RESUMO
During the activation of mitogen-activated protein kinase (MAPK) signaling, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3-5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane that correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.
Assuntos
Cisteína , Proteínas Proto-Oncogênicas c-raf , Sítios de Ligação , Membrana Celular/metabolismo , Cisteína/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Solventes/metabolismoRESUMO
Development of resistance remains a major challenge for drugs to treat HIV-1 infections, including those targeting the essential viral polymerase, HIV-1 reverse transcriptase (RT). Resistance associated with the Tyr181Cys mutation in HIV-1 RT has been a key roadblock in the discovery of nonnucleoside RT inhibitors (NNRTIs). It is the principal point mutation that arises from treatment of HIV-infected patients with nevirapine, the first-in-class drug still widely used, especially in developing countries. We report covalent inhibitors of Tyr181Cys RT (CRTIs) that can completely knock out activity of the resistant mutant and of the particularly challenging Lys103Asn/Tyr181Cys variant. Conclusive evidence for the covalent modification of Cys181 is provided from enzyme inhibition kinetics, mass spectrometry, protein crystallography, and antiviral activity in infected human T-cell assays. The CRTIs are also shown to be selective for Cys181 and have lower cytotoxicity than the approved NNRTI drugs efavirenz and rilpivirine.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Substituição de Aminoácidos , Fármacos Anti-HIV/síntese química , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Humanos , Cinética , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Inibidores da Transcriptase Reversa/síntese química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Human immunodeficiency virus 1 (HIV-1) infection is a global health issue since neither a cure nor a vaccine is available. However, the highly active antiretroviral therapy (HAART) has improved the life expectancy for patients with acquired immunodeficiency syndrome (AIDS). Nucleoside reverse transcriptase inhibitors (NRTIs) are in almost all HAART and target reverse transcriptase (RT), an essential enzyme for the virus. Even though NRTIs are highly effective, they have limitations caused by RT resistance. The main mechanisms of RT resistance to NRTIs are discrimination and excision. Understanding the molecular mechanisms for discrimination and excision are essential to develop more potent and selective NRTIs. Using protein X-ray crystallography, we determined the first crystal structure of RT in its post-catalytic state in complex with emtricitabine, (-)FTC or stavudine (d4T). Our structural studies provide the framework for understanding how RT discriminates between NRTIs and natural nucleotides, and for understanding the requirement of (-)FTC to undergo a conformation change for successful incorporation by RT. The crystal structure of RT in post-catalytic complex with d4T provides a "snapshot" for considering the possible mechanism of how RT develops resistance for d4T via excision. The findings reported herein will contribute to the development of next generation NRTIs.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/química , Catálise , Cristalografia por Raios X , Emtricitabina/química , Emtricitabina/farmacologia , Humanos , Modelos Moleculares , Nucleotídeos/química , Nucleotídeos/farmacologia , Inibidores da Transcriptase Reversa/química , Estavudina/química , Estavudina/farmacologiaRESUMO
The clinical benefits of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are hindered by their unsatisfactory pharmacokinetic (PK) properties along with the rapid development of drug-resistant variants. However, the clinical efficacy of these inhibitors can be improved by developing compounds with enhanced pharmacological profiles and heightened antiviral activity. We used computational and structure-guided design to develop two next-generation NNRTI drug candidates, compounds I and II, which are members of a class of catechol diethers. We evaluated the preclinical potential of these compounds in BALB/c mice because of their high solubility (510 µg/ml for compound I and 82.9 µg/ml for compound II), low cytotoxicity, and enhanced antiviral activity against wild-type (WT) HIV-1 RT and resistant variants. Additionally, crystal structures of compounds I and II with WT RT suggested an optimal binding to the NNRTI binding pocket favoring the high anti-viral potency. A single intraperitoneal dose of compounds I and II exhibited a prolonged serum residence time of 48 hours and concentration maximum (Cmax) of 4000- to 15,000-fold higher than their therapeutic/effective concentrations. These Cmax values were 4- to 15-fold lower than their cytotoxic concentrations observed in MT-2 cells. Compound II showed an enhanced area under the curve (0-last) and decreased plasma clearance over compound I and efavirenz, the standard of care NNRTI. Hence, the overall (PK) profile of compound II was excellent compared with that of compound I and efavirenz. Furthermore, both compounds were very well tolerated in BALB/c mice without any detectable acute toxicity. Taken together, these data suggest that compounds I and II possess improved anti-HIV-1 potency, remarkable in vivo safety, and prolonged in vivo circulation time, suggesting strong potential for further development as new NNRTIs for the potential treatment of HIV infection.
