RESUMO
Hyaluronan (HA) plays important roles in a wide range of biological events. The principal receptor of HA in the human body is the Cluster of Differentiation 44 (CD44). To enhance the binding between HA and CD44, a new approach was designed to take advantage of the four-component Ugi reaction. By modifying the carboxyl group on HA with various amine, aldehyde, and isocyanide moieties through the Ugi reaction, 36 HA like polysaccharides were generated. Two lead compounds were identified with enhanced CD44 binding compared to unmodified HA, which was confirmed by surface plasmon resonance (SPR), cellular studies and an in vivo mouse tumor model. Ski-learn as a machine learning tool was applied to analyze library data and yield predictions with an accuracy over 80 %. In conclusion, modification of HA via the Ugi reaction can be a promising strategy to develop novel binders toward HA receptors such as CD44.
Assuntos
Receptores de Hialuronatos , Ácido Hialurônico , Humanos , Camundongos , Animais , Ácido Hialurônico/química , Receptores de Hialuronatos/metabolismo , PolissacarídeosRESUMO
Since the discovery of the caspase-2 (Casp2)-mediated ∆tau314 cleavage product and its associated impact on tauopathies such as Alzheimer's disease, the design of selective Casp2 inhibitors has become a focus in medicinal chemistry research. In the search for new lead structures with respect to Casp2 selectivity and drug-likeness, we have taken an approach by looking more closely at the specific sites of Casp2-mediated proteolysis. Using seven selected protein cleavage sequences, we synthesized a peptide series of 53 novel molecules and studied them using in vitro pharmacology, molecular modeling, and crystallography. Regarding Casp2 selectivity, AcITV(Dab)D-CHO (23) and AcITV(Dap)D-CHO (26) demonstrated the best selectivity (1-6-fold), although these trends were only moderate. However, some analogous tetrapeptides, most notably AcDKVD-CHO (45), showed significantly increased Casp3 selectivities (>100-fold). Tetra- and tripeptides display decreased or no Casp2 affinity, supporting the assumption that a motif of five amino acids is required for efficient Casp2 inhibition. Overall, the results provide a reasonable basis for the development of both selective Casp2 and Casp3 inhibitors.
Assuntos
Caspase 2 , Caspase 2/metabolismo , Caspase 3/metabolismo , Inibidores de Caspase/farmacologia , Proteólise , Relação Estrutura-AtividadeRESUMO
The first crystal structure of the human cytosolic malate dehydrogenase I (MDH1) is described. Structure determination at a high resolution (1.65 Å) followed production, isolation, and purification of human MDH1 using a bacterial expression system. The structure is a binary complex of MDH1 with only a bound malonate molecule in the substrate binding site. Comparisons of this structure with malate dehydrogenase enzymes from other species confirm that the human enzyme adopts similar secondary, tertiary, and quaternary structures and that the enzyme retains a similar conformation even when nicotinamide adenine dinucleotide (NAD+) is not bound. A comparison to the highly homologous porcine (sus scrofa) MDH1 ternary structures leads to the conclusion that only small conformational differences are needed to accommodate binding by NAD+ or other NAD+ mimetics. Conformational differences observed in the second subunit show that the NAD+ binding elements are nevertheless quite flexible. Comparison of hMDH1 to the human mitochondrial malate dehydrogenase (hMDH2) reveals some key differences in the α7-α8 loop, which lies directly beneath the substrate binding pocket. These differences might be exploited in the structure-assisted design of selective small molecule inhibitors of hMDH1, an emerging target for the development of anticancer therapeutics.
