations

In response to the newly approved antiretroviral therapy (ART) islatravir (ISL), the M184V and A114S resistance mutations have emerged in the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). These mutations markedly hypersensitize RT to the globally administered ART tenofovir disoproxil fumarate (TDF). We have solved six structures - four by X-ray crystallography and two by cryo-EM - that capture the single- and double-mutant RTs during inhibitor incorporation and demonstrate the role of the mutations in altering protein-antiviral interactions. These snapshots reveal that the smaller, more flexible TDF diphosphate (TDF-DP) can better accommodate mutation-induced active site changes than ISL triphosphate (ISL-TP). Structural differences between the two inhibitors are consistent with biochemical determination of inhibitory constants (Kis), highlighting differences at the step of inhibitor incorporation. Virological evaluation of ISL and TDF combinations reveals additive inhibition of HIV-1. Given the converse ISL hypersusceptibility imparted by the TDF-resistant K65R mutation, we propose ISL and TDF as a combination that can inspire future therapeutic options.

biochemistry↗

bioRxiv2026

Structural Basis of Polypurine Track Strand Displacement by HIV-1 Reverse Transcriptase

To complete reverse transcription, HIV-1 reverse transcriptase (RT) must displace the RNase H-resistant polypurine tract (PPT) primers. This enables synthesis of the long terminal repeats and formation of the central cDNA flap. However, the molecular mechanism of this PPT strand displacement (SD) has remained unknown, and no structural data exist on how a retroviral polymerase execute these reactions. We report the first cryo-EM structures of HIV-1 RT bound to nucleic acid substrates containing either a PPTRNA or PPTDNA displacement strand, with incoming dATP positioned at the polymerase catalytic site. These structures reveal key features of the PPT displacement mechanism by RT. Specifically, we observed a binding mode where the template nucleotide (T1) base-paired to the first displacement nucleotide (D1) undergoes a 90{degrees} rotation relative to the preceding template base (T0). This sharp template flip positions D1 [~]30 [A] away from the primers 3-end and is coordinated by RTp66 residues at the SD interface: F61 and R78 contact T1/T0 to drive template translocation, while W24 engages both T1 and D1 to stabilize the displacement strand. Biochemical and virological mutagenesis experiments confirm that interactions with F61 and R78 are essential for both canonical cDNA polymerization and SD, whereas the W24-nucleotide interactions are required exclusively for SD but are dispensable for standard cDNA synthesis. These results contribute to the structural and functional understanding of PPT strand displacement by HIV-1 RT and reveal a distinct mechanistic vulnerability for the design of next-generation antiretrovirals.

biochemistry↗

bioRxiv2026

HIV-1 Reverse Transcriptase interactions with Long-acting NNRTI, Depulfavirine (VM1500A)

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are key components of combination antiretroviral therapy (ART) for the treatment of human immunodeficiency virus type 1 (HIV-1) infection, binding an allosteric pocket of reverse transcriptase (RT) and inhibiting viral replication. Although second-generation NNRTIs have improved potency and resistance profiles compared to first-generation NNRTIs, the continued emergence of resistant viral strains and the need for long-acting therapeutic options underscore the importance of developing next-generation compounds. Depulfavirine (VM1500A) is a potent NNRTI being developed as a long-acting formulation. Its prodrug, elsulfavirine (ESV), is approved for HIV-1 treatment in Eurasian countries as a once-daily oral regimen and has demonstrated favorable antiviral efficacy, pharmacokinetics, and tolerability in clinical studies. Here, we report the 2.4 [A] crystal structure of HIV-1 RT in complex with depulfavirine, revealing an extended binding conformation within the NNRTI pocket that reaches from the back of the binding pocket to the entrance. These interactions may shed light on mechanisms of resistance to the F227C mutation, with and without V106 substitution, and Y188L. Notably, depulfavirine maintains potent inhibition of common NNRTI-resistant RT variants, including K103N and Y181C. Combination studies of ESV with antivirals from diverse inhibitor categories demonstrated additive or near-synergistic activity with islatravir (ISL), cabotegravir (CAB), lenacapavir (LEN), and tenofovir (TDF). These findings highlight the broad resistance profile and potential of the depulfavirine combination.

biochemistry↗

bioRxiv2026

Damaging the conical morphology of HIV-1 capsid by targeting the FG-binding pocket and disfavoring pentameric subunits needed for core closure