Assuntos
Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Infecções por HIV/tratamento farmacológico , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/uso terapêutico , Alcinos , Animais , Benzoxazinas/química , Benzoxazinas/farmacologia , Cristalografia por Raios X , Ciclopropanos , Feminino , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos Endogâmicos BALB C , Inibidores da Transcriptase Reversa/farmacocinética , Inibidores da Transcriptase Reversa/toxicidade , SolubilidadeRESUMO
The endospore forming bacterium Bacillus anthracis causes lethal anthrax disease in humans and animals. The ability of this pathogen to replicate within macrophages is dependent upon the display of bacterial surface proteins attached to the cell wall by the B. anthracis Sortase A ((Ba)SrtA) enzyme. Previously, we discovered that the class A (Ba)SrtA sortase contains a unique N-terminal appendage that wraps around the body of the protein to contact the active site of the enzyme. To gain insight into its function, we determined the NMR structure of (Ba)SrtA bound to a LPXTG sorting signal analog. The structure, combined with dynamics, kinetics, and whole cell protein display data suggest that the N terminus modulates substrate access to the enzyme. We propose that it may increase the efficiency of protein display by reducing the unproductive hydrolytic cleavage of enzyme-protein covalent intermediates that form during the cell wall anchoring reaction. Notably, a key active site loop (ß7/ß8 loop) undergoes a disordered to ordered transition upon binding the sorting signal, potentially facilitating recognition of lipid II.
Assuntos
Aminoaciltransferases/química , Bacillus anthracis/enzimologia , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Sinais Direcionadores de Proteínas , Aminoaciltransferases/metabolismo , Bacillus anthracis/patogenicidade , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Especificidade por SubstratoRESUMO
Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-indolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility. Crystal structures for two complexes enhance the analysis of the structure-activity data.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Compostos Azabicíclicos/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Descoberta de Drogas , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV/efeitos dos fármacos , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Triazinas/farmacologia , Fármacos Anti-HIV/síntese química , Compostos Azabicíclicos/síntese química , Compostos Azabicíclicos/química , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Transcriptase Reversa do HIV/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores da Transcriptase Reversa/síntese química , Solubilidade , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/químicaRESUMO
RAS proteins play a vital role in regulating downstream signaling and essential cellular processes, positioning them as key players in normal cellular physiology and disease development. Among the various isoforms of RAS, KRAS stands out as one of the most frequently mutated genes in human cancer. The prevalence of RAS mutations in cancer often involves single amino acid substitutions at codons 12, 13, or 61. These mutations disrupt the RAS protein's inherent ability to transition between its active and inactive states, resulting in a constant activation signal and driving uncontrolled cell growth. Crystallization and structural analysis of KRAS with inhibitors and RAS-binding proteins play a pivotal role in unraveling the structural and mechanistic details of KRAS function, aiding in drug discovery efforts, and advancing our understanding of KRAS-driven diseases. Here, we present our experimental methodology for crystallizing KRAS in the presence of covalent or non-covalent small molecules and proteins acting as effectors or regulators of RAS. We detail the techniques for successful crystallization and the subsequent optimization of crystallization conditions. The resulting crystals and their structures will provide valuable insights into the key interactions between KRAS and its partner proteins or potential inhibitors, offering a foundation for developing targeted therapies that are more potent and selective against KRAS-driven cancers.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas de Transporte/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Transdução de Sinais , Neoplasias/genética , MutaçãoRESUMO
HIV-1 reverse transcriptase (RT) remains a key target for HIV drug development. As successful management of the disease requires lifelong treatment, the emergence of resistance mutations is inevitable, making development of new RT inhibitors, which remain effective against resistant variants crucial. To this end, previous computationally guided drug design efforts have resulted in catechol diether compounds, which inhibit wildtype RT with picomolar affinities and appear to be promising preclinical candidates. To confirm that these compounds remain potent against Y181C, a widespread mutation conferring resistance to first generation inhibitors, they were screened against the HIV-1 N119 clinical isolate, reported as a Y181C single mutant. In comparison to a molecular clone with the same mutation, N119 appears less susceptible to inhibition by our preclinical candidate compounds. A more detailed sequencing effort determined that N119 was misidentified and carries V106A in combination with Y181C. While both indolizine and naphthalene substituted catechol diethers are potent against the classical Y181C single mutant, the addition of V106A confers more resistance against the indolizine derivatives than the naphthalene derivatives. Crystal structures presented in this study highlight key features of the naphthyl group, which allow these compounds to remain potent in the double mutant, including stronger interactions with F227 and less reliance on V106 for stabilization of the ethoxy-uracil ring, which makes critical hydrogen bonds with other residues in the binding pocket.
