Clinical targeting of HIV capsid protein with a long-acting small molecule.

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1-5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7-9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV.

PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps.

Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA-bound proteins, and basic properties like the elementary step size remain controversial. Using single-molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single-stranded loop. Static disorder limited previous ensemble studies of a PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations, suggesting a mode of action for eliminating potentially deleterious recombination intermediates.

Long Dissociation of Bictegravir from HIV-1 Integrase-DNA Complexes.

The HIV integrase (IN) strand transfer inhibitor (INSTI) bictegravir (BIC) has a long dissociation half-life (t1/2) from wild-type IN-DNA complexes: BIC 163 hr > dolutegravir (DTG) 96 hr > raltegravir (RAL) 10 hr > elvitegravir (EVG) 3.3 hr. In cells, BIC had more durable antiviral activity against wild-type HIV after drug washout than RAL or EVG. BIC also had a longer t1/2 and maintained longer antiviral activity after drug washout than DTG with the clinically relevant resistance IN mutant G140S+Q148H. Structural analyses indicate that BIC makes more contacts with the IN-DNA complex than DTG mainly via its bicyclic ring system which may contribute to more prolonged residence time and resilience against many resistance mutations.

Characterization of a Novel Capsid Assembly Modulator for the Treatment of Chronic Hepatitis B Virus Infection.

The standard of care for the treatment of chronic hepatitis B (CHB) is typically lifelong treatment with nucleos(t)ide analogs (NAs), which suppress viral replication and provide long-term clinical benefits. However, infectious virus can still be detected in patients who are virally suppressed on NA therapy, which may contribute to the failure of these agents to cure most CHB patients. Accordingly, new antiviral treatment options are being developed to enhance the suppression of hepatitis B virus (HBV) replication in combination with NAs ("antiviral intensification"). Here, we describe GS-SBA-1, a capsid assembly modulator (CAM) belonging to class CAM-E, that demonstrates potent inhibition of extracellular HBV DNA in vitro (EC50 [50% effective concentration] = 19 nM) in HBV-infected primary human hepatocytes (PHHs) as well as in vivo in an HBV-infected immunodeficient mouse model. GS-SBA-1 has comparable activities across HBV genotypes and nucleos(t)ide-resistant mutants in HBV-infected PHHs. In addition, GS-SBA-1 demonstrated in vitro additivity in combination with tenofovir alafenamide (TAF). The administration of GS-SBA-1 to PHHs at the time of infection prevents covalently closed circular DNA (cccDNA) formation and, hence, decreases HBV RNA and antigen levels (EC50 = 80 to 200 nM). Furthermore, GS-SBA-1 prevents the production of extracellular HBV RNA-containing viral particles in vitro. Collectively, these data demonstrate that GS-SBA-1 is a potent CAM that has the potential to enhance viral suppression in combination with an NA.

Hepatitis B Virus X Protein Function Requires Zinc Binding.

The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.

Structure-guided discovery of a novel, potent, and orally bioavailable 3,5-dimethylisoxazole aryl-benzimidazole BET bromodomain inhibitor.

The bromodomain and extra-terminal (BET) family of proteins, consisting of the bromodomains containing protein 2 (BRD2), BRD3, BRD4, and the testis-specific BRDT, are key epigenetic regulators of gene transcription and has emerged as an attractive target for anticancer therapy. Herein, we describe the discovery of a novel potent BET bromodomain inhibitor, using a systematic structure-based approach focused on improving potency, metabolic stability, and permeability. The optimized dimethylisoxazole aryl-benzimidazole inhibitor exhibited high potency towards BRD4 and related BET proteins in biochemical and cell-based assays and inhibited tumor growth in two proof-of-concept preclinical animal models.

Antiviral Activity of Bictegravir (GS-9883), a Novel Potent HIV-1 Integrase Strand Transfer Inhibitor with an Improved Resistance Profile.

