102 results found
Maertens G, Barski MS, Vanzo T, et al., 2021, Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures, Nature Communications, Vol: 12, Pages: 1-10, ISSN: 2041-1723
Between 10 and 20 million people worldwide are infected with the human T-celllymphotropic virus type 1 (HTLV-1). Despite causing life-threateningpathologies there is no therapeutic regimen for this deltaretrovirus. Here, wescreened a library of integrase strand transfer inhibitor (INSTI) candidates builtaround several chemical scaffolds to determine their effectiveness in limitingHTLV-1 infection. Naphthyridines with substituents in position 6 emerged as themost potent compounds against HTLV-1, with XZ450 having highest efficacy invitro. Using single-particle cryo-electron microscopy we visualised XZ450 aswell as the clinical HIV-1 INSTIs raltegravir and bictegravir bound to the activesite of the deltaretroviral intasome. The structures reveal subtle differences inthe coordination environment of the Mg2+ ion pair involved in the interactionwith the INSTIs. Our results elucidate the binding of INSTIs to the HTLV-1intasome and support their use for pre-exposure prophylaxis and possiblyfuture treatment of HTLV-1 infection.
Maertens GN, Engelman AN, Cherepanov P, 2021, Structure and function of retroviral integrase, Nature Reviews Microbiology, ISSN: 1740-1526
A hallmark of retroviral replication is establishment of the proviral state, wherein a DNA copy of the viral RNA genome is stably incorporated into a host cell chromosome. Integrase is the viral enzyme responsible for the catalytic steps involved in this process, and integrase strand transfer inhibitors are widely used to treat people living with HIV. Over the past decade, a series of X-ray crystallography and cryogenic electron microscopy studies have revealed the structural basis of retroviral DNA integration. A variable number of integrase molecules congregate on viral DNA ends to assemble a conserved intasome core machine that facilitates integration. The structures additionally informed on the modes of integrase inhibitor action and the means by which HIV acquires drug resistance. Recent years have witnessed the development of allosteric integrase inhibitors, a highly promising class of small molecules that antagonize viral morphogenesis. In this Review, we explore recent insights into the organization and mechanism of the retroviral integration machinery and highlight open questions as well as new directions in the field.
Davis C, Logan N, Tyson G, et al., 2021, Reduced neutralisation of the Delta (B.1.617.2) SARS-CoV-2 variant of concern following vaccination
<jats:title>Abstract</jats:title><jats:p>Vaccines are proving to be highly effective in controlling hospitalisation and deaths associated with SARS-CoV-2 infection but the emergence of viral variants with novel antigenic profiles threatens to diminish their efficacy. Assessment of the ability of sera from vaccine recipients to neutralise SARS-CoV-2 variants will inform the success of strategies for minimising COVID19 cases and the design of effective antigenic formulations. Here, we examine the sensitivity of variants of concern (VOCs) representative of the B.1.617.1 and B.1.617.2 (first associated with infections in India) and B.1.351 (first associated with infection in South Africa) lineages of SARS-CoV-2 to neutralisation by sera from individuals vaccinated with the BNT162b2 (Pfizer/BioNTech) and ChAdOx1 (Oxford/AstraZeneca) vaccines. Across all vaccinated individuals, the spike glycoproteins from B.1.617.1 and B.1.617.2 conferred reductions in neutralisation of 4.31 and 5.11-fold respectively. The reduction seen with the B.1.617.2 lineage approached that conferred by the glycoprotein from B.1.351 (South African) variant (6.29-fold reduction) that is known to be associated with reduced vaccine efficacy. Neutralising antibody titres elicited by vaccination with two doses of BNT162b2 were significantly higher than those elicited by vaccination with two doses of ChAdOx1. Fold decreases in the magnitude of neutralisation titre following two doses of BNT162b2, conferred reductions in titre of 7.77, 11.30 and 9.56-fold respectively to B.1.617.1, B.1.617.2 and B.1.351 pseudoviruses, the reduction in neutralisation of the delta variant B.1.617.2 surpassing that of B.1.351. Fold changes in those vaccinated with two doses of ChAdOx1 were 0.69, 4.01 and 1.48 respectively. The accumulation of mutations in these VOCs, and others, demonstrate the quantifiable risk of antigenic drift and subsequent reduction in vaccine efficacy. Accordingly, booster vaccines b
