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1.
Sci Rep ; 12(1): 16031, 2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36192567

RESUMO

In-cell protein crystallization (ICPC) has been investigated as a technique to support the advancement of structural biology because it does not require protein purification and a complicated crystallization process. However, only a few protein structures have been reported because these crystals formed incidentally in living cells and are insufficient in size and quality for structure analysis. Here, we have developed a cell-free protein crystallization (CFPC) method, which involves direct protein crystallization using cell-free protein synthesis. We have succeeded in crystallization and structure determination of nano-sized polyhedra crystal (PhC) at a high resolution of 1.80 Å. Furthermore, nanocrystals were synthesized at a reaction scale of only 20 µL using the dialysis method, enabling structural analysis at a resolution of 1.95 Å. To further demonstrate the potential of CFPC, we attempted to determine the structure of crystalline inclusion protein A (CipA), whose structure had not yet been determined. We added chemical reagents as a twinning inhibitor to the CFPC solution, which enabled us to determine the structure of CipA at 2.11 Å resolution. This technology greatly expands the high-throughput structure determination method of unstable, low-yield, fusion, and substrate-biding proteins that have been difficult to analyze with conventional methods.


Assuntos
Nanopartículas , Proteínas , Cristalização/métodos , Cristalografia por Raios X , Indóis , Propionatos , Proteínas/química
2.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 10): 348-353, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189718

RESUMO

The small molecule belumosudil was initially identified as a selective inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2) and has recently been approved for the treatment of graft-versus-host disease. However, recent studies have shown that many of the phenotypes displayed upon treatment with belumosudil were due to CK2α inhibition. CK2α is in itself a very promising therapeutic target for a range of conditions and has recently been put forward as a potential treatment for COVID-19. Belumosudil presents a promising starting point for the development of future CK2α inhibitors as it provides a safe, potent and orally bioavailable scaffold. Therefore, several of the major hurdles in drug development have already been overcome. Here, the crystal structure of belumosudil bound to the ATP site of CK2α is presented. This crystal structure combined with modelling studies further elucidates how belumosudil could be developed into a selective and potent CK2α or ROCK2 inhibitor.


Assuntos
COVID-19 , Quinases Associadas a rho , Acetamidas , Trifosfato de Adenosina , Cristalografia por Raios X , Humanos , Quinases Associadas a rho/genética
3.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 10): 354-362, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189719

RESUMO

Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane(s) and manipulate their infected host. To date, 11 secretion systems have been identified, including the type IX secretion system (T9SS) associated with human, avian and farmed-fish diseases. As a bacterial secretion system, the T9SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria. PorX is a highly conserved protein that regulates the transcription of essential T9SS components and additionally mediates the function of T9SS via direct interaction with PorL, the rotary motor protein of the T9SS. PorX is also a member of a two-component system regulatory cascade, where it serves as the response regulator that relays a signal transduced from a conserved sensor histidine kinase, PorY, to a designated sigma factor. Here, the recombinant expression and purification of PorX homologous proteins from the pathogenic bacterium Porphyromonas gingivalis and the nonpathogenic bacterium Flavobacterium johnsoniae are reported. A bioinformatical characterization of the different domains comprising the PorX protein is also provided, and the crystallization and X-ray analysis of PorX from F. johnsoniae are reported.


Assuntos
Proteínas de Bactérias , Fator sigma , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Sistemas de Secreção Bacterianos/metabolismo , Cristalização , Cristalografia por Raios X , Histidina Quinase/metabolismo , Humanos , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , Fator sigma/metabolismo , Fatores de Virulência/metabolismo
4.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 10): 363-370, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189720