Bictegravir (BIC; GS-9883), a novel, potent, once-daily, unboosted inhibitor of HIV-1 integrase (IN), specifically targets IN strand transfer activity (50% inhibitory concentration [IC50] of 7.5 ± 0.3 nM) and HIV-1 integration in cells. BIC exhibits potent and selective in vitro antiretroviral activity in both T-cell lines and primary human T lymphocytes, with 50% effective concentrations ranging from 1.5 to 2.4 nM and selectivity indices up to 8,700 relative to cytotoxicity. BIC exhibits synergistic in vitro antiviral effects in pairwise combinations with tenofovir alafenamide, emtricitabine, or darunavir and maintains potent antiviral activity against HIV-1 variants resistant to other classes of antiretrovirals. BIC displayed an in vitro resistance profile that was markedly improved compared to the integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG), and comparable to that of dolutegravir (DTG), against nine INSTI-resistant site-directed HIV-1 mutants. BIC displayed statistically improved antiviral activity relative to EVG, RAL, and DTG against a panel of 47 patient-derived HIV-1 isolates with high-level INSTI resistance; 13 of 47 tested isolates exhibited >2-fold lower resistance to BIC than DTG. In dose-escalation experiments conducted in vitro, BIC and DTG exhibited higher barriers to resistance than EVG, selecting for HIV-1 variants with reduced phenotypic susceptibility at days 71, 87, and 20, respectively. A recombinant virus with the BIC-selected M50I/R263K dual mutations in IN exhibited only 2.8-fold reduced susceptibility to BIC compared to wild-type virus. All BIC-selected variants exhibited low to intermediate levels of cross-resistance to RAL, DTG, and EVG (<8-fold) but remained susceptible to other classes of antiretrovirals. A high barrier to in vitro resistance emergence for both BIC and DTG was also observed in viral breakthrough studies in the presence of constant clinically relevant drug concentrations. The overall virologic profile of BIC supports its ongoing clinical investigation in combination with other antiretroviral agents for both treatment-naive and -experienced HIV-infected patients.

Functional label-free assays for characterizing the in vitro mechanism of action of small molecule modulators of capsid assembly.

HIV capsid protein is an important target for antiviral drug design. High-throughput screening campaigns have identified two classes of compounds (PF74 and BI64) that directly target HIV capsid, resulting in antiviral activity against HIV-1 and HIV-2 laboratory strains. Using recombinant proteins, we developed a suite of label-free assays to mechanistically understand how these compounds modulate capsid activity. PF74 preferentially binds to the preassembled hexameric capsid form and prevents disruption of higher-order capsid structures by stabilizing capsid intersubunit interactions. BI64 binds only the monomeric capsid and locks the protein in the assembly incompetent monomeric form by disrupting capsid intersubunit interactions. We also used these assays to characterize the interaction between capsid and the host protein cleavage and polyadenylation specific factor 6 (CPSF6). Consistent with recently published results, our assays revealed CPSF6 activates capsid polymerization and preferentially binds to the preassembled hexameric capsid form similar to the small molecule compound, PF74. Furthermore, these label-free assays provide a robust method for facilitating the identification of a different class of small molecule modulators of capsid function.

GS-5806 inhibits pre- to postfusion conformational changes of the respiratory syncytial virus fusion protein.

GS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproduced in vitro using a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity.

Anti-Citrullinated Protein Antibodies With Multiple Specificities Ameliorate Collagen Antibody-Induced Arthritis in a Time-Dependent Manner.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA) and have long been regarded as pathogenic. Despite substantial in vitro evidence supporting this claim, reports investigating the proinflammatory effects of ACPAs in animal models of arthritis are rare and include mixed results. Here, we sequenced the plasmablast antibody repertoire of a patient with RA and functionally characterized the encoded ACPAs. METHODS: We expressed ACPAs from the antibody repertoire of a patient with RA and characterized their autoantigen specificities on antigen arrays and enzyme-linked immunosorbent assays. Binding affinities were estimated by bio-layer interferometry. Select ACPAs (n = 9) were tested in the collagen antibody-induced arthritis (CAIA) mouse model to evaluate their effects on joint inflammation. RESULTS: Recombinant ACPAs bound preferentially and with high affinity (nanomolar range) to citrullinated (cit) autoantigens (primarily histones and fibrinogen) and to auto-cit peptidylarginine deiminase 4 (PAD4). ACPAs were grouped for in vivo testing based on their predominant cit-antigen specificities. Unexpectedly, injections of recombinant ACPAs significantly reduced paw thickness and arthritis severity in CAIA mice as compared with isotype-matched control antibodies (P ≤ 0.001). Bone erosion, synovitis, and cartilage damage were also significantly reduced (P ≤ 0.01). This amelioration of CAIA was observed for all the ACPAs tested and was independent of cit-PAD4 and cit-fibrinogen specificities. Furthermore, disease amelioration was more prominent when ACPAs were injected at earlier stages of CAIA than at later phases of the model. CONCLUSION: Recombinant patient-derived ACPAs ameliorated CAIA. Their antiinflammatory effects were more preventive than therapeutic. This study highlights a potential protective role for ACPAs in arthritis.

