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1.
Proc Natl Acad Sci U S A ; 121(2): e2313326120, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38165934

RESUMO

Our understanding of how human skin cells differ according to anatomical site and tumour formation is limited. To address this, we have created a multiscale spatial atlas of healthy skin and basal cell carcinoma (BCC), incorporating in vivo optical coherence tomography, single-cell RNA sequencing, spatial global transcriptional profiling, and in situ sequencing. Computational spatial deconvolution and projection revealed the localisation of distinct cell populations to specific tissue contexts. Although cell populations were conserved between healthy anatomical sites and in BCC, mesenchymal cell populations including fibroblasts and pericytes retained signatures of developmental origin. Spatial profiling and in silico lineage tracing support a hair follicle origin for BCC and demonstrate that cancer-associated fibroblasts are an expansion of a POSTN+ subpopulation associated with hair follicles in healthy skin. RGS5+ pericytes are also expanded in BCC suggesting a role in vascular remodelling. We propose that the identity of mesenchymal cell populations is regulated by signals emanating from adjacent structures and that these signals are repurposed to promote the expansion of skin cancer stroma. The resource we have created is publicly available in an interactive format for the research community.


Assuntos
Carcinoma Basocelular , Neoplasias Cutâneas , Humanos , Neoplasias Cutâneas/patologia , Pele/patologia , Folículo Piloso
2.
Proc Natl Acad Sci U S A ; 117(12): 6484-6490, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152099

RESUMO

In redox metalloenzymes, the process of electron transfer often involves the concerted movement of a proton. These processes are referred to as proton-coupled electron transfer, and they underpin a wide variety of biological processes, including respiration, energy conversion, photosynthesis, and metalloenzyme catalysis. The mechanisms of proton delivery are incompletely understood, in part due to an absence of information on exact proton locations and hydrogen bonding structures in a bona fide metalloenzyme proton pathway. Here, we present a 2.1-Å neutron crystal structure of the complex formed between a redox metalloenzyme (ascorbate peroxidase) and its reducing substrate (ascorbate). In the neutron structure of the complex, the protonation states of the electron/proton donor (ascorbate) and all of the residues involved in the electron/proton transfer pathway are directly observed. This information sheds light on possible proton movements during heme-catalyzed oxygen activation, as well as on ascorbate oxidation.


Assuntos
Elétrons , Metaloproteínas/química , Prótons , Ascorbato Peroxidases/química , Ascorbato Peroxidases/metabolismo , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Catálise , Heme/química , Ligação de Hidrogênio , Metaloproteínas/metabolismo , Modelos Moleculares , Difração de Nêutrons , Oxirredução
3.
Glycobiology ; 31(2): 151-158, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-32601663

RESUMO

l-Fucose and l-fucose-containing polysaccharides, glycoproteins or glycolipids play an important role in a variety of biological processes. l-Fucose-containing glycoconjugates have been implicated in many diseases including cancer and rheumatoid arthritis. Interest in fucose and its derivatives is growing in cancer research, glyco-immunology, and the study of host-pathogen interactions. l-Fucose can be extracted from bacterial and algal polysaccharides or produced (bio)synthetically. While deuterated glucose and galactose are available, and are of high interest for metabolic studies and biophysical studies, deuterated fucose is not easily available. Here, we describe the production of perdeuterated l-fucose, using glyco-engineered Escherichia coli in a bioreactor with the use of a deuterium oxide-based growth medium and a deuterated carbon source. The final yield was 0.2 g L-1 of deuterated sugar, which was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We anticipate that the perdeuterated fucose produced in this way will have numerous applications in structural biology where techniques such as NMR, solution neutron scattering and neutron crystallography are widely used. In the case of neutron macromolecular crystallography, the availability of perdeuterated fucose can be exploited in identifying the details of its interaction with protein receptors and notably the hydrogen bonding network around the carbohydrate binding site.


Assuntos
Escherichia coli/metabolismo , Polissacarídeos/biossíntese , Polissacarídeos/química
4.
Biomacromolecules ; 22(4): 1600-1613, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33749252

RESUMO

Commercial mucin glycoproteins are routinely used as a model to investigate the broad range of important functions mucins fulfill in our bodies, including lubrication, protection against hostile germs, and the accommodation of a healthy microbiome. Moreover, purified mucins are increasingly selected as building blocks for multifunctional materials, i.e., as components of hydrogels or coatings. By performing a detailed side-by-side comparison of commercially available and lab-purified variants of porcine gastric mucins, we decipher key molecular motifs that are crucial for mucin functionality. As two main structural features, we identify the hydrophobic termini and the hydrophilic glycosylation pattern of the mucin glycoprotein; moreover, we describe how alterations in those structural motifs affect the different properties of mucins-on both microscopic and macroscopic levels. This study provides a detailed understanding of how distinct functionalities of gastric mucins are established, and it highlights the need for high-quality mucins-for both basic research and the development of mucin-based medical products.