RESUMO

Succinyl-CoA synthetase (SCS) catalyzes a three-step reaction in the citric acid cycle with succinyl-phosphate proposed as a catalytic intermediate. However, there are no structural data to show the binding of succinyl-phosphate to SCS. Recently, the catalytic mechanism underlying acetyl-CoA production by ATP-citrate lyase (ACLY) has been debated. The enzyme belongs to the family of acyl-CoA synthetases (nucleoside diphosphate-forming) for which SCS is the prototype. It was postulated that the amino-terminal portion catalyzes the full reaction and the carboxy-terminal portion plays only an allosteric role. This interpretation was based on the partial loss of the catalytic activity of ACLY when Glu599 was mutated to Gln or Ala, and on the interpretation that the phospho-citryl-CoA intermediate was trapped in the 2.85 Šresolution structure from cryogenic electron microscopy (cryo-EM). To better resolve the structure of the intermediate bound to the E599Q mutant, the equivalent mutation, E105αQ, was made in human GTP-specific SCS. The structure of the E105αQ mutant shows succinyl-phosphate bound to the enzyme at 1.58 Šresolution when the mutant, after phosphorylation in solution by Mg2+-ATP, was crystallized in the presence of magnesium ions, succinate and desulfo-CoA. The E105αQ mutant is still active but has a specific activity that is 120-fold less than that of the wild-type enzyme, with apparent Michaelis constants for succinate and CoA that are 50-fold and 11-fold higher, respectively. Based on this high-resolution structure, the cryo-EM maps of the E599Q ACLY complex reported previously should have revealed the binding of citryl-phosphate and CoA and not phospho-citryl-CoA.


Assuntos
ATP Citrato (pro-S)-Liase , Succinato-CoA Ligases , ATP Citrato (pro-S)-Liase/química , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Acetilcoenzima A , Acil Coenzima A , Trifosfato de Adenosina/metabolismo , Cristalografia por Raios X , Difosfatos , Guanosina Trifosfato/metabolismo , Humanos , Magnésio , Complexos Multienzimáticos , Nucleosídeos , Oxo-Ácido-Liases , Succinato-CoA Ligases/química , Succinatos , Ácido Succínico/metabolismo
5.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 10): 371-377, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189721

RESUMO

The infectious disease human monkeypox is spreading rapidly in 2022, causing a global health crisis. The genomics of Monkeypox virus (MPXV) have been extensively analyzed and reported, although little is known about the virus-encoded proteome. In particular, there are no reported experimental MPXV protein structures other than computational models. Here, a 1.52 Šresolution X-ray structure of the MPXV protein A42R, the first MPXV-encoded protein with a known structure, is reported. A42R shows structural similarity to profilins, which are cellular proteins that are known to function in the regulation of actin cytoskeletal assembly. However, structural comparison of A42R with known members of the profilin family reveals critical differences that support prior biochemical findings that A42R only weakly binds actin and does not bind poly(L-proline). In addition, the analysis suggests that A42R may make distinct interactions with phosphatidylinositol lipids. Overall, the data suggest that the role of A42R in the replication of orthopoxviruses may not be readily determined by comparison to cellular profilins. Furthermore, these findings support the need for increased efforts to determine high-resolution structures of other MPXV proteins to inform physiological studies of the poxvirus infection cycle and to reveal potential new strategies to combat human monkeypox should this emerging infectious disease with pandemic potential become more common in the future.


Assuntos
Varíola dos Macacos , Profilinas , Actinas/química , Actinas/metabolismo , Cristalografia por Raios X , Humanos , Vírus da Varíola dos Macacos/metabolismo , Fosfatidilinositóis , Profilinas/química , Profilinas/genética , Profilinas/metabolismo , Proteoma
6.
Acta Crystallogr D Struct Biol ; 78(Pt 10): 1192-1193, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189739
7.
Acta Crystallogr D Struct Biol ; 78(Pt 10): 1210-1220, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189741

RESUMO

The recombination directionality factors from Mesorhizobium spp. (RdfS) are involved in regulating the excision and transfer of integrative and conjugative elements. Here, solution small-angle X-ray scattering, and crystallization and preliminary structure solution of RdfS from Mesorhizobium japonicum R7A are presented. RdfS crystallizes in space group P212121, with evidence of eightfold rotational crystallographic/noncrystallographic symmetry. Initial structure determination by molecular replacement using ab initio models yielded a partial model (three molecules), which was completed after manual inspection revealed unmodelled electron density. The finalized crystal structure of RdfS reveals a head-to-tail polymer forming left-handed superhelices with large solvent channels. Additionally, RdfS has significant disorder in the C-terminal region of the protein, which is supported by the solution scattering data and the crystal structure. The steps taken to finalize structure determination, as well as the scattering and crystallographic characteristics of RdfS, are discussed.