The HIV-1 capsid is an essential viral component, targeted by the long-acting antiretroviral Lenacapavir (LEN). LEN binds to the HIV-1 capsid protein (CA) at the phenylalanine-glycine (FG) binding pocket (FGBP), a site for multiple host-factor and antiviral interactions in CA hexamers (CAHEX). Previously, we generated a chemical library to investigate the FGBP; ZW-1261, a lead compound, exhibits potent antiviral activity and strong inter-subunit interactions within CAHEX. Here, we report the molecular mechanism by which ZW-1261 affects the morphology and integrity of capsid lattice. ZW-1261 alone rapidly induces tubular CA assemblies; simultaneous addition of ZW-1261 with the assembly cofactor inositol hexaphosphate (IP6) forms morphologically distinct tubes. In mature virions, IP6 is required for the assembly of both CAHEX and CA pentamers (CAPENT). Cryogenic-electron microscopy analysis of in vitro assembled capsid-like particles (CLPs) with IP6 suggests that ZW-1261 leads to the absence of CAPENT and damages the pre-formed conical lattice. To elucidate how this FGBP-targeting antiviral impacts CAPENT, we further solved structures of CAPENT-only icosahedral assemblies (T = 1), formed by reported mutations, that were treated with ZW-1261. We find that ZW-1261 binding in these constrained T = 1 assemblies converts CAPENT to a CAHEX-like conformation. Collectively, this suggests a mechanism by which addition of FGBP-binding inhibitor to native cores leads to the absence of CAPENT, impacting capsid closure and core integrity.

molecular biology↗

bioRxiv2025

Structural, biophysical, and virological mechanistic characterization of HIV-1 capsid-targeting antivirals

Due to its significant role in virus replication, the HIV capsid is an attractive antiviral target. This is validated by the recent clinical approval of lenacapavir for both treatment and pre-exposure prophylaxis (PrEP). PF74 is a well-characterized capsid-targeting antiviral that was discontinued in further study due to potency and metabolic issues. We hypothesized that making chemical modifications at certain sites of PF74 could result in capsid-targeting antivirals with improved potency and bioavailability. Our cumulative studies show that making changes at the R1 and R3 positions of PF74 results in compounds with increased antiviral potency, increased stability of wild-type HIV capsid hexamers and virions, tighter binding to wild-type HIV capsid hexamer compared to PF74, and different interactions at the "FG" binding site of capsid compared to PF74. These data provide insights into the design of future capsid-targeting antivirals relevant for clinical use.

biochemistry↗

bioRxiv2025

Design, synthesis and profiling of highly potent antivirals targeting emerging drug-resistant HIV-1 variants

AbstractLenacapavir (LEN), the first-in-class HIV capsid inhibitor (CAI), is approved by FDA as a long acting injectable (LAI) for both treatment and pre-exposure prophylaxis (PrEP). Despite its exceptional potency and long pharmacokinetics (PK), a few major resistant mutations have been selected in LEN-treated patients, underscoring the need to develop second-generation LEN analogs to mitigate resistance. Particularly, the M66I mutation confers an extraordinarily high-level LEN resistance, essentially abrogating LEN potency. In this work, we have designed and synthesized LEN analogs featuring a cycloalkyl R2 in subunit B drastically different from known analogs. Against wild-type HIV-1, the potency of our analog 3 (EC50 = 0.073 nM) was 2.6-fold higher than LEN (EC50 = 0.19 nM). More importantly, against the M66I mutant, 3 (EC50 = 5.8 nM) was decisively more potent than LEN (EC50 > 15 M) or any known analogs. We have also shown that the size of the R2 cycloalkyl ring is a major pharmacophore factor as a smaller (cyclopropyl, analog 1) or bigger (cyclohexyl, analog 4) ring confers weaker antiviral potency against both WT HIV-1 and M66I. The vastly improved profile of our lead 3 against M66I was confirmed in the target binding thermal shift assay. These results strongly validate our design and may represent a breakthrough in LEN-based HIV therapy and prophylaxis.

microbiology↗

bioRxiv2025

Translocation of HIV capsid core through the Nuclear Pore Complex by affinity gradient