Assuntos
Glicoproteínas , Mucinas , Animais , Glicoproteínas/metabolismo , Glicosilação , Hidrogéis , Lubrificação , Mucinas/metabolismo , Suínos
5.
J Struct Biol ; 205(2): 147-154, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30639924

RESUMO

Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IXmimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IXmimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.


Assuntos
Anidrases Carbônicas/metabolismo , Cristalografia por Raios X/métodos , Sacarina/farmacologia , Domínio Catalítico , Nêutrons , Ligação Proteica
6.
Biochemistry ; 57(12): 1833-1837, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29517905

RESUMO

As one of the main receptors of a second messenger, cGMP, cGMP-dependent protein kinase (PKG) isoforms I and II regulate distinct physiological processes. The design of isoform-specific activators is thus of great biomedical importance and requires detailed structural information about PKG isoforms bound with activators, including accurate positions of hydrogen atoms and a description of the hydrogen bonding and water architecture. Here, we determined a 2.2 Å room-temperature joint X-ray/neutron (XN) structure of the human PKG II carboxyl cyclic nucleotide binding (CNB-B) domain bound with a potent PKG II activator, 8-pCPT-cGMP. The XN structure directly visualizes intermolecular interactions and reveals changes in hydrogen bonding patterns upon comparison to the X-ray structure determined at cryo-temperatures. Comparative analysis of the backbone hydrogen/deuterium exchange patterns in PKG II:8-pCPT-cGMP and previously reported PKG Iß:cGMP XN structures suggests that the ability of these agonists to activate PKG is related to how effectively they quench dynamics of the cyclic nucleotide binding pocket and the surrounding regions.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo II/química , GMP Cíclico/análogos & derivados , Ativadores de Enzimas/química , Tionucleotídeos/química , GMP Cíclico/química , Humanos , Ligação de Hidrogênio , Difração de Nêutrons , Domínios Proteicos , Espalhamento a Baixo Ângulo
7.
Angew Chem Int Ed Engl ; 55(16): 4924-7, 2016 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-26958828

RESUMO

Neutron crystallography was used to directly locate two protons before and after a pH-induced two-proton transfer between catalytic aspartic acid residues and the hydroxy group of the bound clinical drug darunavir, located in the catalytic site of enzyme HIV-1 protease. The two-proton transfer is triggered by electrostatic effects arising from protonation state changes of surface residues far from the active site. The mechanism and pH effect are supported by quantum mechanics/molecular mechanics (QM/MM) calculations. The low-pH proton configuration in the catalytic site is deemed critical for the catalytic action of this enzyme and may apply more generally to other aspartic proteases. Neutrons therefore represent a superb probe to obtain structural details for proton transfer reactions in biological systems at a truly atomic level.


Assuntos
Cristalografia/métodos , Protease de HIV/metabolismo , Eletricidade Estática , Domínio Catalítico , Protease de HIV/química , Prótons , Teoria Quântica , Especificidade por Substrato
8.
Biochemistry ; 53(43): 6725-7, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25271401

RESUMO

High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG Iß CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). The XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted, explaining its low affinity for cAMP.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Ativadores de Enzimas/química , Nêutrons , Espalhamento de Radiação , Animais , AMP Cíclico/química , GMP Cíclico/química , Desenho de Fármacos , Ativação Enzimática , Humanos , Ligação de Hidrogênio
9.
Cell Rep Phys Sci ; 5(2)2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38645802

RESUMO

Pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B6, is an essential cofactor in many biosynthetic pathways. The emergence of PLP-dependent enzymes as drug targets and biocatalysts, such as tryptophan synthase (TS), has underlined the demand to understand PLP-dependent catalysis and reaction specificity. The ability of neutron diffraction to resolve the positions of hydrogen atoms makes it an ideal technique to understand how the electrostatic environment and selective protonation of PLP regulates PLP-dependent activities. Facilitated by microgravity crystallization of TS with the Toledo Crystallization Box, we report the 2.1 Å joint X-ray/neutron (XN) structure of TS with PLP in the internal aldimine form. Positions of hydrogens were directly determined in both the α- and ß-active sites, including PLP cofactor. The joint XN structure thus provides insight into the selective protonation of the internal aldimine and the electrostatic environment of TS necessary to understand the overall catalytic mechanism.