Assuntos
Polímeros , Recombinação Genética , Cristalografia , Cristalografia por Raios X , Solventes , Raios X
8.
Acta Crystallogr D Struct Biol ; 78(Pt 10): 1221-1234, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189742

RESUMO

Enzymes catalyze reactions by binding and orienting substrates with dynamic interactions. Horse liver alcohol dehydrogenase catalyzes hydrogen transfer with quantum-mechanical tunneling that involves fast motions in the active site. The structures and B factors of ternary complexes of the enzyme with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or NAD+ and 2,2,2-trifluoroethanol were determined to 1.1-1.3 Šresolution below the `glassy transition' in order to extract information about the temperature-dependent harmonic motions, which are reflected in the crystallographic B factors. The refinement statistics and structures are essentially the same for each structure at all temperatures. The B factors were corrected for a small amount of radiation decay. The overall B factors for the complexes are similar (13-16 Å2) over the range 25-100 K, but increase somewhat at 150 K. Applying TLS refinement to remove the contribution of pseudo-rigid-body displacements of coenzyme binding and catalytic domains provided residual B factors of 7-10 Å2 for the overall complexes and of 5-10 Å2 for C4N of NAD+ and the methylene carbon of the alcohols. These residual B factors have a very small dependence on temperature and include local harmonic motions and apparently contributions from other sources. Structures at 100 K show complexes that are poised for hydrogen transfer, which involves atomic displacements of ∼0.3 Šand is compatible with the motions estimated from the residual B factors and molecular-dynamics simulations. At 298 K local conformational changes are also involved in catalysis, as enzymes with substitutions of amino acids in the substrate-binding site have similar positions of NAD+ and pentafluorobenzyl alcohol and similar residual B factors, but differ by tenfold in the rate constants for hydride transfer.


Assuntos
Álcool Desidrogenase , NAD , Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Aminoácidos/química , Animais , Álcoois Benzílicos/química , Álcoois Benzílicos/metabolismo , Sítios de Ligação , Carbono , Cristalografia por Raios X , Fluorbenzenos , Fluorcarbonetos , Cavalos , Hidrogênio/química , Cinética , Fígado , NAD/química , Conformação Proteica , Temperatura , Trifluoretanol/química , Trifluoretanol/metabolismo
9.
Acta Crystallogr D Struct Biol ; 78(Pt 10): 1235-1248, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189743

RESUMO

Elaiophylin (Ela), a unique 16-membered symmetric macrodiolide antibiotic, displays broad biological activity. Two rare 2-deoxy-L-fucose moieties at the ends of Ela are critical for its activity. Previously, elaiophylin glycosyltransferase (ElaGT) was identified as the enzyme that is responsible for the symmetric glycosylation of Ela, acting as a potential enzymatic tool for enhancing the diversity and activity of Ela. However, a symmetric catalytic mechanism has never been reported for a glycosyltransferase (GT). To explore the catalytic mechanism, the structure of ElaGT was determined in four forms: the apo form and Ela-bound, thymidine diphosphate-bound and uridine diphosphate-bound forms. In the Ela-bound structure, two ElaGTs form a `face-to-face' C2-symmetric homodimer with a continuous acceptor-binding pocket, allowing a molecule of Ela to shuffle through. Interestingly, this dimer interface resembles that of the activator-dependent GT EryCIII with its activator EryCII. Sequence analysis also indicates that ElaGT belongs to the activator-dependent GT family, but no putative activator has been identified in the Ela gene cluster. It was then found that the ElaGT homodimer may utilize this `face-to-face' arrangement to stabilize the Ela-binding loops on the interface and to simultaneously allosterically regulate the catalytic center. Therefore, these structures present a novel self-activating model for symmetric sugar transfer in the GT family and a new potential regulation site for substrate specificity.