Smith SJ, Zhao XZ, Passos DO, et al., 2021, HIV-1 Integrase Inhibitors with Modifications That Affect Their Potencies against Drug Resistant Integrase Mutants, ACS INFECTIOUS DISEASES, Vol: 7, Pages: 1469-1482, ISSN: 2373-8227
Rosa A, Pye VE, Graham C, et al., 2021, SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity, SCIENCE ADVANCES, Vol: 7, ISSN: 2375-2548
Rees-Spear C, Muir L, Griffith SA, et al., 2021, The effect of spike mutations on SARS-CoV-2 neutralization, CELL REPORTS, Vol: 34, ISSN: 2211-1247
Alrubayyi A, Gea-Mallorquí E, Touizer E, et al., 2021, Characterization of humoral and SARS-CoV-2 specific T cell responses in people living with HIV., Res Sq
There is an urgent need to understand the nature of immune responses against SARS-CoV-2, to inform risk-mitigation strategies for people living with HIV (PLWH). We show that the majority of PLWH, controlled on ART, mount a functional adaptive immune response to SARS-CoV-2. Humoral and SARS-CoV-2-specific T cell responses are comparable between HIV-positive and negative subjects and persist 5-7 months following predominately mild COVID-19 disease. T cell responses against Spike, Membrane and Nucleocapsid are the most prominent, with SARS-CoV-2-specific CD4 T cells outnumbering CD8 T cells. We further show that the overall magnitude of SARS-CoV-2-specific T cell responses relates to the size of the naive CD4 T cell pool and the CD4:CD8 ratio in PLWH, in whom disparate antibody and T cell responses are observed. These findings suggest that inadequate immune reconstitution on ART, could hinder immune responses to SARS-CoV-2 with implications for the individual management and vaccine effectiveness in PLWH.
Hughes EC, Amat JAR, Haney J, et al., 2021, Severe Acute Respiratory Syndrome Coronavirus 2 Serosurveillance in a Patient Population Reveals Differences in Virus Exposure and Antibody-Mediated Immunity According to Host Demography and Healthcare Setting, JOURNAL OF INFECTIOUS DISEASES, Vol: 223, Pages: 971-980, ISSN: 0022-1899
El Bouzidi K, Pirani T, Rosadas C, et al., 2021, Severe Acute Respiratory Syndrome Coronavirus-2 Infections in Critical Care Staff: Beware the Risks Beyond the Bedside, CRITICAL CARE MEDICINE, Vol: 49, Pages: 428-436, ISSN: 0090-3493
Alrubayyi A, Gea-Mallorquí E, Touizer E, et al., 2021, Characterization of humoral and SARS-CoV-2 specific T cell responses in people living with HIV., bioRxiv
There is an urgent need to understand the nature of immune responses generated against SARS-CoV-2, to better inform risk-mitigation strategies for people living with HIV (PLWH). Although not all PLWH are considered immunosuppressed, residual cellular immune deficiency and ongoing inflammation could influence COVID-19 disease severity, the evolution and durability of protective memory responses. Here, we performed an integrated analysis, characterizing the nature, breadth and magnitude of SARS-CoV-2-specific immune responses in PLWH, controlled on ART, and HIV negative subjects. Both groups were in the convalescent phase of predominately mild COVID-19 disease. The majority of PLWH mounted SARS-CoV-2 Spike- and Nucleoprotein-specific antibodies with neutralizing activity and SARS-CoV-2-specific T cell responses, as measured by ELISpot, at levels comparable to HIV negative subjects. T cell responses against Spike, Membrane and Nucleocapsid were the most prominent, with SARS-CoV-2-specific CD4 T cells outnumbering CD8 T cells. Notably, the overall magnitude of SARS-CoV-2-specific T cell responses related to the size of the naive CD4 T cell pool and the CD4:CD8 ratio in PLWH, in whom disparate antibody and T cell responses were observed. Both humoral and cellular responses to SARS-CoV-2 were detected at 5-7 months post-infection, providing evidence of medium-term durability of responses irrespective of HIV serostatus. Incomplete immune reconstitution on ART and a low CD4:CD8 ratio could, however, hamper the development of immunity to SARS-CoV-2 and serve as a useful tool for risk stratification of PLWH. These findings have implications for the individual management and potential effectiveness of vaccination against SARS-CoV-2 in PLWH. One Sentence Summary: Adaptive immune responses to SARS-CoV-2 in the setting of HIV infection.
Graham C, Seow J, Huettner I, et al., 2021, Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS-CoV-2 Spike., bioRxiv
The interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, the N-terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike-reactive monoclonal antibodies from SARS-CoV-2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD-specific. None of the S2-specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD-specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD-specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.
