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1.
J Chem Inf Model ; 62(3): 472-485, 2022 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-35029985

RESUMO

Nuclear magnetic resonance (NMR) data from NOESY (nuclear Overhauser enhancement spectroscopy) and ROESY (rotating frame Overhauser enhancement spectroscopy) experiments can easily be combined with distance geometry (DG) based conformer generators by modifying the molecular distance bounds matrix. In this work, we extend the modern DG based conformer generator ETKDG, which has been shown to reproduce experimental crystal structures from small molecules to large macrocycles well, to include NOE-derived interproton distances. In noeETKDG, the experimentally derived interproton distances are incorporated into the distance bounds matrix as loose upper (or lower) bounds to generate large conformer sets. Various subselection techniques can subsequently be applied to yield a conformer bundle that best reproduces the NOE data. The approach is benchmarked using a set of 24 (mostly) cyclic peptides for which NOE-derived distances as well as reference solution structures obtained by other software are available. With respect to other packages currently available, the advantages of noeETKDG are its speed and that no prior force-field parametrization is required, which is especially useful for peptides with unnatural amino acids. The resulting conformer bundles can be further processed with the use of structural refinement techniques to improve the modeling of the intramolecular nonbonded interactions. The noeETKDG code is released as a fully open-source software package available at www.github.com/rinikerlab/customETKDG.


Assuntos
Peptídeos Cíclicos , Peptídeos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Proteica
2.
Biochemistry ; 55(39): 5587-5594, 2016 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-27607918

RESUMO

Nitric oxide is produced in Gram-positive pathogens Bacillus anthracis and Staphylococcus aureus by the bacterial isoform of nitric oxide synthase (NOS). Inhibition of bacterial nitric oxide synthase (bNOS) has been identified as a promising antibacterial strategy for targeting methicillin-resistant S. aureus [Holden, J. K., et al. (2015) Chem. Biol. 22, 785-779]. One class of NOS inhibitors that demonstrates antimicrobial efficacy utilizes an aminoquinoline scaffold. Here we report on a variety of aminoquinolines that target the bacterial NOS active site, in part, by binding to a hydrophobic patch that is unique to bNOS. Through mutagenesis and crystallographic studies, our findings demonstrate that aminoquinolines are an excellent scaffold for further aiding in the development of bNOS specific inhibitors.


Assuntos
Aminoquinolinas/farmacologia , Bacillus anthracis/enzimologia , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/metabolismo , Staphylococcus aureus/enzimologia , Cristalografia por Raios X , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/química
3.
Proc Natl Acad Sci U S A ; 110(45): 18127-31, 2013 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-24145412

RESUMO

Nitric oxide (NO) produced by bacterial NOS functions as a cytoprotective agent against oxidative stress in Staphylococcus aureus, Bacillus anthracis, and Bacillus subtilis. The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds impede the growth of B. subtilis under oxidative stress, and crystal structures show that each compound exhibits a unique binding mode. Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Modelos Moleculares , Óxido Nítrico Sintase/antagonistas & inibidores , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Domínio Catalítico/genética , Clonagem Molecular , Cristalização , Heme/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Mutagênese Sítio-Dirigida , Óxido Nítrico Sintase/química , Estresse Oxidativo/fisiologia , Ligação Proteica , Ratos , Análise de Regressão
4.
Biochemistry ; 54(26): 4075-82, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26062720

RESUMO

Nitric oxide generated by bacterial nitric oxide synthase (NOS) increases the susceptibility of Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis to oxidative stress, including antibiotic-induced oxidative stress. Not surprisingly, NOS inhibitors also improve the effectiveness of antimicrobials. Development of potent and selective bacterial NOS inhibitors is complicated by the high active site sequence and structural conservation shared with the mammalian NOS isoforms. To exploit bacterial NOS for the development of new therapeutics, recognition of alternative NOS surfaces and pharmacophores suitable for drug binding is required. Here, we report on a wide number of inhibitor-bound bacterial NOS crystal structures to identify several compounds that interact with surfaces unique to the bacterial NOS. Although binding studies indicate that these inhibitors weakly interact with the NOS active site, many of the inhibitors reported here provide a revised structural framework for the development of new antimicrobials that target bacterial NOS. In addition, mutagenesis studies reveal several key residues that unlock access to bacterial NOS surfaces that could provide the selectivity required to develop potent bacterial NOS inhibitors.


