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1.
Chem Sci ; 15(22): 8557-8568, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38846383

RESUMO

Protein modification has garnered increasing interest over the past few decades and has become an important tool in many aspects of chemical biology. In recent years, much effort has focused on site-selective modification strategies that generate more homogenous bioconjugates, and this is particularly so in the antibody modification space. Modifying native antibodies by targeting solvent-accessible cysteines liberated by interchain disulfide reduction is, perhaps, the predominant strategy for achieving more site-selectivity on an antibody scaffold. This is evidenced by numerous approved antibody therapeutics that have utilised cysteine-directed conjugation reagents and the plethora of methods/strategies focused on antibody cysteine modification. However, all of these methods have a common feature in that after the reduction of native solvent-accessible cystines, the liberated cysteines are all reacted in the same manner. Herein, we report the discovery and application of dehydroalanine forming reagents (including novel reagents) capable of regio- and chemo-selectively modifying these cysteines (differentially) on a clinically relevant antibody fragment and a full antibody. We discovered that these reagents could enable differential reactivity between light chain C-terminal cysteines, heavy chain hinge region cysteines (cysteines with an adjacent proline residue, Cys-Pro), and other heavy chain internal cysteines. This differential reactivity was also showcased on small molecules and on the peptide somatostatin. The application of these dehydroalanine forming reagents was exemplified in the preparation of a dually modified antibody fragment and full antibody. Additionally, we discovered that readily available amide coupling agents can be repurposed as dehydroalanine forming reagents, which could be of interest to the broader field of chemical biology.

2.
Chem Commun (Camb) ; 58(35): 5359-5362, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35394478

RESUMO

Herein we report a fundamental discovery on the use of tris(dialkylamino)phosphine reagents for peptide and protein modification. We discovered that C-terminal thiophosphonium species, which are uniquely stable, could be selectively and rapidly generated from their disulfide counterparts. In sharp and direct contrast, internal thiophosphonium species rapidly degrade to dehydroalanine. We demonstrate this remarkable chemoselectivity on a bis-cysteine model peptide, and the formation of a stable C-terminal-thiophosphonium adduct on an antibody fragment, as well as characterise the species in various small molecule/peptide studies.


Assuntos
Cisteína , Proteínas , Dissulfetos , Peptídeos , Processamento de Proteína Pós-Traducional
3.
Brain ; 144(8): 2284-2290, 2021 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-33742656

RESUMO

A next generation of tau PET tracers for the imaging of Alzheimer's disease and other dementias has recently been developed. Whilst the new compounds have now entered clinical studies, there is limited information available to assess their suitability for clinical applications. Head-to-head comparisons are urgently needed to understand differences in the radiotracer binding profiles. We characterized the binding of the tau tracers PI2620, RO948, MK6240 and JNJ067 in human post-mortem brain tissue from a cohort of 25 dementia cases and age-matched controls using quantitative phosphorimaging with tritium-labelled radiotracers in conjunction with phospho-tau specific immunohistochemistry. The four radiotracers depicted tau inclusions composed of paired helical filaments with high specificity, both in cases with Alzheimer's disease and in primary tauopathy cases with concomitant Alzheimer's disease pathology. In contrast, cortical binding to primary tauopathy in cases without paired helical filament tau was found to be within the range of age-matched controls. Off-target binding to monoamine oxidase B has been overcome, as demonstrated by heterologous blocking studies in basal ganglia tissue. The high variability of cortical tracer binding within the Alzheimer's disease group followed the same pattern with each tracer, suggesting that all compounds are suited to differentiate Alzheimer's disease from other dementias.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/metabolismo , Tomografia por Emissão de Pósitrons , Tauopatias/diagnóstico por imagem , Tauopatias/metabolismo
4.
Chem Commun (Camb) ; 56(75): 11090-11093, 2020 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-32812554

RESUMO

The goal of "personalised" medicine has seen a growing interest in the development of theranostic agents. Bifunctional, and targeted-trifunctional, theranostic water-soluble porphyrins with a histidine-like chelating group have been synthesised via copper-catalysed azide-alkyne cycloaddition (CuAAC) "click" chemistry in high yield and purity. They are capable of photodynamic treatment and [99mTc(CO)3]+ complexation for single-photon emission computed tomography (SPECT) imaging, with a radiochemical yield of >95%. The toxicity and phototoxicity were evaluated on HT-29 cells, DU145, and DU145-PSMA cell lines, with the targeted theranostic showing more potent phototoxicity towards DU145-PSMA expressing cells.

5.
Chemistry ; 26(34): 7602-7608, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32068310

RESUMO

In this study a bispidine ligand has been applied to the complexation of gallium(III) and radiolabelled with gallium-68 for the first time. Despite its 5-coordinate nature, the resulting complex is stable in serum for over two hours, demonstrating a ligand system well matched to the imaging window of gallium-68 positron emission tomography (PET). To show the versatility of the bispidine ligand and its potential use in PET, the bifunctional chelator was conjugated to a porphyrin, producing a PET/PDT-theranostic, which showed the same level of stability to serum as the non-conjugated gallium-68 complex. The PET/PDT complex killed >90 % of HT-29 cells upon light irradiation at 50 µm. This study shows bispidines have the versatility to be used as a ligand system for gallium-68 in PET.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Quelantes/química , Gálio/química , Porfirinas/química , Compostos Bicíclicos Heterocíclicos com Pontes/análise , Radioisótopos de Gálio , Humanos , Ligantes , Tomografia por Emissão de Pósitrons/métodos , Nanomedicina Teranóstica/métodos
6.
Chem Commun (Camb) ; 54(57): 7952-7954, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-29956694

RESUMO

A theranostic conjugate for use as a positron emission tomography (PET) radiotracer and as a photosensitiser for photodynamic therapy (PDT) has been synthesised. A water-soluble porphyrin was coupled with the bifunctional chelate, H4Dpaa.ga. This conjugate is capable of rapid 68Ga complexation under physiological conditions; with 93% and 80% radiochemical yields achieved, at pH 4.5 and pH 7.4 respectively, in 15 min at 25 °C. Photocytotoxicity was evaluated on HT-29 cells and showed the conjugate was capable of >50% cell death at 50 µM upon irradiation with light, while causing minimal toxicity in the absence of light (>95% cell survival).

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