Balestrini S, Koepp MJ, Gandhi S, et al., 2021, Clinical outcomes of COVID-19 in long-term care facilities for people with epilepsy, EPILEPSY & BEHAVIOR, Vol: 115, ISSN: 1525-5050
Rosa A, Pye VE, Graham C, et al., 2021, SARS-CoV-2 recruits a haem metabolite to evade antibody immunity., medRxiv
The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that the virus co-opts the haem metabolite for the evasion of humoral immunity via allosteric shielding of a sensitive epitope and demonstrate the remarkable structural plasticity of the NTD.
Ng KW, Faulkner N, Cornish GH, et al., 2020, Preexisting and de novo humoral immunity to SARS-CoV-2 in humans, Science, Vol: 370, Pages: 1339-1343, ISSN: 0036-8075
Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detected preexisting humoral immunity. SARS-CoV-2 spike glycoprotein (S)–reactive antibodies were detectable using a flow cytometry–based method in SARS-CoV-2–uninfected individuals and were particularly prevalent in children and adolescents. They were predominantly of the immunoglobulin G (IgG) class and targeted the S2 subunit. By contrast, SARS-CoV-2 infection induced higher titers of SARS-CoV-2 S–reactive IgG antibodies targeting both the S1 and S2 subunits, and concomitant IgM and IgA antibodies, lasting throughout the observation period. SARS-CoV-2–uninfected donor sera exhibited specific neutralizing activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes. Distinguishing preexisting and de novo immunity will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.
O'Nions J, Muir L, Zheng J, et al., 2020, SARS-CoV-2 antibody responses in patients with acute leukaemia, LEUKEMIA, Vol: 35, Pages: 289-292, ISSN: 0887-6924
Barski M, Minnell J, Pye V, et al., 2020, Cryo-EM structure of the deltaretroviral intasome in complex with the PP2A regulatory subunit B56γ, Nature Communications, Vol: 11, ISSN: 2041-1723
Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.
Flower B, Brown JC, Simmons B, et al., 2020, Clinical and laboratory evaluation of SARS-CoV-2 lateral flow assays for use in a national COVID-19 sero-prevalence survey, Thorax, Vol: 75, Pages: 1082-1088, ISSN: 0040-6376
BackgroundAccurate antibody tests are essential to monitor the SARS-CoV-2 pandemic. Lateral flow immunoassays (LFIAs) can deliver testing at scale. However, reported performance varies, and sensitivity analyses have generally been conducted on serum from hospitalised patients. For use in community testing, evaluation of finger-prick self-tests, in non-hospitalised individuals, is required.MethodsSensitivity analysis was conducted on 276 non-hospitalised participants. All had tested positive for SARS-CoV-2 by RT-PCR and were ≥21d from symptom-onset. In phase I we evaluated five LFIAs in clinic (with finger-prick) and laboratory (with blood and sera) in comparison to a) PCR-confirmed infection and b) presence of SARS-CoV-2 antibodies on two “in-house” ELISAs. Specificity analysis was performed on 500 pre-pandemic sera. In phase II, six additional LFIAs were assessed with serum.Findings95% (95%CI [92.2, 97.3]) of the infected cohort had detectable antibodies on at least one ELISA. LFIA sensitivity was variable, but significantly inferior to ELISA in 8/11 assessed. Of LFIAs assessed in both clinic and laboratory, finger-prick self-test sensitivity varied from 21%-92% vs PCR-confirmed cases and 22%-96% vs composite ELISA positives. Concordance between finger-prick and serum testing was at best moderate (kappa 0.56) and, at worst, slight (kappa 0.13). All LFIAs had high specificity (97.2% - 99.8%).InterpretationLFIA sensitivity and sample concordance is variable, highlighting the importance of evaluations in setting of intended use. This rigorous approach to LFIA evaluation identified a test with high specificity (98.6% (95%CI [97.1, 99.4])), moderate sensitivity (84.4% with fingerprick (95%CI [70.5, 93.5])), and moderate concordance, suitable for seroprevalence surveys.
Dick SD, Federico S, Hughes SM, et al., 2020, Structural basis for the activation and target site specificity of CDC7kinase, Structure, Vol: 28, Pages: 954-962.e4, ISSN: 0969-2126
CDC7 is an essential Ser/Thr kinase that acts upon the replicative helicase throughout the S phase of the cell cycle and is activated by DBF4. Here, we present crystal structures of a highly active human CDC7-DBF4 construct. The structures reveal a zinc-finger domain at the end of the kinase insert 2 that pins the CDC7 activation loop to motif M of DBF4 and the C lobe of CDC7. These interactions lead to ordering of the substrate-binding platform and full opening of the kinase active site. In a co-crystal structure with a mimic of MCM2 Ser40 phosphorylation target, the invariant CDC7 residues Arg373 and Arg380 engage phospho-Ser41 at substrate P+1 position, explaining the selectivity of the S-phase kinase for Ser/Thr residues followed by a pre-phosphorylated or an acidic residue. Our results clarify the role of DBF4 in activation of CDC7 and elucidate the structural basis for recognition of its preferred substrates.
