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1.
J Am Chem Soc ; 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32412754

RESUMO

With a resurgence in interest in covalent drugs, there is need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as cysteine reactive warhead is employed to target Cys788 in c-KIT where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification whilst avoiding some of the limitations generally associated with established moieties.

2.
Bioconjug Chem ; 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32375474

RESUMO

The chemistry of diazo compounds has generated a huge breadth of applications in the field of organic synthesis. Their versatility combined with their tunable reactivity, stability, and chemoselectivity makes diazo compounds desirable reagents for chemical biologists. Here, we describe a method for the precise installation of diazo handles on proteins and antibodies in a mild and specific approach. Subsequent 1,3-cycloaddition reactions with strained alkynes enable both bioimaging through an in-cell "click" reaction and probing of the cysteine proteome in cell lysates. The selectivity and efficiency of these processes makes these suitable reagents for chemical biology studies.

3.
Cell Metab ; 2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32402266

RESUMO

Anti-obesity drugs in the amphetamine (AMPH) class act in the brain to reduce appetite and increase locomotion. They are also characterized by adverse cardiovascular effects with origin that, despite absence of any in vivo evidence, is attributed to a direct sympathomimetic action in the heart. Here, we show that the cardiac side effects of AMPH originate from the brain and can be circumvented by PEGylation (PEGyAMPH) to exclude its central action. PEGyAMPH does not enter the brain and facilitates SNS activity via theß2-adrenoceptor, protecting mice against obesity by increasing lipolysis and thermogenesis, coupled to higher heat dissipation, which acts as an energy sink to increase energy expenditure without altering food intake or locomotor activity. Thus, we provide proof-of-principle for a novel class of exclusively peripheral anti-obesity sympathofacilitators that are devoid of any cardiovascular and brain-related side effects.

5.
Bioorg Chem ; 100: 103927, 2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32422389

RESUMO

Protein tyrosine phosphatase 1B (PTP1B) is considered a potential therapeutic target for the treatment of type 2 diabetes mellitus (T2DM), since this enzyme plays a significant role to down-regulate insulin and leptin signalling and its over expression has been implicated in the development of insulin resistance, T2DM and obesity. Some thiazolidinediones (TZD) derivatives have been reported as promising PTP1B inhibitors with anti hyperglycemic effects. Recently, lobeglitazone, a new TZD, was described as an antidiabetic drug that targets the PPAR-γ (peroxisome γ proliferator-activated receptor) pathway, but no information on its effects on PTP1B have been reported to date. We investigated the effects of lobeglitazone on PTP1B activity in vitro. Surprisingly, lobeglitazone led to moderate inhibition on PTP1B (IC50 42.8 ± 3.8 µM) activity and to a non-competitive reversible mechanism of action. As lobeglitazone inhibits PTP1B activity in vitro, we speculate that it could also target PTP1B signalling pathway in vivo and thus contribute to potentiate its antidiabetic effects.

6.
Chembiochem ; 2020 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-32289201

RESUMO

The introduction of chemical reporter groups into glycan structures through metabolic oligosaccharide engineering (MOE) followed by bio-orthogonal ligation is an important tool to study glycosylation. We show the incorporation of synthetic galactose derivatives that bear terminal alkene groups in hepatic cells, with and without infection by Plasmodium berghei parasites, the causative agent of malaria. Additionally, we demonstrated the contribution of GLUT1 to the transport of these galactose derivatives, and observed a consistent increase in the uptake of these compounds going from naïve to P. berghei-infected cells. Finally, we used MOE to study the interplay between Plasmodium parasites and their mosquito hosts, to reveal a possible transfer of galactose building blocks from the latter to the former. This strategy has the potential to provide new insights into Plasmodium glycobiology as well as for the identification and characterization of key glycan structures for further vaccine development.

7.
Chemistry ; 26(27): 5965-5969, 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32237164

RESUMO

Nanoparticles are widely studied as carrier vehicles in biological systems because their size readily allows access through cellular membranes. Moreover, they have the potential to carry cargo molecules and as such, these factors make them especially attractive for intravenous drug delivery purposes. Interest in protein-based nanoparticles has recently gained attraction due to particle biocompatibility and lack of toxicity. However, the production of homogeneous protein nanoparticles with high encapsulation efficiencies, without the need for additional cross-linking or further engineering of the molecule, remains challenging. Herein, we present a microfluidic 3D co-flow device to generate human serum albumin/celastrol nanoparticles by co-flowing an aqueous protein solution with celastrol in ethanol. This microscale co-flow method resulted in the formation of nanoparticles with a homogeneous size distribution and an average size, which could be tuned from ≈100 nm to 1 µm by modulating the flow rates used. We show that the high stability of the particles stems from the covalent cross-linking of the naturally present cysteine residues within the particles formed during the assembly step. By choosing optimal flow rates during synthesis an encapsulation efficiency of 75±24 % was achieved. Finally, we show that this approach achieves significantly enhanced solubility of celastrol in the aqueous phase and, crucially, reduced cellular toxicity.