New class of HIV-1 integrase (IN) inhibitors with a dual mode of action.

tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction.

Impact of primary elvitegravir resistance-associated mutations in HIV-1 integrase on drug susceptibility and viral replication fitness.

Elvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG.

Discovery of GS-5245 (Obeldesivir), an Oral Prodrug of Nucleoside GS-441524 That Exhibits Antiviral Efficacy in SARS-CoV-2-Infected African Green Monkeys.

Remdesivir 1 is an phosphoramidate prodrug that releases the monophosphate of nucleoside GS-441524 (2) into lung cells, thereby forming the bioactive triphosphate 2-NTP. 2-NTP, an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP. Here, we describe the discovery of a 5'-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3) that has low cellular cytotoxicity and 3-7-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved presystemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP, leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2, which supports development of 3 as a promising COVID-19 treatment.

A trimer of dimers is the basic building block for human immunodeficiency virus-1 capsid assembly.

Human immunodeficiency virus-1 (HIV-1) capsid protein (CA) has become a target of antiviral drug design in recent years. The recognition that binding of small molecules to the CA protein can result in the perturbation of capsid assembly or disassembly has led to mathematical modeling of the process. Although a number of capsid assembly models have been developed using biophysical parameters of the CA protein obtained experimentally, there is currently no model of CA polymerization that can be practically used to analyze in vitro CA polymerization data to facilitate drug discovery. Herein, we describe an equilibrium model of CA polymerization for the kinetic analysis of in vitro assembly of CA into polymer tubes. This new mathematical model has been used to assess whether a triangular trimer of dimers rather than a hexagonal hexamer can be the basic capsomere building block of CA polymer. The model allowed us to quantify for the first time the affinity for each of the four crucial interfaces involved in the polymerization process and indicated that the trimerization of CA dimers is a relatively slow step in CA polymerization in vitro. For wild-type CA, these four interfaces include the interface between two monomers of a CA dimer (K(D) = 6.6 µM), the interface between any two dimers within a CA trimer of dimers (K(D) = 32 nM), and two types of interfaces between neighboring trimers of dimers, either within the same ring around the perimeter of the polymer tube (K(D) = 438 nM) or from two adjacent rings (K(D) = 147 nM). A comparative analysis of the interface dissociation constants between wild-type and two mutant CA proteins, cross-linked hexamer (A14C/E45C/W184A/M185A) and A14C/E45C, yielded results that are consistent with the trimer of dimers with a triangular geometry being the capsomere building block involved in CA polymer growth. This work provides additional insights into the mechanism of HIV-1 CA assembly and may prove useful in elucidating how small molecule CA binding agents may disturb this essential step in the HIV-1 life cycle.

Self-assembly of Escherichia coli MutL and its complexes with DNA.

The Escherichia coli MutL protein regulates the activity of several enzymes, including MutS, MutH, and UvrD, during methyl-directed mismatch repair of DNA. We have investigated the self-association properties of MutL and its binding to DNA using analytical sedimentation velocity and equilibrium. Self-association of MutL is quite sensitive to solution conditions. At 25 °C in Tris at pH 8.3, MutL assembles into a heterogeneous mixture of large multimers. In the presence of potassium phosphate at pH 7.4, MutL forms primarily stable dimers, with the higher-order assembly states suppressed. The weight-average sedimentation coefficient of the MutL dimer in this buffer ( ̅s(20,w)) is equal to 5.20 ± 0.08 S, suggesting a highly asymmetric dimer (f/f(o) = 1.58 ± 0.02). Upon binding the nonhydrolyzable ATP analogue, AMPPNP/Mg(2+), the MutL dimer becomes more compact ( ̅s(20,w) = 5.71 ± 0.08 S; f/f(o) = 1.45 ± 0.02), probably reflecting reorganization of the N-terminal ATPase domains. A MutL dimer binds to an 18 bp duplex with a 3'-(dT(20)) single-stranded flanking region, with apparent affinity in the micromolar range. AMPPNP binding to MutL increases its affinity for DNA by a factor of ∼10. These results indicate that the presence of phosphate minimizes further MutL oligomerization beyond a dimer and that differences in solution conditions likely explain apparent discrepancies in previous studies of MutL assembly.