Assuntos
Fármacos Anti-HIV , HIV-1 , Indolizinas , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/química , Transcriptase Reversa do HIV/química , Indolizinas/farmacologia , Catecóis/química , Catecóis/farmacologia , Naftalenos/farmacologia , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/química , Relação Estrutura-AtividadeRESUMO
Localized dynamics of RAS, including regions distal to the nucleotide-binding site, is of high interest for elucidating the mechanisms by which RAS proteins interact with effectors and regulators and for designing inhibitors. Among several oncogenic mutants, methyl relaxation dispersion experiments reveal highly synchronized conformational dynamics in the active (GMPPNP-bound) KRASG13D, which suggests an exchange between two conformational states in solution. Methyl and 31P NMR spectra of active KRASG13D in solution confirm a two-state ensemble interconverting on the millisecond timescale, with a major Pγ atom peak corresponding to the dominant State 1 conformation and a secondary peak indicating an intermediate state different from the known State 2 conformation recognized by RAS effectors. High-resolution crystal structures of active KRASG13D and KRASG13D-RAF1 RBD complex provide snapshots of the State 1 and 2 conformations, respectively. We use residual dipolar couplings to solve and cross-validate the structure of the intermediate state of active KRASG13D, showing a conformation distinct from those of States 1 and 2 outside the known flexible switch regions. The dynamic coupling between the conformational exchange in the effector lobe and the breathing motion in the allosteric lobe is further validated by a secondary mutation in the allosteric lobe, which affects the conformational population equilibrium.
Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Proteínas ras/metabolismo , Conformação Proteica , Espectroscopia de Ressonância MagnéticaRESUMO
Reverse transcriptase (RT) from the human immunodeficiency virus continues to be an attractive drug target for antiretroviral therapy. June 2022 will commemorate the 30th anniversary of the first Human Immunodeficiency Virus (HIV) RT crystal structure complex that was solved with non-nucleoside reverse transcriptase inhibitor nevirapine. The release of this structure opened opportunities for designing many families of non-nucleoside reverse transcriptase inhibitors (NNRTIs). In paying tribute to the first RT-nevirapine structure, we have developed several compound classes targeting the non-nucleoside inhibitor binding pocket of HIV RT. Extensive analysis of crystal structures of RT in complex with the compounds informed iterations of structure-based drug design. Structures of seven additional complexes were determined and analyzed to summarize key interactions with residues in the non-nucleoside inhibitor binding pocket (NNIBP) of RT. Additional insights comparing structures with antiviral data and results from molecular dynamics simulations elucidate key interactions and dynamics between the nucleotide and non-nucleoside binding sites.
RESUMO
The appeal of multiscale modeling approaches is predicated on the promise of combinatorial synergy. However, this promise can only be realized when distinct scales are combined with reciprocal consistency. Here, we consider multiscale molecular dynamics (MD) simulations that combine the accuracy and macromolecular flexibility accessible to fixed-charge all-atom (AA) representations with the sampling speed accessible to reductive, coarse-grained (CG) representations. AA-to-CG conversions are relatively straightforward because deterministic routines with unique outcomes are achievable. Conversely, CG-to-AA conversions have many solutions due to a surge in the number of degrees of freedom. While automated tools for biomolecular CG-to-AA transformation exist, we find that one popular option, called Backward, is prone to stochastic failure and the AA models that it does generate frequently have compromised protein structure and incorrect stereochemistry. Although these shortcomings can likely be circumvented by human intervention in isolated instances, automated multiscale coupling requires reliable and robust scale conversion. Here, we detail an extension to Multiscale Machine-learned Modeling Infrastructure (MuMMI), including an improved CG-to-AA conversion tool called sinceCG. This tool is reliable (â¼98% weakly correlated repeat success rate), automatable (no unrecoverable hangs), and yields AA models that generally preserve protein secondary structure and maintain correct stereochemistry. We describe how the MuMMI framework identifies CG system configurations of interest, converts them to AA representations, and simulates them at the AA scale while on-the-fly analyses provide feedback to update CG parameters. Application to systems containing the peripheral membrane protein RAS and proximal components of RAF kinase on complex eight-component lipid bilayers with â¼1.5 million atoms is discussed in the context of MuMMI.