RESUMO
Alzheimer's disease (AD) was first described by Alois Alzheimer over 100 years ago, but there is still no overarching theory that can explain its cause in detail. There are also no effective therapies to treat either the cause or the associated symptoms of this devastating disease. A potential approach to better understand the pathogenesis of AD could be the development of selective caspase-2 (Casp2) probes, as we have shown that a Casp2-mediated cleavage product of tau (Δtau314) reversibly impairs cognitive and synaptic function in animal models of tauopathies. In this article, we map out the Casp2 binding site through the preparation and assay of a series of 35 pentapeptide inhibitors with the goal of gaining selectivity against caspase-3 (Casp3). We also employed computational docking methods to understand the key interactions in the binding pocket of Casp2 and the differences predicted for binding at Casp3. Moreover, we crystallographically characterized the binding of selected pentapeptides with Casp3. Furthermore, we engineered and expressed a series of recombinant tau mutants and investigated them in an in vitro cleavage assay. These studies resulted in simple peptidic inhibitors with nanomolar affinity, for example, AcVDV(Dab)D-CHO (24) with up to 27.7-fold selectivity against Casp3. Our findings provide a good basis for the future development of selective Casp2 probes and inhibitors that can serve as pharmacological tools in planned in vivo studies and as lead compounds for the design of bioavailable and more drug-like small molecules.
RESUMO
Inherited peripheral neuropathies are a group of neurodegenerative disorders that clinically affect 1 in 2500 individuals. Recently, genetic mutations in human histidine nucleotide-binding protein 1 (hHint1) have been strongly and most frequently associated with patients suffering from axonal neuropathy with neuromyotonia. However, the correlation between the impact of these mutations on the hHint1 structure, enzymatic activity and in vivo function has remained ambiguous. Here, we provide detailed biochemical characterization of a set of these hHint1 mutations. Our findings indicate that half of the mutations (R37P, G93D and W123*) resulted in a destabilization of the dimeric state and a significant decrease in catalytic activity and HINT1 inhibitor binding affinity. The H112N mutant was found to be dimeric, but devoid of catalytic activity, due to the loss of the catalytically essential histidine; nevertheless, it exhibited high affinity to AMP and a HINT1 inhibitor. In contrast to the active-site mutants, the catalytic activity and dimeric structure of the surface mutants, C84R and G89V, were found to be similar to the wild-type enzyme. Taken together, our results suggest that the pathophysiology of inherited axonal neuropathy with neuromyotonia can be induced by conversion of HINT1 from a homodimer to monomer, by modification of select surface residues or by a significant reduction of the enzyme's catalytic efficiency.
Assuntos
Síndrome de Isaacs/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Histidina/metabolismo , Humanos , Proteínas Mutantes/genética , Proteínas do Tecido Nervoso/genética , Conformação Proteica , Homologia de SequênciaRESUMO
Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide.
Assuntos
Proteínas do Tecido Nervoso/química , Nucleotídeos/química , Amidas/química , Cristalografia por Raios X , Humanos , Ácidos Fosfóricos/química , Especificidade por SubstratoRESUMO
Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidase and adenylate hydrolase that has emerged as a potential therapeutic target for the management of pain. However, the molecular mechanism of Hint1 in the signaling pathway has remained less clear. The role of metal ions in regulating postsynaptic transmission is well known, and the active site of hHint1 contains multiple histidines. Here we have investigated the effect of divalent metal ions (Cd2+, Cu2+, Mg2+, Mn2+, Ni2+, and Zn2+) on the structural integrity and catalytic activity of hHint1. With the exception of Mg2+, all the divalent ions inhibited hHint1, the rank of order was found to be Cu2+ >Zn2+ >Cd2+ ≥Ni2+ >Mn2+ based on their IC50 and kin/KI values. A crystal structure of hHint1 with bound Cu2+ is described to explain the competitive reversible inactivation of hHint1 by divalent cations. All the metal ions exhibited time- and concentration- dependent inhibition, with the rate of inactivation highly dependent on alterations of the C-terminus. With the exception of Cu2+; restoration of inhibition was observed for all the metal ions after treatment with EDTA. Our studies reveal a loss in secondary structure and aggregation of hHint1 upon incubation with 10-fold excess of copper. Thus, hHint1 appears to be structurally sensitive to irreversible inactivation by copper, which may be of neurotoxicological and pharmacological significance.
Assuntos
Metais/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/ultraestrutura , Analgésicos Opioides/química , Animais , Sítios de Ligação , Catálise , Cátions Bivalentes/química , Cobre/química , Humanos , Íons , Neuralgia/metabolismo , Ligação Proteica , Desdobramento de ProteínaRESUMO
Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES1) and product (EP2) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes.