The HIV capsid core encapsulates the viral genome for subsequent integration into host cellular DNA. Prior to nuclear entry, the core must translocate through the Nuclear Pore Complex (NPC). This transit involves interactions between the capsid core and phenylalanine-glycine (FG) repeats found in nucleoporins within the NPC. Despite this critical role in the viral replication cycle, the molecular mechanism of capsid core translocation remains unclear. FG repeats consist of three classes of canonical motifs: FG, GLFG, and FxFG motifs. These are segregated within the NPC to define distinct zones of the gating machinery. FG- and FxFG-type motifs are enriched in the cytoplasmic and nuclear ("nuclear basket") peripheries of the NPC while GLFG motifs are enriched in regions adjacent to the core of the NPC. To investigate the capsid core translocation, we use biochemical, biophysical, and structural approaches to study FG-capsid interactions in a quantitative manner. We show that the capsid (CA) interacts with a range of diverse FG repeats with varying affinities. GLFG motifs of core NUP98 exhibit increased affinity to CA proteins compared to other conventional FG/FxFG. However, the non-canonical FxFG motif of NUP153 at the "nuclear basket" significantly increases binding affinity to CA compared to canonical FxFG, therefore called FG super-motif. In addition, C-terminal motif of NUP153 consists of a stretch of basic residues, which enhances the affinity of this non-canonical FG super-motif to capsid core at the NPC nuclear periphery. We identified other binding enhancers of the NPC core FG-NUPs, NUP58 and POM121. The relationship between the binding strength of FG/FxFG binding enhancers of NUP58, POM121, NUP153 and their position within the NPC also shows capsid core binding affinity increases with increasing proximity to the "nuclear basket." Based on our data, the difference in binding affinities between the canonical FxFG motif and the enhancer-coupled FG super-motif of NUP153 to capsid cores at the "nuclear basket" is approximately 1,000-fold. Therefore, the diverse FG motifs and binding enhancers, which are naturally distributed within the NPC into distinct zones, create an avidity gradient--with changes in both concentration and binding affinity--along the cytoplasmic-nuclear axis. We suggest that HIV capsid translocation into the nucleus is potentiated by this gradient in a unidirectional manner (outside- to-inside) within the NPC.

microbiology↗

bioRxiv2025

Characterization of antiviral compounds using Bio-Layer Interferometry

Small molecule-protein interactions underpin many biological functions and play an integral role in the treatment and prevention of several human diseases. These interactions can be key to understanding the mechanism of action of these compounds. Previous methods of determining protein-protein or protein-antibody interactions have been well established; however, the use of BLI in antiviral discovery is a promising and relatively new avenue. The high-throughput nature of this method in tandem with its pM sensitivity allows for quick and seamless identification of hit compounds. Here we discuss ways to overcome common pitfalls that can occur while using BLI such as nonspecific binding (NSB) and ligand drift while offering possible solutions. Characterizing small molecule-protein interactions is not trivial and optimizing the experimental conditions is imperative. To address this gap in knowledge, we present optimized BLI protocols for the study of three cases of protein-small molecule interactions: PF74 or Lenacapavir (LEN) with HIV-1 capsid protein (CA), and Nirmatrelvir (NIR) with SARS-CoV-2 Mpro. LEN and NIR are of particular interest because they are clinically relevant, and PF74, a well-studied control, was the first compound reported to target the LEN binding site. We demonstrate that BLI can be a powerful and effective tool in calculating the binding affinities between a protein and small molecule. These newly designed methods enabled calculation of KD values, the affinity between ligand and analyte, ranging from the micro to the sub-nanomolar range for CA binding events and confirmed the covalent interaction between NIR and Mpro. These protocols will facilitate efficient testing of new antivirals or derivatives in a high- throughput format. SummaryBio-Layer Interferometry (BLI) is a multifunctional technology that is used to determine valuable information on real-time kinetics including association and dissociation. Optimizing experimental conditions to acquire data about protein-ligand interactions can be challenging. We provide three example methods of collecting binding data that characterize how viral proteins interact with antivirals.

biochemistry↗

bioRxiv2024

Disruption of LEDGF/p75-directed integration derepresses antisensetranscription of the HIV-1 genome

AbstractDisruption of HIV-1 Integrase (IN) interactions with the host-factor Lens Epithelium-Derived Growth Factor (LEDGF)/p75 leads to decreased, random integration, increased latent infection, and described here, accumulation of HIV-1 antisense RNA (asRNA). asRNA increase was observed following interruptions of IN-LEDGF/p75 interactions either through pharmacologic perturbations of IN-LEDGF/p75 by treatment with allosteric HIV-1 integrase inhibitors (ALLINIs) or in cell lines with LEDGF genetic knockout. Additionally, by impairing Tat-dependent HIV transcription, asRNA abundance markedly increases. Illumina sequencing characterization of asRNA transcripts in primary T cells infected in the presence of ALLINIs showed that most initiate from within the HIV-1. Overall, loss of IN-LEDGF/p75 interactions increase asRNA abundance. Understanding the relationship between ALLINIs, integration sites, asRNA, and latency could aid in future therapeutic strategies.