10.
Commun Chem ; 6(1): 162, 2023 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-37532884

RESUMO

Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize a vitamin B6-derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine.

11.
ACS Omega ; 8(32): 29101-29112, 2023 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-37599915

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen's large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

12.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 9): 1201-6, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22948921

RESUMO

D-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni(2+) cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg(2+) ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni(2+) ions occupying the catalytic metal site (M2) were found at two locations, while Mg(2+) in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH.


Assuntos
Aldose-Cetose Isomerases/química , Inibidores Enzimáticos/química , Polímeros/química , Domínios e Motivos de Interação entre Proteínas , Streptococcus/enzimologia , Aldose-Cetose Isomerases/antagonistas & inibidores , Cristalografia por Raios X , Modelos Moleculares , Difração de Nêutrons
13.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 1): 35-41, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22194331

RESUMO

Neutron crystallography is a powerful technique for experimental visualization of the positions of light atoms, including hydrogen and its isotope deuterium. In recent years, structural biologists have shown increasing interest in the technique as it uniquely complements X-ray crystallographic data by revealing the positions of D atoms in macromolecules. With this regained interest, access to macromolecular neutron crystallography beamlines is becoming a limiting step. In this report, it is shown that a rapid data-collection strategy can be a valuable alternative to longer data-collection times in appropriate cases. Comparison of perdeuterated rubredoxin structures refined against neutron data sets collected over hours and up to 5 d shows that rapid neutron data collection in just 14 h is sufficient to provide the positions of 269 D atoms without ambiguity.


Assuntos
Hidrogênio/análise , Difração de Nêutrons/métodos , Proteínas/química , Hidrogênio/química , Modelos Moleculares , Estrutura Terciária de Proteína , Fatores de Tempo
14.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 68(Pt 12): 1482-7, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23192028

RESUMO

Inorganic pyrophosphatase (IPPase) from the archaeon Thermococcus thioreducens was cloned, overexpressed in Escherichia coli, purified and crystallized in restricted geometry, resulting in large crystal volumes exceeding 5 mm3. IPPase is thermally stable and is able to resist denaturation at temperatures above 348 K. Owing to the high temperature tolerance of the enzyme, the protein was amenable to room-temperature manipulation at the level of protein preparation, crystallization and X-ray and neutron diffraction analyses. A complete synchrotron X-ray diffraction data set to 1.85 Šresolution was collected at room temperature from a single crystal of IPPase (monoclinic space group C2, unit-cell parameters a=106.11, b=95.46, c=113.68 Å, α=γ=90.0, ß=98.12°). As large-volume crystals of IPPase can be obtained, preliminary neutron diffraction tests were undertaken. Consequently, Laue diffraction images were obtained, with reflections observed to 2.1 Šresolution with I/σ(I) greater than 2.5. The preliminary crystallographic results reported here set in place future structure-function and mechanism studies of IPPase.


Assuntos
Proteínas Arqueais/química , Pirofosfatase Inorgânica/química , Thermococcus/enzimologia , Proteínas Arqueais/isolamento & purificação , Cristalização , Cristalografia por Raios X , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/isolamento & purificação , Difração de Nêutrons/métodos , Difração de Raios X/métodos
15.
Nat Commun ; 13(1): 194, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017516

RESUMO

The opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low-barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.


Assuntos
Aderência Bacteriana/genética , Fucose/metabolismo , Lectinas/química , Pseudomonas aeruginosa/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Clonagem Molecular , Infecção Hospitalar/microbiologia , Cristalografia por Raios X , Deutério/química , Escherichia coli/genética , Escherichia coli/metabolismo , Fucose/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ligação de Hidrogênio , Lectinas/genética , Lectinas/metabolismo , Ligantes , Nêutrons , Ligação Proteica , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Água/metabolismo
16.
Adv Healthc Mater ; 11(2): e2101719, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34710279

RESUMO

The importance of lubrication between oral surfaces provided by the salivary film is most acutely apparent when it is disrupted, a prevalent consequence of salivary gland hypofunction experienced with aging, a symptom of certain diseases, or a side effect of some medical interventions. Sufferers report difficulty with speech and oral food processing and collectively is detrimental to quality of life. Polyethylene glycol (PEG) is widely employed as a successful biocompatible boundary lubricant in engineering and biomedical applications. It is hypothesized that the immobilization of PEG to biological materials such as oral epithelial cells and tissue can mimic the salivary film and provide durable relief from the symptoms of mucosal dryness. To do so, PEG is functionalized with a sugar binding lectin (wheat germ agglutinin) to enhance epithelial adhesion through lectin-sugar interactions. Retention and lubricity are characterized on an ex vivo oral tissue tribology rig. WGA-PEG coats and retains on mucin films, oral epithelial cells, and porcine tongue tissue, and offers sustained reduction in coefficient of friction (COF). WGA-PEG could be developed into a useful topical treatment for reducing oral friction and the perception of dry mouth.