Assuntos
Antivirais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sofosbuvir/metabolismo , Ativação Metabólica , Domínio Catalítico , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Mutação Puntual , Especificidade por SubstratoRESUMO
The pyridoxal 5'-phosphate (PLP)-dependent transaminase BioA catalyzes the second step in the biosynthesis of biotin in Mycobacterium tuberculosis (Mtb) and is an essential enzyme for bacterial survival and persistence in vivo. A promising BioA inhibitor 6 containing an N-aryl, N'-benzoylpiperazine scaffold was previously identified by target-based whole-cell screening. Here, we explore the structure-activity relationships (SAR) through the design, synthesis, and biological evaluation of a systematic series of analogues of the original hit using a structure-based drug design strategy, which was enabled by cocrystallization of several analogues with BioA. To confirm target engagement and discern analogues with off-target activity, each compound was evaluated against wild-type (WT) Mtb in biotin-free and -containing medium as well as BioA under- and overexpressing Mtb strains. Conformationally constrained derivative 36 emerged as the most potent analogue with a KD of 76 nM against BioA and a minimum inhibitory concentration of 1.7 µM (0.6 µg/mL) against Mtb in biotin-free medium.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Biotina/biossíntese , Mycobacterium tuberculosis/efeitos dos fármacos , Piperazinas/farmacologia , Fosfato de Piridoxal/metabolismo , Transaminases/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Biocatálise , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/metabolismo , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-Atividade , Transaminases/metabolismoRESUMO
Mechanism-based inhibitors (MBIs) are widely employed in chemistry, biology, and medicine because of their exquisite specificity and sustained duration of inhibition. Optimization of MBIs is complicated because of time-dependent inhibition resulting from multistep inactivation mechanisms. The global kinetic parameters kinact and KI have been used to characterize MBIs, but they provide far less information than is commonly assumed, as shown by derivation and simulation of these parameters. We illustrate an alternative and more rigorous approach for MBI characterization through determination of the individual microscopic rate constants. Kinetic analysis revealed the rate-limiting step of inactivation of the PLP-dependent enzyme BioA by dihydro-(1,4)-pyridone 1. This knowledge was subsequently applied to rationally design a second-generation inhibitor scaffold with a nearly optimal maximum inactivation rate (0.48 min-1).
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Piridonas/farmacologia , Transaminases/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/química , Cinética , Estrutura Molecular , Piridonas/química , Transaminases/metabolismoRESUMO
Hint1 has recently emerged to be an important target of interest due to its involvement in the regulation of a broad range of CNS functions including opioid signaling, tolerance, neuropathic pain, and nicotine dependence. A series of inhibitors were rationally designed, synthesized, and tested for their inhibitory activity against hHint1 using isothermal titration calorimetry (ITC). The studies resulted in the development of the first small-molecule inhibitors of hHint1 with submicromolar binding affinities. A combination of thermodynamic and high-resolution X-ray crystallographic studies provides an insight into the biomolecular recognition of ligands by hHint1. These novel inhibitors have potential utility as molecular probes to better understand the role and function of hHint1 in the CNS.
RESUMO
The Bacillus anthracis lethal factor (LF) is one component of a tripartite exotoxin partly responsible for persistent anthrax cytotoxicity after initial bacterial infection. Inhibitors of the zinc metalloproteinase have been investigated as potential therapeutic agents, but LF is a challenging target because inhibitors lack sufficient selectivity or possess poor pharmaceutical properties. These structural studies reveal an alternate conformation of the enzyme, induced upon binding of specific inhibitors, that opens a previously unobserved deep pocket termed S1'(∗) which might afford new opportunities to design selective inhibitors that target this subsite.
Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Inibidores de Metaloproteinases de Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Sítios de Ligação , Ligantes , Inibidores de Metaloproteinases de Matriz/química , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Tirosina/metabolismoRESUMO
The lethal factor (LF) enzyme secreted by Bacillus anthracis is a zinc hydrolase that is chiefly responsible for anthrax-related cell death. Although many studies of the design of small molecule LF inhibitors have been conducted, no LF inhibitor is yet available as a therapeutic agent. Inhibitors with considerable chemical diversity have been developed and investigated; however, the LF S2' subsite has not yet been systematically explored as a potential target for lead optimization. Here we present synthesis, experimental evaluation, modeling, and structural biology for a novel series of sulfonamide hydroxamate LF inhibitor analogues specifically designed to extend into, and probe chemical preferences of, this S2' subsite. We discovered that this region accommodates a wide variety of chemical functionalities and that a broad selection of ligand structural modifications directed to this area can be incorporated without significant deleterious alterations in biological activity. We also identified key residues in this subsite that can potentially be targeted to improve inhibitor binding.
Assuntos
Antraz/microbiologia , Antígenos de Bactérias/química , Bacillus anthracis/enzimologia , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/química , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Alquilação , Antraz/tratamento farmacológico , Antígenos de Bactérias/metabolismo , Bacillus anthracis/química , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Inibidores Enzimáticos/química , Humanos , Ácidos Hidroxâmicos/química , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Mycobacterium tuberculosis (Mtb), responsible for both latent and symptomatic tuberculosis (TB), remains the second leading cause of mortality among infectious diseases worldwide. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. We report the synthesis and evaluation of a systematic series of potent nucleoside-based inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with K(D)s ≤ 2 nM. Additionally, we obtained high-resolution cocrystal structures for a majority of the compounds. Despite fairly uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MIC) ranging from 0.78 to >100 µM. Cellular accumulation studies showed a nearly 10-fold enhancement in accumulation of a C-2'-α analogue over the corresponding C-2'-ß analogue, consistent with their differential whole-cell activity.
Assuntos
Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Carbono-Nitrogênio Ligases/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Nucleosídeos/química , Antituberculosos/síntese química , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Biotinilação , Carbono-Nitrogênio Ligases/metabolismo , Cristalografia por Raios X , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Nucleosídeos/síntese química , Nucleosídeos/farmacologia , Conformação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The PLP-dependent transaminase (BioA) of Mycobacterium tuberculosis and other pathogens that catalyzes the second step of biotin biosynthesis is a now well-validated target for antibacterial development. Fragment screening by differential scanning fluorimetry has been performed to discover new chemical scaffolds and promote optimization of existing inhibitors. Calorimetry confirms binding of six molecules with high ligand efficiency. Thermodynamic data identifies which molecules bind with the enthalpy driven stabilization preferred in compounds that represent attractive starting points for future optimization. Crystallographic characterization of complexes with these molecules reveals the dynamic nature of the BioA active site. Different side chain conformational states are stabilized in response to binding by different molecules. A detailed analysis of conformational diversity in available BioA structures is presented, resulting in the identification of two states that might be targeted with molecular scaffolds incorporating well-defined conformational attributes. This new structural data can be used as part of a scaffold hopping strategy to further optimize existing inhibitors or create new small molecules with improved therapeutic potential.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Transaminases/química , Transaminases/metabolismo , Tuberculose/tratamento farmacológico , Sítios de Ligação , Varredura Diferencial de Calorimetria , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , TermodinâmicaRESUMO
Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counterscreen in biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counterscreen proved crucial to filter out compounds whose whole-cell activity was off target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were cocrystallized with BioA to provide a framework for future structure-based drug design efforts.
Assuntos
Biotina/biossíntese , Mycobacterium tuberculosis/metabolismo , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biotina/antagonistas & inibidores , Calorimetria , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Triagem em Larga Escala , Ligação de Hidrogênio , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Transaminases/antagonistas & inibidores , Transaminases/metabolismoRESUMO
The secreted anthrax toxin consists of three components: the protective antigen (PA), edema factor (EF) and lethal factor (LF). LF, a zinc metalloproteinase, compromises the host immune system primarily by targeting mitogen-activated protein kinase kinases in macrophages. Peptide substrates and small-molecule inhibitors bind LF in the space between domains 3 and 4 of the hydrolase. Domain 3 is attached on a hinge to domain 2 via residues Ile300 and Pro385, and can move through an angular arc of greater than 35° in response to the binding of different ligands. Here, multiple LF structures including five new complexes with co-crystallized inhibitors are compared and three frequently populated LF conformational states termed `bioactive', `open' and `tight' are identified. The bioactive position is observed with large substrate peptides and leaves all peptide-recognition subsites open and accessible. The tight state is seen in unliganded and small-molecule complex structures. In this state, domain 3 is clamped over certain substrate subsites, blocking access. The open position appears to be an intermediate state between these extremes and is observed owing to steric constraints imposed by specific bound ligands. The tight conformation may be the lowest-energy conformation among the reported structures, as it is the position observed with no bound ligand, while the open and bioactive conformations are likely to be ligand-induced.