Assuntos
Bacillus subtilis/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/química , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Bacillus subtilis/química , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Óxido Nítrico Sintase/metabolismo , Conformação Proteica , Alinhamento de Sequência , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química
5.
J Biol Chem ; 289(42): 29437-45, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-25194416

RESUMO

Production of nitric oxide (NO) by nitric oxide synthase (NOS) requires electrons to reduce the heme iron for substrate oxidation. Both FAD and FMN flavin groups mediate the transfer of NADPH derived electrons to NOS. Unlike mammalian NOS that contain both FAD and FMN binding domains within a single polypeptide chain, bacterial NOS is only composed of an oxygenase domain and must rely on separate redox partners for electron transfer and subsequent activity. Here, we report on the native redox partners for Bacillus subtilis NOS (bsNOS) and a novel chimera that promotes bsNOS activity. By identifying and characterizing native redox partners, we were also able to establish a robust enzyme assay for measuring bsNOS activity and inhibition. This assay was used to evaluate a series of established NOS inhibitors. Using the new assay for screening small molecules led to the identification of several potent inhibitors for which bsNOS-inhibitor crystal structures were determined. In addition to characterizing potent bsNOS inhibitors, substrate binding was also analyzed using isothermal titration calorimetry giving the first detailed thermodynamic analysis of substrate binding to NOS.


Assuntos
Antibacterianos/química , Bacillus subtilis/enzimologia , NADH NADPH Oxirredutases/metabolismo , NADP/metabolismo , Óxido Nítrico Sintase/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Calorimetria , Clonagem Molecular , Citocromos c/metabolismo , Elétrons , Escherichia coli/metabolismo , Ferredoxina-NADP Redutase/genética , Flavodoxina/genética , Humanos , Imidazóis/química , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Oxirredução , Ratos
6.
Nat Commun ; 15(1): 2625, 2024 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-38521763

RESUMO

Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.


Assuntos
Sistemas CRISPR-Cas , Reparo de DNA por Recombinação , Humanos , Edição de Genes/métodos , Reparo do DNA , Reparo do DNA por Junção de Extremidades
7.
Front Mol Biosci ; 9: 848689, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35495632

RESUMO

Technologies for discovering peptides as potential therapeutics have rapidly advanced in recent years with significant interest from both academic and pharmaceutical labs. These advancements in turn drive the need for new computational tools to design peptides for purposes of advancing lead molecules into the clinic. Here we report the development and application of a new automated tool, AutoRotLib, for parameterizing a diverse set of non-canonical amino acids (NCAAs), N-methyl, or peptoid residues for use with the computational design program Rosetta. In addition, we developed a protocol for designing thioether-cyclized macrocycles within Rosetta, due to their common application in mRNA display using the RaPID platform. To evaluate the utility of these new computational tools, we screened a library of canonical and NCAAs on both a linear peptide and a thioether macrocycle, allowing us to quickly identify mutations that affect peptide binding and subsequently measure our results against previously published data. We anticipate in silico screening of peptides against a diverse chemical space will be a fundamental component for peptide design and optimization, as more amino acids can be explored in a single in silico screen than an in vitro screen. As such, these tools will enable maturation of peptide affinity for protein targets of interest and optimization of peptide pharmacokinetics for therapeutic applications.

8.
Nat Commun ; 13(1): 5222, 2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064790

RESUMO

The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric inhibition mechanism of HTRA1 by a clinical Fab fragment, currently being evaluated for GA treatment. Using cryo-EM, X-ray crystallography and biochemical assays we identify the exposed LoopA of HTRA1 as the sole Fab epitope, which is approximately 30 Å away from the active site. The cryo-EM structure of the HTRA1:Fab complex in combination with molecular dynamics simulations revealed that Fab binding to LoopA locks HTRA1 in a non-competent conformational state, incapable of supporting catalysis. Moreover, grafting the HTRA1-LoopA epitope onto HTRA2 and HTRA3 transferred the allosteric inhibition mechanism. This suggests a conserved conformational lock mechanism across the HTRA family and a critical role of LoopA for catalysis, which was supported by the reduced activity of HTRA1-3 upon LoopA deletion or perturbation. This study reveals the long-range inhibition mechanism of the clinical Fab and identifies an essential function of the exposed LoopA for activity of HTRA family proteases.


Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A , Degeneração Macular , Serina Endopeptidases , Cristalografia por Raios X , Epitopos , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
9.
Cell Rep ; 31(12): 107809, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32579935

RESUMO

The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.


Assuntos
Lipídeos/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Cristalografia por Raios X , Humanos , Lipoilação , Camundongos , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Fatores de Transcrição/agonistas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Cancers (Basel) ; 10(3)2018 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-29558384

RESUMO

The Hippo pathway is a critical transcriptional signaling pathway that regulates cell growth, proliferation and organ development. The transcriptional enhanced associate domain (TEAD) protein family consists of four paralogous transcription factors that function to modulate gene expression in response to the Hippo signaling pathway. Transcriptional activation of these proteins occurs upon binding to the co-activator YAP/TAZ whose entry into the nucleus is regulated by Lats1/2 kinase. In recent years, it has become apparent that the dysregulation and/or overexpression of Hippo pathway effectors is implicated in a wide range of cancers, including prostate, gastric and liver cancer. A large body of work has been dedicated to understanding the therapeutic potential of modulating the phosphorylation and localization of YAP/TAZ. However, YAP/TAZ are considered to be natively unfolded and may be intractable as drug targets. Therefore, TEAD proteins present themselves as an excellent therapeutic target for intervention of the Hippo pathway. This review summarizes the functional role of TEAD proteins in cancer and assesses the therapeutic potential of antagonizing TEAD function in vivo.

11.
Protein Sci ; 25(2): 374-82, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26448477

RESUMO

Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing L-Arg to NO and L-citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH-derived electrons are transferred to the heme-containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN-to-heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme-FMN-calmodulin NOS complex based on the recent biophysical studies using a 105-ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis.


Assuntos
Simulação de Dinâmica Molecular , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Calmodulina/metabolismo , Transporte de Elétrons , Mononucleotídeo de Flavina/metabolismo , Heme/metabolismo , Humanos , NADP/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Domínios e Motivos de Interação entre Proteínas
12.
J Med Chem ; 58(2): 994-1004, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25522110

RESUMO

Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial ( Holden , , Proc. Natl. Acad. Sci. U.S.A. 2013 , 110 , 18127 ). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.


Assuntos
Antibacterianos/síntese química , Bactérias/enzimologia , Inibidores Enzimáticos/síntese química , Óxido Nítrico Sintase/antagonistas & inibidores , Aminopiridinas/síntese química , Aminopiridinas/farmacologia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Sítios de Ligação , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Simulação de Dinâmica Molecular , Óxido Nítrico Sintase/química , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/farmacologia
13.
Chem Biol ; 22(6): 785-92, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26091171

RESUMO

Bacterial infections associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major economic burden to hospitals, and confer high rates of morbidity and mortality among those infected. Exploitation of novel therapeutic targets is thus necessary to combat this dangerous pathogen. Here, we report on the identification and characterization, including crystal structures, of two nitric oxide synthase (NOS) inhibitors that function as antimicrobials against MRSA. These data provide the first evidence that bacterial NOS (bNOS) inhibitors can work synergistically with oxidative stress to enhance MRSA killing. Crystal structures show that each inhibitor contacts an active site Ile residue in bNOS that is Val in the mammalian NOS isoforms. Mutagenesis studies show that the additional nonpolar contacts provided by the Ile in bNOS contribute to tighter binding toward the bacterial enzyme.


Assuntos
Proteínas de Bactérias/metabolismo , Staphylococcus aureus Resistente à Meticilina/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Bases de Dados de Proteínas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Cinética , Camundongos , Simulação de Acoplamento Molecular , Mutagênese , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo
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