8.
Angew Chem Int Ed Engl ; 59(15): 6196-6200, 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-31981460

RESUMO

An azanorbornadiene bromovinyl sulfone reagent for cysteine-selective bioconjugation has been developed. Subsequent reaction with dipyridyl tetrazine leads to bond cleavage and formation of a pyrrole-linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene-tagged protein through inverse electron demand Diels-Alder cycloaddition with subsequent double retro-Diels-Alder reactions to form a stable pyrrole linkage. The sequence of site-selective bioconjugation followed by bioorthogonal bond cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which has potential for the routine preparation of protein conjugates for chemical biology studies.

9.
Chembiochem ; 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31802583

RESUMO

Rapid detection of cysteine oxidation in living cells is critical in advancing our understanding of responses to reactive oxygen species (ROS) and oxidative stress. Accordingly, there is a need to develop chemical probes that facilitate proteome-wide detection of cysteine's many oxidation states. Herein, we report the first whole-cell proteomics analysis using a norbornene probe to detect the initial product of cysteine oxidation: cysteine sulfenic acid. The oxidised proteins identified in the HeLa cell model represent the first targets of the ROS hydrogen peroxide. The panel of protein hits provides new and important information about the targets of oxidative stress, including 148 new protein members of the sulfenome. These findings provide new leads for the study and understanding of redox signalling and diseases associated with oxidative stress.

10.
Curr Opin Chem Biol ; 56: 16-22, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31734566

RESUMO

The discovery of macromolecular targets for bioactive agents is currently a bottleneck for the informed design of chemical probes and drug leads. Typically, activity profiling against genetically manipulated cell lines or chemical proteomics is pursued to shed light on their biology and deconvolute drug-target networks. By taking advantage of the ever-growing wealth of publicly available bioactivity data, learning algorithms now provide an attractive means to generate statistically motivated research hypotheses and thereby prioritize biochemical screens. Here, we highlight recent successes in machine intelligence for target identification and discuss challenges and opportunities for drug discovery.

11.
ACS Cent Sci ; 5(8): 1417-1424, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31482124

RESUMO

Protein behavior is closely regulated by a plethora of post-translational modifications (PTMs). It is therefore desirable to develop approaches to design rational PTMs to modulate specific protein functions. Here, we report one such method, and we illustrate its successful implementation by potentiating the anti-aggregation activity of a molecular chaperone. Molecular chaperones are a multifaceted class of proteins essential to protein homeostasis, and one of their major functions is to combat protein misfolding and aggregation, a phenomenon linked to a number of human disorders. In this work, we conjugated a small-molecule inhibitor of the aggregation of α-synuclein, a process associated with Parkinson's disease (PD), to a specific cysteine residue on human Hsp70, a molecular chaperone with five free cysteines. We show that this regioselective conjugation augments in vitro the anti-aggregation activity of Hsp70 in a synergistic manner. This Hsp70 variant also displays in vivo an enhanced suppression of α-synuclein aggregation and its associated toxicity in a Caenorhabditis elegans model of PD.

12.
Chem Commun (Camb) ; 55(68): 10128-10131, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31386708

RESUMO

Fueled by the therapeutic potential of the epigenetic machinery, BET bromodomains have seen high interest as drug targets. Herein, we introduce different linkers to a BET bromodomain benzodiazepine ligand (I-BET762) to gauge its implications in the development of hybrid drugs, imaging probes and small molecule drug conjugates. Biophysical studies confirmed minimal disruption to binding of the BRD4 cavity by the synthesized entities, which includes imaging probes. Target engagement was confirmed in a cellular context, but poor membrane diffusion was found despite efficient localization in the nuclei after membrane disruption. Our study highlights challenges and opportunities for the successful design of benzodiazepine-derived drug-delivery systems.