Assuntos
Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Humanos , Bicamadas Lipídicas/química , Estrutura Secundária de Proteína , Proteínas/químicaRESUMO
The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.
Assuntos
Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas ras/química , Proteínas ras/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Cisteína/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Human immunodeficiency virus (HIV)-1 remains as a global health issue that is primarily treated with highly active antiretroviral therapy, a combination of drugs that target the viral life cycle. One class of these drugs are non-nucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral reverse transcriptase (RT). First generation NNRTIs were troubled with poor pharmacological properties and drug resistance, incentivizing the development of improved compounds. One class of developed compounds are the 2-naphthyl phenyl ethers, showing promising efficacy against the Y181C RT mutation. Further biochemical and structural work demonstrated differences in potency against the Y181C mutation and binding mode of the compounds. This work aims to understand the relationship between the binding mode and ability to overcome drug resistance using macromolecular x-ray crystallography. Comparison of 2-naphthyl phenyl ethers bound to Y181C RT reveal that compounds that interact with the invariant W229 are more capable of retaining efficacy against the resistance mutation. Additional modifications to these compounds at the 4-position, computationally designed to compensate for the Y181C mutation, do not demonstrate improved potency. Ultimately, we highlight important considerations for the development of future HIV-1 drugs that are able to combat drug resistance.
Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , HIV-1/enzimologia , Mutação de Sentido Incorreto , Éteres Fenílicos/química , Inibidores da Transcriptase Reversa/química , Substituição de Aminoácidos , Sítios de Ligação , Transcriptase Reversa do HIV/genética , HIV-1/genéticaRESUMO
The retrovirus HIV-1 has been a major health issue since its discovery in the early 80s. In 2017, over 37 million people were infected with HIV-1, of which 1.8 million were new infections that year. Currently, the most successful treatment regimen is the highly active antiretroviral therapy (HAART), which consists of a combination of three to four of the current 26 FDA-approved HIV-1 drugs. Half of these drugs target the reverse transcriptase (RT) enzyme that is essential for viral replication. One class of RT inhibitors is nucleoside reverse transcriptase inhibitors (NRTIs), a crucial component of the HAART. Once incorporated into DNA, NRTIs function as a chain terminator to stop viral DNA replication. Unfortunately, treatment with NRTIs is sometimes linked to toxicity caused by off-target side effects. NRTIs may also target the replicative human mitochondrial DNA polymerase (Pol γ), causing long-term severe drug toxicity. The goal of this work is to understand the discrimination mechanism of different NRTI analogues by RT. Crystal structures and kinetic experiments are essential for the rational design of new molecules that are able to bind selectively to RT and not Pol γ. Structural comparison of NRTI-binding modes with both RT and Pol γ enzymes highlights key amino acids that are responsible for the difference in affinity of these drugs to their targets. Therefore, the long-term goal of this research is to develop safer, next generation therapeutics that can overcome off-target toxicity.
Assuntos
DNA Polimerase gama/química , Emtricitabina/farmacologia , Transcriptase Reversa do HIV/química , Lamivudina/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Motivos de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , DNA Polimerase gama/metabolismo , Emtricitabina/efeitos adversos , Emtricitabina/química , Transcriptase Reversa do HIV/metabolismo , Humanos , Lamivudina/efeitos adversos , Lamivudina/química , Modelos Moleculares , Conformação Proteica , Inibidores da Transcriptase Reversa/efeitos adversos , Inibidores da Transcriptase Reversa/química , Relação Estrutura-AtividadeRESUMO
Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48â¯h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.
