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1.
Proc Natl Acad Sci U S A ; 121(19): e2318003121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38691588

RESUMO

Peptides presented by HLA-E, a molecule with very limited polymorphism, represent attractive targets for T cell receptor (TCR)-based immunotherapies to circumvent the limitations imposed by the high polymorphism of classical HLA genes in the human population. Here, we describe a TCR-based bispecific molecule that potently and selectively binds HLA-E in complex with a peptide encoded by the inhA gene of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans. We reveal the biophysical and structural bases underpinning the potency and specificity of this molecule and demonstrate its ability to redirect polyclonal T cells to target HLA-E-expressing cells transduced with mycobacterial inhA as well as primary cells infected with virulent Mtb. Additionally, we demonstrate elimination of Mtb-infected cells and reduction of intracellular Mtb growth. Our study suggests an approach to enhance host T cell immunity against Mtb and provides proof of principle for an innovative TCR-based therapeutic strategy overcoming HLA polymorphism and therefore applicable to a broader patient population.


Assuntos
Antígenos de Histocompatibilidade Classe I , Mycobacterium tuberculosis , Receptores de Antígenos de Linfócitos T , Linfócitos T , Mycobacterium tuberculosis/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Linfócitos T/imunologia , Antígenos HLA-E , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Tuberculose/imunologia
2.
Eur J Immunol ; 52(4): 618-632, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35108401

RESUMO

The nonpolymorphic class Ib molecule, HLA-E, primarily presents peptides from HLA class Ia leader peptides, providing an inhibitory signal to NK cells via CD94/NKG2 interactions. Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral (HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes while maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E-specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs.


Assuntos
Aminoácidos , Antígenos HLA , Antígenos de Histocompatibilidade Classe I , Peptídeos , Receptores de Antígenos de Linfócitos T , Antígenos HLA-E
3.
Angew Chem Int Ed Engl ; 55(48): 14978-14982, 2016 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-27783466

RESUMO

Glycoside hydrolases (GHs) have attracted considerable attention as targets for therapeutic agents, and thus mechanism-based inhibitors are of great interest. We report the first structural analysis of a carbocyclic mechanism-based GH inactivator, the results of which show that the two Michaelis complexes are in 2 H3 conformations. We also report the synthesis and reactivity of a fluorinated analogue and the structure of its covalently linked intermediate (flattened 2 H3 half-chair). We conclude that these inactivator reactions mainly involve motion of the pseudo-anomeric carbon atom, knowledge that should stimulate the design of new transition-state analogues for use as chemical biology tools.

4.
Mol Microbiol ; 89(1): 84-95, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23672584

RESUMO

Bacteria often produce toxins which kill competing bacteria. Colicins, produced by and toxic to Escherichia coli bacteria are three-domain proteins so efficient that one molecule can kill a cell. The C-terminal domain carries the lethal activity and the central domain is required for surface receptor binding. The N-terminal domain, required for translocation across the outer membrane, is always intrinsically unstructured. It has always been assumed therefore that the C-terminal cytotoxic domain is required for the bactericidal activity. Here we report the unexpected finding that in isolation, the 90-residue unstructured N-terminal domain of colicin N is cytotoxic. Furthermore it causes ion leakage from cells but, unlike known antimicrobial peptides (AMPs) with this property, shows no membrane binding behaviour. Finally, its activity remains strictly dependent upon the same receptor proteins (OmpF and TolA) used by full-length colicin N. This mechanism of rapid membrane disruption, via receptor mediated binding of a soluble peptide, may reveal a new target for the development of highly specific antibacterials.


Assuntos
Colicinas/toxicidade , Escherichia coli/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Análise Mutacional de DNA , Escherichia coli/fisiologia , Proteínas de Escherichia coli/metabolismo , Porinas/metabolismo , Estrutura Terciária de Proteína
5.
J Clin Invest ; 130(5): 2673-2688, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32310221

RESUMO

Tumor-associated peptide-human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface-expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.


Assuntos
Antígenos HLA/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Especificidade de Anticorpos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Cristalografia por Raios X , Antígenos HLA/química , Antígenos HLA/genética , Humanos , Indicadores e Reagentes , Modelos Moleculares , Simulação de Dinâmica Molecular , Mimetismo Molecular/genética , Mimetismo Molecular/imunologia , Peptídeos/química , Peptídeos/genética , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia
6.
Nat Commun ; 9(1): 3243, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30104598

RESUMO

Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate's structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair (2H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair (2H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.


Assuntos
Carbaçúcares/farmacologia , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Biocatálise , Carbaçúcares/síntese química , Carbaçúcares/química , Domínio Catalítico , Cicloexenos/síntese química , Cicloexenos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Galactose/análogos & derivados , Glicosídeo Hidrolases/química , Cinética , Simulação de Dinâmica Molecular , Teoria Quântica , Thermotoga maritima/enzimologia
7.
Nat Commun ; 9(1): 3700, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194298

RESUMO

In the originally published version of this Article, the affiliation details for Tracey M. Gloster were incorrectly given as 'Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada'. The correct affiliation is 'Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9ST, UK'. This has now been corrected in both the PDF and HTML versions of the Article.

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