Dithiothreitol causes HIV-1 integrase dimer dissociation while agents interacting with the integrase dimer interface promote dimer formation.

We have developed a homogeneous time-resolved fluorescence resonance energy transfer (FRET)-based assay that detects the formation of HIV-1 integrase (IN) dimers. The assay utilizes IN monomers that express two different epitope tags that are recognized by their respective antibodies, coupled to distinct fluorophores. Surprisingly, we found that dithiothreitol (DTT), a reducing agent essential for in vitro enzymatic activity of IN, weakened the interaction between IN monomers. This effect of DTT on IN is dependent on its thiol groups, since the related chemical threitol, which contains hydroxyls in place of thiols, had no effect on IN dimer formation. By studying mutants of IN, we determined that cysteines in IN appear to be dispensable for the dimer dissociation effect of DTT. Peptides derived from the IN binding domain (IBD) of lens epithelium derived growth factor/transcriptional coactivator p75 (LEDGF), a cellular cofactor that interacts with the IN dimer interface, were tested in this IN dimerization assay. These peptides, which compete with LEDGF for binding to IN, displayed an intriguing equilibrium binding dose-response curve characterized by a plateau rising to a peak, then descending to a second plateau. Mathematical modeling of this binding system revealed that these LEDGF-derived peptides promote IN dimerization and block subunit exchange between IN dimers. This dose-response behavior was also observed with a small molecule that interacts with the IN dimer interface and inhibits LEDGF binding to IN. In conclusion, this novel IN dimerization assay revealed that peptide and small molecule inhibitors of the IN-LEDGF interaction also stabilize IN dimers and promote their formation.

Discovery of Potent and Selective MTH1 Inhibitors for Oncology: Enabling Rapid Target (In)Validation.

We describe the discovery of three structurally differentiated potent and selective MTH1 inhibitors and their subsequent use to investigate MTH1 as an oncology target, culminating in target (in)validation. Tetrahydronaphthyridine 5 was rapidly identified as a highly potent MTH1 inhibitor (IC50 = 0.043 nM). Cocrystallization of 5 with MTH1 revealed the ligand in a Φ-cis-N-(pyridin-2-yl)acetamide conformation enabling a key intramolecular hydrogen bond and polar interactions with residues Gly34 and Asp120. Modification of literature compound TH287 with O- and N-linked aryl and alkyl aryl substituents led to the discovery of potent pyrimidine-2,4,6-triamine 25 (IC50 = 0.49 nM). Triazolopyridine 32 emerged as a highly selective lead compound with a suitable in vitro profile and desirable pharmacokinetic properties in rat. Elucidation of the DNA damage response, cell viability, and intracellular concentrations of oxo-NTPs (oxidized nucleoside triphosphates) as a function of MTH1 knockdown and/or small molecule inhibition was studied. Based on our findings, we were unable to provide evidence to further pursue MTH1 as an oncology target.

The KN-93 Molecule Inhibits Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity by Binding to Ca2+/CaM.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93-based mechanism-of-action studies and drug discovery efforts.

A highly potent long-acting small-molecule HIV-1 capsid inhibitor with efficacy in a humanized mouse model.

People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options1-3. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently4. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection.

Regulation of UvrD Helicase Activity by MutL.

Escherichia coli UvrD is a superfamily 1 helicase/translocase involved in multiple DNA metabolic processes including methyl-directed mismatch DNA repair. Although a UvrD monomer can translocate along single-stranded DNA, a UvrD dimer is needed for processive helicase activity in vitro. E. coli MutL, a regulatory protein involved in methyl-directed mismatch repair, stimulates UvrD helicase activity; however, the mechanism is not well understood. Using single-molecule fluorescence and ensemble approaches, we find that a single MutL dimer can activate latent UvrD monomer helicase activity. However, we also find that MutL stimulates UvrD dimer helicase activity. We further find that MutL enhances the DNA-unwinding processivity of UvrD. Hence, MutL acts as a processivity factor by binding to and presumably moving along with UvrD to facilitate DNA unwinding.