Assuntos
Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacocinética , Fármacos Anti-HIV/farmacologia , Cristalografia por Raios X , Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Infecções por HIV/virologia , Transcriptase Reversa do HIV/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/uso terapêutico , Nanopartículas/virologia , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacocinética , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
Staphylococcus aureus is a leading cause of hospital-acquired infections in the USA and is a major health concern as methicillin-resistant S. aureus and other antibiotic-resistant strains are common. Compounds that inhibit the S. aureus sortase (SrtA) cysteine transpeptidase may function as potent anti-infective agents as this enzyme attaches virulence factors to the bacterial cell wall. While a variety of SrtA inhibitors have been discovered, the vast majority of these small molecules have not been optimized using structure-based approaches. Here we have used NMR spectroscopy to determine the molecular basis through which pyridazinone-based small molecules inhibit SrtA. These inhibitors covalently modify the active cysteine thiol and partially mimic the natural substrate of SrtA by inducing the closure of an active site loop. Computational and synthetic chemistry methods led to second-generation analogues that are ~70-fold more potent than the lead molecule. These optimized molecules exhibit broad-spectrum activity against other types of class A sortases, have reduced cytotoxicity, and impair SrtA-mediated protein display on S. aureus cell surface. Our work shows that pyridazinone analogues are attractive candidates for further development into anti-infective agents, and highlights the utility of employing NMR spectroscopy and solubility-optimized small molecules in structure-based drug discovery.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Piridazinas/química , Staphylococcus aureus/enzimologia , Aminoaciltransferases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Parede Celular/enzimologia , Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Conformação Molecular , Simulação de Acoplamento Molecular , Piridazinas/farmacologia , Piridazinas/toxicidade , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Catechol diethers that incorporate a 7-cyano-2-naphthyl substituent are reported as non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Many of the compounds have 1-10 nM potencies toward wild-type HIV-1. An interesting conformational effect allows two unique conformers for the naphthyl group in complexes with HIV-RT. X-ray crystal structures for 4a and 4f illustrate the alternatives.
RESUMO
Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdH(N2N3), A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Hemoglobinas/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Many species of Gram-positive bacteria use sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of sortases (class A to F enzymes); however, class E enzymes have not been extensively studied. Class E sortases are used by soil and freshwater-dwelling Actinobacteria to display proteins that contain a non-canonical LAXTG sorting signal, which differs from 90% of known sorting signals by substitution of alanine for proline. Here we report the first crystal structure of a class E sortase, the 1.93 Å resolution structure of the SrtE1 enzyme from Streptomyces coelicolor. The active site is bound to a tripeptide, providing insight into the mechanism of substrate binding. SrtE1 possesses ß3/ß4 and ß6/ß7 active site loops that contact the LAXTG substrate and are structurally distinct from other classes. We propose that SrtE1 and other class E sortases employ a conserved tyrosine residue within their ß3/ß4 loop to recognize the amide nitrogen of alanine at position P3 of the sorting signal through a hydrogen bond, as seen here. Incapability of hydrogen-bonding with canonical proline-containing sorting signals likely contributes to class E substrate specificity. Furthermore, we demonstrate that surface anchoring of proteins involved in aerial hyphae formation requires an N-terminal segment in SrtE1 that is presumably positioned within the cytoplasm. Combined, our results reveal unique features within class E enzymes that enable them to recognize distinct sorting signals, and could facilitate the development of substrate-based inhibitors of this important enzyme family.
Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Streptomyces coelicolor/enzimologia , Sequência de Aminoácidos , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Cisteína Endopeptidases/metabolismo , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Conformação Proteica , Sinais Direcionadores de Proteínas , Streptomyces coelicolor/química , Streptomyces coelicolor/metabolismo , Especificidade por SubstratoRESUMO
Staphylococcus aureus is the leading cause of hospital-acquired infections in the United States. The emergence of multidrug-resistant strains of S. aureus has created an urgent need for new antibiotics. Staphylococcus aureus uses the sortase A enzyme to display surface virulence factors suggesting that compounds that inhibit its activity will function as potent anti-infective agents. Here, we report the identification of several inhibitors of sortase A using virtual screening methods that employ the relaxed complex scheme, an advanced computer-docking methodology that accounts for protein receptor flexibility. Experimental testing validates that several compounds identified in the screen inhibit the activity of sortase A. A lead compound based on the 2-phenyl-2,3-dihydro-1H-perimidine scaffold is particularly promising, and its binding mechanism was further investigated using molecular dynamics simulations and conducting preliminary structure-activity relationship studies.