microbiology↗

bioRxiv2023

Use of TSAR, Thermal Shift Analysis in R, to identify Folic Acid as a Molecule that Interacts with HIV-1 Capsid

The thermal shift assay (TSA) is a versatile biophysical technique for studying protein-ligand interactions in vitro. Here, we report a free, open-source software tool, TSAR ("Thermal Shift Analysis in R"), to expedite the analysis of TSA data. The TSAR package incorporates multiple workflows that facilitate TSA analyses, returns publication-ready graphics, and includes an optional graphic user interface. The package is available at https://bioconductor.org/packages/TSAR/. Applying TSAR, we screened two chemical libraries and found multiple molecules that potentially interact in vitro with the capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1). First, a library of vitamins exemplifies the different graphic outputs of TSAR, and we report a change in the 50% melting temperature ({Delta}Tm) for folic acid-treated CA hexamers (CAHEX). Since HIV-1 CAHEX interacts with host-derived acids like inositol hexaphosphate (IP6) or dNTPs, a second library was screened containing 96 organic, acidic metabolites; multiple anionic ligands caused a {Delta}Tm for CAHEX. Subsequent investigation of these interactions includes biolayer interferometry, antiviral activity against pseudotyped HIV-1, and endogenous reverse transcriptase assays that were used to validate and investigate the biological impact of these native ligands that thermally-stabilize CAHEX. One compound hit, gallic acid, exhibited anti-HIV-1 activity as previously reported, and we show interacts with CAHEX as a potentially novel mechanism. Overall, the TSAR package facilitated quick analysis of TSA data from multiple libraries to help identify a biologically relevant hit, gallic acid, as a molecule that can inhibit HIV-1 replication and targets CAHEX. ImportanceThe TSAR package is freely available (AGPL-3) and is designed for both experienced or new R users, having command-line code for handling large and challenging datasets while also including an optional GUI that enables easy use by non-programmers. This is the first TSA analysis program written in R, a free and open-source language; TSAR simplifies TSA analysis while maintaining diverse visualization options for small-to-large libraries and multidimensional analysis. Additionally, we report multiple endogenous metabolites that potentially interact with the HIV-1 capsid protein hexamers (CAHEX) in vitro, including folic acid, gallic acid, and multiple others, primarily from the tricarboxylic acid (TCA) cycle. Various methods validate gallic acid interactions with CAHEX, leading to a novel suggested mechanism of action, and in-line with previous reports, this metabolite has potential for natural-based treatments of HIV-1. Further, we advance the understanding of the potential mechanism(s) for HIV-1 inhibition by gallic acid treatment.

biochemistry↗

bioRxiv2021

Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication

Replicon-based technologies were used to develop reagents and assays for advanced drug discovery efforts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and for examining all facets of the SARS-CoV-2 replication cycle at reduced biocontainment level. Specifically: a) 21 replicons were cloned in bacterial artificial chromosomes (BACs) and delivered as transfectable plasmid DNA or transcribed RNA in various cell types. Replicons carrying mutations that affect the activity or antiviral susceptibility of SARS-CoV-2 enzymes were used to establish utility for mechanistic studies while reducing the community risks associated with gain-of-function studies in fully infectious virus. b) A BHK-21 stable cell line harboring SARS-CoV-2 replicon was generated and characterized in robust high/ultra-high throughput assays of antiviral efficacy with orthogonal SARS-CoV-2 replication reporter genes (Nano luciferase and enhanced green fluorescent protein-eGFP); the estimated antiviral potencies in the fully infectious SARS-CoV-2 system and in the transient or stable replicon systems were similar. HEK293 and Calu1 stable cell lines expressing SARS-CoV-2 replicon have also been prepared. Finally, c) we generated trans-encapsidated replicons by co-expression with SARS-CoV-2 structural proteins, thus producing single-round infectious SARS-CoV-2 virus-like particles that are able to transduce susceptible cell types and have expanded utility to enable study of virion assembly and entry into target cells. Hence, these SARS-CoV-2 replicon-based reagents include a novel approach to replicon-harboring cell line generation and are valuable tools that can be used at lower biosafety level (BSL2) for drug discovery efforts, characterization of SARS-CoV-2 and variant evolution in the COVID-19 pandemic, mechanisms of inhibition and resistance, and studies on the role of SARS-CoV-2 genes and host dependency factors.

microbiology↗