Assuntos
Difosfatos , Glicosiltransferases , Antibacterianos/química , Cristalografia por Raios X , Dimerização , Glicosilação , Glicosiltransferases/química , Macrolídeos , Modelos Moleculares , Açúcares , Timidina , Difosfato de Uridina
10.
Int J Mol Sci ; 23(17)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36077400

RESUMO

The trinitromethyl moiety is a useful group for the design and development of novel energetic compounds with high nitrogen and oxygen content. In this work, by using an improved nitration method, the dinitromethyl precursor was successfully nitrated to the trinitromethyl product (2), and its structure was thoroughly characterized by FTIR, NMR, elemental analysis, differential scanning calorimetry, and single-crystal X-ray diffraction. Compound 2 has a high density (1.897 g cm-3), high heat of formation (984.8 kJ mmol-1), and a high detonation performance (D: 9351 m s-1, P: 37.46 GPa) that may find useful applications in the field of high energy density materials.


Assuntos
Substâncias Explosivas , Oxidiazóis , Cristalografia por Raios X , Substâncias Explosivas/química , Nitrogênio , Oxidiazóis/química , Oxigênio/química
11.
Molecules ; 27(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36080329

RESUMO

Reactions of isocyanates/isothiocyanates with primary and secondary phosphines without solvent at room temperature afforded phosphinecarboxamide/phosphinecarbothioamide, respectively, in excellent yields. Furthermore, palladium complex Pd(COD)Cl2 was allowed to react with Ph2PC(O)NHPh (1a) to afford [Pd{Ph2PC(O)NHPh-κP}2Cl2] (3). On the other hand, the reaction of Pd(COD)Cl2 with 1 eq. of Ph2PC(S)NHPh (2a) afforded [PdCl2{Ph2PC(S)NHPh-κP,S}] (4). In the case of a 1:2 molar ratio, [PdCl{Ph2PC(S)NHPh-κP,S}{Ph2PC(S)NHPh-κP}]Cl (5) was formed. The newly obtained compounds were fully characterized using multielement NMR measurements and elemental analyses. In addition, the molecular structures of Ph2PC(O)NH(CH2)2Cl (1j), Ph2PC(S)NHPh(4-Cl) (2c), and 3-5 were determined using single-crystal X-ray diffraction.


Assuntos
Compostos Organometálicos , Fosfinas , Cristalografia por Raios X , Estrutura Molecular , Compostos Organometálicos/química , Paládio/química , Fosfinas/química
12.
Acta Crystallogr C Struct Chem ; 78(Pt 9): 462-469, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36063373

RESUMO

Two new 1,2,4-triazine-containing sulfonamide derivatives, namely, 4-bromo-N-(5,6-diphenyl-2H-1,2,4-triazin-3-ylidene)benzenesulfonamide, C21H15BrN4O4S, 3a, and methyl 2-{[(5,6-diphenyl-1,2,4-triazin-3-yl)sulfamoyl]methyl}benzoate, C24H20N4O4S, 3b, which crystallize in the different sulfonimide and sulfonamide tautomeric forms, respectively, were synthesized and characterized by spectroscopic, X-ray diffraction and theoretical calculation methods. Both molecules adopt a very similar conformation of the common part of the structure and the differences occur within the substituents on the sulfonamide group. The amino groups characteristic for the existing tautomeric forms are involved in strong intermolecular N-H...N and N-H...O hydrogen bonds in 3a and 3b, respectively. The Hirshfeld surface analysis showed that H...H contacts constitute a high percentage of the intermolecular interactions. Theoretical calculations at the ab initio DFT/B3LYP/6-311++G(d,p) level showed that the two tautomeric forms observed for 3a and 3b can co-exist in chloroform, ethanol and water solutions, with a distinct predominance of the sulfonamide form; the participation of the sulfonimide form increases with increasing solvent polarity.