Houlihan CF, Vora N, Byrne T, et al., 2020, Pandemic peak SARS-CoV-2 infection and seroconversion rates in London frontline health-care workers, LANCET, Vol: 396, Pages: E6-E7, ISSN: 0140-6736
Aitken J, Ambrose K, Barrell S, et al., 2020, Scalable and robust SARS-CoV-2 testing in an academic center (vol 47, pg 613, 2020), NATURE BIOTECHNOLOGY, Vol: 38, Pages: 1000-1000, ISSN: 1087-0156
Engelman AN, Cherepanov P, 2020, Close-up: HIV/SIV intasome structures shed new light on integrase inhibitor binding and viral escape mechanisms, FEBS JOURNAL, Pages: 1-7, ISSN: 1742-464X
Integrase strand transfer inhibitors (INSTIs) are important components of drug formulations that are used to treat people living with HIV, and second‐generation INSTIs dolutegravir and bictegravir impart high barriers to the development of drug resistance. Reported 10 years ago, X‐ray crystal structures of prototype foamy virus (PFV) intasome complexes explained how INSTIs bind integrase to inhibit strand transfer activity and provided initial glimpses into mechanisms of drug resistance. However, comparatively low sequence identity between PFV and HIV‐1 integrases limited the depth of information that could be gleaned from the surrogate model system. Recent high‐resolution structures of HIV‐1 intasomes as well as intasomes from a closely related strain of simian immunodeficiency virus (SIV), which were determined using single‐particle cryogenic electron microscopy, have overcome this limitation. The new structures reveal the binding modes of several advanced INSTI compounds to the HIV/SIV integrase active site and critically inform the structural basis of drug resistance. These findings will help guide the continued development of this important class of antiretroviral therapeutics.
Aitken J, Ambrose K, Barrell S, et al., 2020, Scalable and robust SARS-CoV-2 testing in an academic center, Nature Biotechnology, Vol: 38, Pages: 927-931, ISSN: 1087-0156
Cook NJ, Li W, Berta D, et al., 2020, Structural basis of second-generation HIV integrase inhibitor action and viral resistance, Science, Vol: 367, Pages: 806-810, ISSN: 0036-8075
Despite worldwide prescription, the mechanistic basis for superiority of second-generation HIV integrase (IN) strand transfer inhibitors (INSTIs) is poorly understood. We use single-particle cryo-electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near atomic resolution. Q148H/G140S amino acid substitutions in IN that pervade clinical INSTI failure perturb optimal magnesium ion coordination in the enzyme active site. The expanded chemical scaffolds of second-generation compounds mediate interactions with the protein backbone, which are critical for antagonizing Q148H/G140S mutant virus. Our results reveal that binding to magnesium ions underpins a fundamental weakness of the INSTI pharmacophore that is exploited by the virus to engender resistance and provide a structural framework for the development of this important class of anti-HIV/AIDS therapeutics.
Pye VE, Rosa A, Bertelli C, et al., 2020, A bipartite structural organization defines the SERINC family of HIV-1 restriction factors, Nature Structural and Molecular Biology, Vol: 27, Pages: 78-83, ISSN: 1545-9985
The human integral membrane protein SERINC5 potently restricts HIV-1 infectivity and sensitizes the virus to antibody-mediated neutralization. Here, using cryo-EM, we determine the structures of human SERINC5 and its orthologue from Drosophila melanogaster at subnanometer and near-atomic resolution, respectively. The structures reveal a novel fold comprised of ten transmembrane helices organized into two subdomains and bisected by a long diagonal helix. A lipid binding groove and clusters of conserved residues highlight potential functional sites. A structure-based mutagenesis scan identified surface-exposed regions and the interface between the subdomains of SERINC5 as critical for HIV-1-restriction activity. The same regions are also important for viral sensitization to neutralizing antibodies, directly linking the antiviral activity of SERINC5 with remodeling of the HIV-1 envelope glycoprotein.
Wilson MD, Renault L, Maskell DP, et al., 2019, Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer, Nature Communications, Vol: 10, ISSN: 2041-1723
Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the histone octamer at the site of integration. To clarify the mechanism of DNA looping by integrase, we determined a 3.9 Å resolution structure of the prototype foamy virus intasome engaged with a nucleosome core particle. The structural data along with complementary single-molecule Förster resonance energy transfer measurements reveal twisting and sliding of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA by approximately two base pairs along the histone octamer accommodates the necessary DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for nucleosomal DNA repositioning, bearing unexpected similarities to chromatin remodelers.