Assuntos
Benzodiazepinas/farmacologia , Fluoresceínas/farmacologia , Corantes Fluorescentes/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Benzodiazepinas/síntese química , Benzodiazepinas/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Desenho de Fármacos , Fluoresceínas/síntese química , Fluoresceínas/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Ligantes , Estrutura Molecular , Proteínas Nucleares/química , Domínios Proteicos
13.
Methods Mol Biol ; 2033: 25-37, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31332745

RESUMO

This protocol details a novel bioconjugation strategy that uses a methanesulfonyl acrylate reagent that is directed to the most reactive lysine on human serum albumin, which enables the construction of chemically defined and stable bioconjugates. The reaction proceeds rapidly and a regioselective modification is achieved using a single molar equivalent of the reagent under biocompatible conditions (37 °C, pH 8.0). Importantly, the bioconjugate retains both the secondary structural content and function of the unmodified protein. During the reaction of the amino group of lysine and the sulfonyl acrylate reagent, methanesulfinic acid is released after the conjugate addition, which then generates an electrophilic acrylate moiety on the protein. This acrylate can be further used for site-specific protein labeling using a synthetic molecule bearing a reactive amine under biocompatible conditions (21 °C, pH 8.0).

14.
Biochemistry ; 58(26): 2861-2866, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31243992

RESUMO

The recent discovery of the osmosensitive calcium (Ca2+) channel OSCA has revealed the potential mechanism by which plant cells sense diverse stimuli. Osmosensory transporters and mechanosensitive channels can detect and respond to osmotic shifts that play an important role in active cell homeostasis. Members of the TMEM63 family of proteins are described as the closest homologues of OSCAs. Here, we characterize TMEM63B, a mammalian homologue of OSCAs, recently classified as mechanosensitive. In HEK293T cells, TMEM63B localizes to the plasma membrane and is associated with F-actin. This Ca2+-permeable channel specifically induces Ca2+ influx across the membrane in response to extracellular Ca2+ concentration and hyperosmolarity. In addition, overexpression of TMEM63B in HEK293T cells significantly enhanced cell migration and wound healing. The link between Ca2+ osmosensitivity and cell migration might help to establish TMEM63B's pathogenesis, for example, in cancer in which it is frequently overexpressed.

15.
Org Biomol Chem ; 17(23): 5725-5730, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31135016

RESUMO

Bioorthogonal decaging reactions are a promising strategy for prodrug activation because they involve bond cleavage to release a molecule of interest. The trans-cyclooctene (TCO)-tetrazine inverse electron-demand Diels-Alder reaction has been widely applied in vivo for decaging of amine prodrugs, however, the release of alcohol-containing bioactive compounds has been less well studied. Here, we report a TCO-carbamate benzyl ether self-immolative linker for the release of OH-molecules upon reaction with a tetrazine trigger. The benzyl ether linker proved to be highly stable and can rapidly liberate alcohols under physiological conditions upon reaction with tetrazines. The mechanism and decaging yield were systematically examined by fluorescence and HPLC analysis by using a fluorogenic TCO-benzyl ether-coumarin probe and different 3,6-substituted tetrazine derivatives. This study revealed that decaging occurs rapidly (t1/2 = 27 min) and the cycloaddition step happens within seconds (t1/2 = 7 s) with reaction rates of ≈100 M-1 s-1. Importantly, the reaction is compatible with living organisms as demonstrated by the decaging of a prodrug of the antibacterial compound triclosan in the presence of live E. Coli, that resulted in complete cell killing by action of the released "OH-active drug". Overall, this work describes a new linker for masking alcohol functionality that can be rapidly reinstated through tetrazine-triggered decaging.


Assuntos
Álcoois/metabolismo , Escherichia coli/efeitos dos fármacos , Triclosan/farmacologia , Escherichia coli/metabolismo , Estrutura Molecular , Pró-Fármacos/química , Pró-Fármacos/farmacologia
16.
Chem Sci ; 10(16): 4515-4522, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31057781

RESUMO

We have developed [2.2.1]azabicyclic vinyl sulfone reagents that simultaneously enable cysteine-selective protein modification and introduce a handle for further bioorthogonal ligation. The reaction is fast and selective for cysteine relative to other amino acids that have nucleophilic side-chains, and the formed products are stable in human plasma and are moderately resistant to retro Diels-Alder degradation reactions. A model biotinylated [2.2.1]azabicyclic vinyl sulfone reagent was shown to efficiently label two cysteine-tagged proteins, ubiquitin and C2Am, under mild conditions (1-5 equiv. of reagent in NaPi pH 7.0, room temperature, 30 min). The resulting thioether-linked conjugates were stable and retained the native activity of the proteins. Finally, the dienophile present in the azabicyclic moiety on a functionalised C2Am protein could be fluorescently labelled through an inverse electron demand Diels-Alder reaction in cells to allow selective apoptosis imaging. The combined advantages of directness, site-specificity and easy preparation mean [2.2.1]azabicyclic vinyl sulfones can be used for residue-specific dual protein labelling/construction strategies with minimal perturbation of native function based simply on the attachment of an [2.2.1]azabicyclic moiety to cysteine.