Assuntos
Sulfonamidas , Triazinas , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares
13.
Acta Crystallogr C Struct Chem ; 78(Pt 9): 493-506, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36063377

RESUMO

The novel compound N-(2-hydroxy-5-methylphenyl)-2,3-dimethoxybenzamide, C16H17NO4, I, was prepared by a two-step reaction and then characterized by elemental analysis and X-ray diffraction (XRD) methods. Moreover, its spectroscopic properties were investigated by FT-IR and 1H and 13C NMR. Compound I crystallized in the monoclinic space group P21/c and the molecular geometry is not planar, being divided into three planar regions. Supramolecular structures are formed by connecting units via hydrogen bonds. The ground-state molecular structure of I was optimized by the DFT-B3LYP/6-31G(d,p) method and the theoretical structure was compared with that obtained by X-ray diffraction. Intermolecular interactions in the crystal network were studied by two-dimensional (2D) and three-dimensional (3D) Hirshfeld analyses. The calculated electronic transition results were examined and the molecular electrostatic potentials (MEPs) were also determined. The in vitro antimicrobial activities of I against three Gram-positive bacteria, three Gram-negative bacteria and two fungi were determined. The compound was compared with several control drugs and showed better activity than the amoxicillin standard against Gram-positive bacteria B. subtilis, S. aureus and E. faecalis, and Gram-negative bacteria E. coli, K. pneumoniae and P. aeruginosa. The density functional theory (DFT)-optimized structure of the small molecule was used to perform molecular docking studies with proteins from experimentally studied bacterial and fungal organisms using AutoDock to determine the most preferred binding mode of the ligand within the protein cavity. A druglikeness assay and ADME (absorption, distribution, metabolism and excretion) and toxicology studies were carried out and predict a good drug-like character.


Assuntos
Anti-Infecciosos , Proteínas de Escherichia coli , Anti-Infecciosos/farmacologia , Bactérias , Cristalografia por Raios X , Cisteína Endopeptidases , Escherichia coli , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus aureus
14.
Acta Crystallogr C Struct Chem ; 78(Pt 9): 507-514, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36063378

RESUMO

Both trans and cis iron-CTMC complexes, namely, trans-dichlorido[(5SR,7RS,12RS,14SR)-5,7,12,14-tetramethyl-1,4,8,11-tetraazacyclotetradecane]iron(III) tetrachloridoferrate, [Fe(C14H32N4)Cl2][FeCl4] (1a), the analogous chloride methanol monosolvate, [Fe(C14H32N4)Cl2]Cl·CH3OH (1b), and cis-dichlorido[(5SR,7RS,12SR,14RS)-5,7,12,14-tetramethyl-1,4,8,11-tetraazacyclotetradecane]iron(III) chloride, [Fe(C14H32N4)Cl2]Cl (2), were successfully synthesized and structurally characterized using X-ray diffraction. The coordination geometry of the macrocycle is dependent on the stereoisomerism of CTMC. The packing of these complexes appears to be strongly influenced by extensive hydrogen-bonding interactions, which are in turn determined by the nature of the counter-anions (1a versus 1b) and/or the coordination geometry of the macrocycle (1a/1b versus 2). These observations are extended to related ferric cis- and trans-dichloro macrocyclic complexes.


Assuntos
Ciclamos , Cloretos , Cristalografia por Raios X , Compostos Férricos , Ligação de Hidrogênio , Ferro , Ligantes
15.
Acta Crystallogr D Struct Biol ; 78(Pt 9): 1143-1155, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36048154

RESUMO

Phthalonitrile derivatives are generally reported to crystallize in space groups P21/c and P1 in the literature. In this study, 7-hydroxy-4,8-dimethyl-3-pentylcoumarin (2) and its phthalonitrile derivative (2d) were crystallized; 2d crystallized in the rare trigonal space group R3. In the phthalonitrile derivative (2d), weak C-H...O hydrogen-bonding interactions promoted the formation of supramolecular double helices, and these supramolecular P and M double helices came together to form a honeycomb-like architectural motif involving one-dimensional tubular channels. In silico molecular-docking studies were performed to support the experimental processes and the results agree with each other. In vitro studies of compounds 2 and 2d were performed in LoVo colorectal adenocarcinoma and CCD18Co healthy human cell lines using flow cytometry. For compounds 2 and 2d, there was a statistically significant increase (p < 0.001) in both early and late apoptosis with respect to the control in a dose-dependent manner.