Assuntos
Saliva , Xerostomia , Animais , Lectinas/análise , Lectinas/metabolismo , Polietilenoglicóis/metabolismo , Qualidade de Vida , Saliva/metabolismo , Suínos , Xerostomia/metabolismo
17.
NPJ Microgravity ; 8(1): 13, 2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35508463

RESUMO

Biologically active vitamin B6-derivative pyridoxal 5'-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (≥0.5 mm3). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1 Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15.

18.
Chem Sci ; 13(34): 10057-10065, 2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36128223

RESUMO

Pyridoxal 5'-phosphate (PLP)-dependent enzymes have been extensively studied for their ability to fine-tune PLP cofactor electronics to promote a wide array of chemistries. Neutron crystallography offers a straightforward approach to studying the electronic states of PLP and the electrostatics of enzyme active sites, responsible for the reaction specificities, by enabling direct visualization of hydrogen atom positions. Here we report a room-temperature joint X-ray/neutron structure of aspartate aminotransferase (AAT) with pyridoxamine 5'-phosphate (PMP), the cofactor product of the first half reaction catalyzed by the enzyme. Between PMP NSB and catalytic Lys258 Nζ amino groups an equally shared deuterium is observed in an apparent low-barrier hydrogen bond (LBHB). Density functional theory calculations were performed to provide further evidence of this LBHB interaction. The structural arrangement and the juxtaposition of PMP and Lys258, facilitated by the LBHB, suggests active site preorganization for the incoming ketoacid substrate that initiates the second half-reaction.

19.
Res Sq ; 2022 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-35169792

RESUMO

The COVID-19 pandemic continues to disrupt everyday life, with constantly emerging SARS-CoV-2 variants threatening to render current vaccines ineffective. Small-molecule antivirals can provide an important therapeutic treatment option that is subject to challenges caused by the virus variants. The viral main protease (M pro ) is critical for the virus replication and thus is considered an attractive drug target for specific protease inhibitors. We performed the design and characterization of three reversible covalent hybrid inhibitors BBH-1, BBH-2 and NBH-2, whose structures were derived from those of hepatitis C protease inhibitors boceprevir and narlaprevir. A joint X-ray/neutron structure of the M pro /BBH-1 complex demonstrated that a Cys145 thiolate reaction with the inhibitor’s keto-warhead creates a negatively charged oxyanion, similar to that proposed for the M pro -catalyzed peptide bond hydrolysis. Protonation states of the ionizable residues in the M pro active site adapt to the inhibitor, which appears to be an intrinsic property of M pro . Structural comparisons of the hybrid inhibitors with PF-07321332 revealed unconventional interactions of PF-07321332 with M pro which may explain its more favorable enthalpy of binding and consequently higher potency. BBH-1, BBH-2 and NBH-2 demonstrated comparable antiviral properties in vitro relative to PF-07321332, making them good candidates for further design of improved antivirals.

20.
Nat Commun ; 13(1): 2268, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35477935

RESUMO

Emerging SARS-CoV-2 variants continue to threaten the effectiveness of COVID-19 vaccines, and small-molecule antivirals can provide an important therapeutic treatment option. The viral main protease (Mpro) is critical for virus replication and thus is considered an attractive drug target. We performed the design and characterization of three covalent hybrid inhibitors BBH-1, BBH-2 and NBH-2 created by splicing components of hepatitis C protease inhibitors boceprevir and narlaprevir, and known SARS-CoV-1 protease inhibitors. A joint X-ray/neutron structure of the Mpro/BBH-1 complex demonstrates that a Cys145 thiolate reaction with the inhibitor's keto-warhead creates a negatively charged oxyanion. Protonation states of the ionizable residues in the Mpro active site adapt to the inhibitor, which appears to be an intrinsic property of Mpro. Structural comparisons of the hybrid inhibitors with PF-07321332 reveal unconventional F···O interactions of PF-07321332 with Mpro which may explain its more favorable enthalpy of binding. BBH-1, BBH-2 and NBH-2 exhibit comparable antiviral properties in vitro relative to PF-07321332, making them good candidates for further design of improved antivirals.


Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Antivirais/química , Antivirais/farmacologia , Vacinas contra COVID-19 , Proteases 3C de Coronavírus , Ciclopropanos , Humanos , Lactamas , Leucina/análogos & derivados , Nitrilas , Prolina/análogos & derivados , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Sulfonas , Ureia
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