Jang S, Cook NJ, Pye VE, et al., 2019, Differential role for phosphorylation in alternative polyadenylation function versus nuclear import of SR-like protein CPSF6, Nucleic Acids Research, Vol: 47, Pages: 4663-4683, ISSN: 0305-1048
Cleavage factor I mammalian (CFIm) complex, composed of cleavage and polyadenylation specificity factor 5 (CPSF5) and serine/arginine-like protein CPSF6, regulates alternative polyadenylation (APA). Loss of CFIm function results in proximal polyadenylation site usage, shortening mRNA 3' untranslated regions (UTRs). Although CPSF6 plays additional roles in human disease, its nuclear translocation mechanism remains unresolved. Two β-karyopherins, transportin (TNPO) 1 and TNPO3, can bind CPSF6 in vitro, and we demonstrate here that while the TNPO1 binding site is dispensable for CPSF6 nuclear import, the arginine/serine (RS)-like domain (RSLD) that mediates TNPO3 binding is critical. The crystal structure of the RSLD-TNPO3 complex revealed potential CPSF6 interaction residues, which were confirmed to mediate TNPO3 binding and CPSF6 nuclear import. Both binding and nuclear import were independent of RSLD phosphorylation, though a hyperphosphorylated mimetic mutant failed to bind TNPO3 and mislocalized to the cell cytoplasm. Although hypophosphorylated CPSF6 largely supported normal polyadenylation site usage, a significant number of mRNAs harbored unnaturally extended 3' UTRs, similar to what is observed when other APA regulators, such as CFIIm component proteins, are depleted. Our results clarify the mechanism of CPSF6 nuclear import and highlight differential roles for RSLD phosphorylation in nuclear translocation versus regulation of APA.
Bellelli R, Belan O, Pye VE, et al., 2018, POLE3-POLE4 is a Histone H3-H4 chaperone that maintains chromatin integrity during DNA replication, Molecular Cell, Vol: 72, Pages: 112-126.e5, ISSN: 1097-2765
Maintenance of epigenetic integrity relies on coordinated recycling and partitioning of parental histones and deposition of newly synthesized histones during DNA replication. This process depends upon a poorly characterized network of histone chaperones, remodelers, and binding proteins. Here we implicate the POLE3-POLE4 subcomplex of the leading-strand polymerase, Polε, in replication-coupled nucleosome assembly through its ability to selectively bind to histones H3-H4. Using hydrogen/deuterium exchange mass spectrometry and physical mapping, we define minimal domains necessary for interaction between POLE3-POLE4 and histones H3-H4. Biochemical analyses establish that POLE3-POLE4 is a histone chaperone that promotes tetrasome formation and DNA supercoiling in vitro. In cells, POLE3-POLE4 binds both newly synthesized and parental histones, and its depletion hinders helicase unwinding and chromatin PCNA unloading and compromises coordinated parental histone retention and new histone deposition. Collectively, our study reveals that POLE3-POLE4 possesses intrinsic H3-H4 chaperone activity, which facilitates faithful nucleosome dynamics at the replication fork.
Cherepanov P, 2018, Cryo-EM structures of lentiviral intasomes, Publisher: WILEY, Pages: 32-32, ISSN: 2211-5463
Zhao XZ, Smith SJ, Maskell DP, et al., 2017, Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors, JOURNAL OF MEDICINAL CHEMISTRY, Vol: 60, Pages: 7315-7332, ISSN: 0022-2623
Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistance-causing mutations that diminish the efficacy of DTG have appeared. Our current findings support and extend the substrate envelope concept that broadly effective INSTIs can be designed by filling the envelope defined by the DNA substrates. Previously, we explored 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides as an INSTI scaffold, making a limited set of derivatives, and concluded that broadly effective INSTIs can be developed using this scaffold. Herein, we report an extended investigation of 6-substituents as well the first examples of 7-substituted analogues of this scaffold. While 7-substituents are not well-tolerated, we have identified novel substituents at the 6-position that are highly effective, with the best compound (6p) retaining better efficacy against a broad panel of known INSTI resistant mutants than any analogues we have previously described.
Frigola J, He J, Kinkelin K, et al., 2017, Cdt1 stabilizes an open MCM ring for helicase loading, Nature Communications, Vol: 8, ISSN: 2041-1723
ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2–5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2–5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC–Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.