Assuntos
Adenocarcinoma , Neoplasias Colorretais , Adenocarcinoma/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Cumarínicos/farmacologia , Cristalografia por Raios X , Humanos , Hidrogênio
16.
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
17.
Chem Commun (Camb) ; 58(76): 10695-10698, 2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36069049

RESUMO

Bibrocathol is an active pharmaceutical ingredient that has been used to treat eyelid diseases for over a century, yet its structure has remained unknown. 3D electron diffraction on crystals from a commercial ointment revealed two structures. These results highlight the technique's potential in structure elucidation from microcrystalline mixtures.


Assuntos
Anti-Infecciosos Locais , Elétrons , Anti-Infecciosos Locais/farmacologia , Catecóis , Cristalografia/métodos , Cristalografia por Raios X , Pomadas
18.
J Med Chem ; 65(18): 12319-12333, 2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36101934

RESUMO

Fragment-based drug discovery (FBDD) has become an established method for the identification of efficient starting points for drug discovery programs. In recent years, electrophilic fragment screening has garnered increased attention from both academia and industry to identify novel covalent hits for tool compound or drug development against challenging drug targets. Herein, we describe the design and characterization of an acrylamide-focused electrophilic fragment library and screening campaign against extracellular signal-regulated kinase 2 (ERK2) using high-throughput protein crystallography as the primary hit-finding technology. Several fragments were found to have covalently modified the adenosine triphosphate (ATP) binding pocket Cys166 residue. From these hits, 22, a covalent ATP-competitive inhibitor with improved potency (ERK2 IC50 = 7.8 µM), was developed.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno , Inibidores de Proteínas Quinases , Acrilamidas/química , Trifosfato de Adenosina/química , Cristalografia por Raios X , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Raios X
19.
J Mol Graph Model ; 117: 108315, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36108568

RESUMO

Antiretroviral drug resistance is a therapeutic obstacle for people with HIV. HIV protease inhibitors darunavir and lopinavir are recommended for resistant infections. We characterized a protease mutant (PR10x) derived from a highly resistant clinical isolate including 10 mutations associated with resistance to lopinavir and darunavir. Compared to the wild-type protease, PR10x exhibits ∼3-fold decrease in catalytic efficiency and Ki values of 2-3 orders of magnitude worse for darunavir, lopinavir, and potent investigational inhibitor GRL-519. Crystal structures of the mutant were solved in a ligand-free form and in complex with GRL-519. The structures show altered interactions in the active site, flap-core interface, hydrophobic core, hinge region, and 80s loop compared to the corresponding wild-type protease structures. The ligand-free crystal structure exhibits a highly curled flap conformation which may amplify drug resistance. Molecular dynamics simulations performed for 1 µs on ligand-free dimers showed extremely large fluctuations in the flaps for PR10x compared to equivalent simulations on PR with a single L76V mutation or wild-type protease. This analysis offers insight about the synergistic effects of mutations in highly resistant variants.


Assuntos
Inibidores da Protease de HIV , Cristalografia por Raios X , Darunavir/farmacologia , Farmacorresistência Viral/genética , Protease de HIV/química , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Humanos , Lopinavir/farmacologia , Simulação de Dinâmica Molecular , Mutação
20.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 9): 324-329, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36048082

RESUMO

Chickpea is a crop that is known as a source of high-quality proteins. CL-AI, which belongs to the 11S globulin and cupin superfamily, was initially identified in chickpea seeds. CL-AI has recently been shown to inhibit various types of α-amylases. To determine its molecular mechanism, the crystal structure of CL-AI was solved at a final resolution of 2.2 Å. Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association, and each molecule is divided into an α-chain and a ß-chain. CL-AI has high structural similarity to other 11S globulins and canonical metal-dependent enzyme-related cupin proteins, whereas its stimilarity to α-amylase inhibitor from Phaseolus vulgaris is quite low. The structure presented here will provide insight into the function of CL-AI.


Assuntos
Cicer , Globulinas , Cicer/metabolismo , Cristalização , Cristalografia por Raios X , Globulinas/análise , Globulinas/química , Proteínas de Plantas/química , Sementes/química
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