Assuntos
Antígenos de Bactérias/química , Bacillus anthracis/química , Toxinas Bacterianas/química , Metaloendopeptidases/química , Antraz/microbiologia , Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Ligantes , Metaloendopeptidases/metabolismo , Modelos Moleculares , Peptídeos , Conformação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Selective inhibitors of hyaluronan (HA) binding to the cell surface receptor CD44 will have value as probes of CD44-mediated signaling and have potential as therapeutic agents in chronic inflammation, cardiovascular disease, and cancer. Using biophysical binding assays, fragment screening, and crystallographic characterization of complexes with the CD44 HA binding domain, we have discovered an inducible pocket adjacent to the HA binding groove into which small molecules may bind. Iterations of fragment combination and structure-driven design have allowed identification of a series of 1,2,3,4-tetrahydroisoquinolines as the first nonglycosidic inhibitors of the CD44-HA interaction. The affinity of these molecules for the CD44 HA binding domain parallels their ability to interfere with CD44 binding to polymeric HA in vitro. X-ray crystallographic complexes of lead compounds are described and compared to a new complex with a short HA tetrasaccharide, to establish the tetrahydroisoquinoline pharmacophore as an attractive starting point for lead optimization.
Assuntos
Carboidratos , Receptores de Hialuronatos/efeitos dos fármacos , Proteínas/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Indicadores e Reagentes , Camundongos , Modelos Moleculares , Conformação Molecular , Polissacarídeos/química , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
7,8-Diaminopelargonic acid synthase (BioA) of Mycobacterium tuberculosis is a recently validated target for therapeutic intervention in the treatment of tuberculosis (TB). Using biophysical fragment screening and structural characterization of compounds, we have identified a potent aryl hydrazine inhibitor of BioA that reversibly modifies the pyridoxal-5'-phosphate (PLP) cofactor, forming a stable quinonoid. Analogous hydrazides also form covalent adducts that can be observed crystallographically but are incapable of inactivating the enzyme. In the X-ray crystal structures, small molecules induce unexpected conformational remodeling in the substrate binding site. We compared these conformational changes to those induced upon binding of the substrate (7-keto-8-aminopelargonic acid), and characterized the inhibition kinetics and the X-ray crystal structures of BioA with the hydrazine compound and analogues to unveil the mechanism of this reversible covalent modification.
Assuntos
Proteínas de Bactérias/metabolismo , Hidrazinas/metabolismo , Mycobacterium tuberculosis/enzimologia , Transaminases/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Hidrazinas/química , Cinética , Simulação de Acoplamento Molecular , Transaminases/antagonistas & inibidoresRESUMO
A software tool and workflow based on distance geometry is presented that can be used to search for local similarity in substructures in a comprehensive database of experimentally derived macromolecular structure. The method does not rely on fold annotation, specific secondary structure assignments, or sequence homology and may be used to locate compound substructures of multiple segments spanning different macromolecules that share a queried backbone geometry. This generalized substructure searching capability is intended to allow users to play an active part in exploring the role specific substructures play in larger protein domains, quaternary assemblies of proteins, and macromolecular complexes of proteins and polynucleotides. The user may select any portion or portions of an existing structure or complex to serve as a template for searching, and other structures that share the same structural features are identified, retrieved and overlaid to emphasize substructural likeness. Matching structures may be compared using a variety of integrated tools including molecular graphics for structure visualization and matching substructure sequence logos. A number of examples are provided that illustrate how generalized substructure searching may be used to understand both the similarity, and individuality of specific macromolecular structures. Web-based access to our substructure searching services is freely available at https://drugsite.msi.umn.edu.