17.
Chem Commun (Camb) ; 55(45): 6369-6372, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31089616

RESUMO

By coalescing bespoke machine learning and bioinformatics analyses with cell-based assays, we unveil the pharmacology of celastrol. Celastrol is a direct modulator of the progesterone and cannabinoid receptors, and its effects correlate with the antiproliferative activity. We demonstrate how in silico methods may drive systems biology studies for natural products.


Assuntos
Aprendizado de Máquina , Progesterona/metabolismo , Receptores de Canabinoides/metabolismo , Triterpenos/farmacologia , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , Humanos , Biologia de Sistemas
18.
Org Biomol Chem ; 17(22): 5633-5638, 2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31120093

RESUMO

Small synthetic molecules capable of inducing transmembrane anion transport have received a lot of attention as potential anti-cancer agents due to their ability to interfere with intracellular pH homeostasis. A series of triaminopyrimidine-based anion transporters have been synthesised, and they are found to diminish proton gradients across lipid bilayers at physiologically relevant pH. The compounds have pKa values (≈7.2) that allow protonation/deprotonation processes coupled with anion binding/unbinding events in physiologically relevant conditions. Synthetic vesicle transport experiments as well as solid state structures indicate synergistic binding of HCl. Cell assays show that the transporters induce apoptosis in various cancerous cell lines.


Assuntos
Antineoplásicos/farmacologia , Ácido Clorídrico/metabolismo , Pirimidinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Transporte Biológico/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Ácido Clorídrico/química , Transporte de Íons/efeitos dos fármacos , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química
19.
Dalton Trans ; 48(17): 5574-5584, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30941385

RESUMO

Three new manganese carbonyl compounds with heavy atom donors were synthesized and their potential use as photoCORMS was evaluated. Interestingly, all compounds had an elusive binding mode, in which the ligands adopted a κ2-X coordination (where X = S or Se), confirmed both by X-ray crystallography and IR spectroscopy. The stability of the title compounds in the dark was determined by monitoring the changes in the UV spectra of the compounds in both dichloromethane and acetonitrile. These studies show that in coordinating solvents there is an exchange of the bromide bonded to the metal centre by a solvent molecule, which is evidenced by the changes in the UV and IR spectra and by DFT analysis. EDA and natural bond order analyses were conducted to evaluate the influence of the heavy atom donors in the first coordination sphere of the compounds. Photoexcitation at 380 nm demonstrated that all compounds showed release of all three COs, as seen in the photoproducts detected by IR spectroscopy. Furthermore, CO release was observed when the photoCORMs were incubated with living cells, and we observed a CO-dependent inhibition of cell viability.

20.
Nat Chem ; 11(5): 402-418, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988417

RESUMO

Small molecule effectors are essential for drug discovery. Specific molecular recognition, reversible binding and dose-dependency are usually key requirements to ensure utility of a novel chemical entity. However, artefactual frequent-hitter and assay interference compounds may divert lead optimization and screening programmes towards attrition-prone chemical matter. Colloidal aggregates are the prime source of false positive readouts, either through protein sequestration or protein-scaffold mimicry. Nevertheless, assessment of colloidal aggregation remains somewhat overlooked and under-appreciated. In this Review, we discuss the impact of aggregation in drug discovery by analysing select examples from the literature and publicly-available datasets. We also examine and comment on technologies used to experimentally identify these potentially problematic entities. We focus on evidence-based computational filters and machine learning algorithms that may be swiftly deployed to flag chemical matter and mitigate the impact of aggregates in discovery programmes. We highlight the tools that can be used to scrutinize libraries, and identify and eliminate these problematic compounds.


Assuntos
Coloides/metabolismo , Descoberta de Drogas , Aprendizado de Máquina , Compostos Orgânicos/metabolismo , Proteínas/metabolismo , Coloides/química , Compostos Orgânicos/química , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
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