59 research outputs found
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Advances in chemical protein modification.
Chemical protein modification has emerged as an invaluable tool for the development of modified proteins. The complementary use of both genetic and chemical methods has provided a large toolbox that allows the preparation of almost unlimited protein constructs with either natural or synthetically modified residues. Such a protein chemodiversity, usually achieved after translation and commonly referred to as post-translational protein modifications (PTMs), is often responsible for the vast biodiversity found in nature. These modifications include acylation, methylation, phosphorylation, sulfation, farnesylation, ubiquitination, and glycosylation, among others, and play a pivotal role in important cellular processes including trafficking, differentiation, migration, and signaling. Consequently, reproducing in a highly efficient and controlled way such natural modifications of proteins (by introducing natural PTMs) would provide an invaluable tool to study their precise function. Additionally, the possibility offered by the introduction and (bio)orthogonal modification of unnatural moieties/amino acids (usually improving the properties of natural PTMs during isolation, analysis, and processing) makes site-selective modification of proteins a key tool for interrogating and intervening biological systems both in vitro and in vivo.
Given the range of chemical modification methods available, it is now possible to decide which residue to target and which modification to attach in order to confer the desired property/function (affinity probes, fluorophores, reactive tags, etc.). For example, increasing the circulation half-life of a therapeutic protein may be achieved by the addition of polyethylene glycol (PEG). On the other hand, the use of a spectroscopic label to monitor biomolecule distribution in vivo enables the construction of highly selective imaging agents. Despite the vast progress in the field of bioconjugation chemistry, scientists still face many challenges, not only synthetically but also from a processing, manufacturing, safety, and stability perspective. A number of methods have been developed and applied for the modification of particular proteins and therefore may not be applicable to any protein of interest. Thus, there remains a need for the development of complementary reactions for the site-selective chemical modification of proteins that are mild, efficient, and robust. Several reviews covering different aspects of the chemical synthesis of proteins, from general native chemical ligation strategies and the modification of endogenous amino acids to more specialized topics such as click modification protocols, the introduction of particular PTMs including glycosylation, PEGylation, and polymerization of protein-based initiators, and the challenging labeling of a specific protein of interest in a complex cellular mixture using the so-called “bioorthogonal” reactions, have been published during the past decade.
While the later publications describe different protein syntheses/modifications in detail, the aim of the present review is not to be an exhaustive survey of all available bioconjugation methodologies but to discuss recent chemical strategies for the site-selective modification of proteins such as fast sulfur exchange or stable thioether formation, photo and metal-free cycloadditions, and other particularly challenging metal-mediated protocols. This review will be divided into two sections: transition metal-free and transition metal-mediated approaches. For clarity, we will use the following terminology throughout this manuscript: residue/amino acid/site-selective (or simply site-selective) reactions are those transformations that preferentially modify one amino acid residue over the others (e.g., cysteine versus lysine) and, thus, can be considered examples of chemoselective reactions; on the other hand transformations described as regioselective preferentially modify only one of a set of the same amino acid, in particular when more than one is present in the same molecule (e.g., solvent-exposed lysine versus internal lysine).O.B. thanks the European Commission (Marie Curie CIG) and Ministerio de Ciencia e Innovación, Spain (Juan de la Cierva Fellowship). G.J.L.B. thanks his generous sources of funding: Royal Society, FCT Portugal (FCT Investigator), European Commission (Marie Curie CIG), and the EPSRC. G.J.L.B. is a Royal Society University Research Fellow. The authors thank Paula Boutureira Regla and Francisco Pinteus da Cruz Lopes Bernardes for inspiration.This article was originally published in Chemical Reviews, 2015, 115 (5), pp 2174–2195 DOI: 10.1021/cr500399p. This is the final published version
A Sweet Galactose Transfer: Metabolic Oligosaccharide Engineering as a Tool To Study Glycans in Plasmodium Infection.
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
Machine learning for target discovery in drug development.
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.T.R. is an Investigador Auxiliar supported by FCT Portugal (CEECIND/00887/2017). T.R. acknowledges the H2020 (TWINN-2017 ACORN, Grant 807281) and FCT/FEDER (02/SAICT/2017, Grant 28333) for funding. G.J.L.B. is a Royal Society University Research Fellow (URF\R\180019) and a FCT Investigator (IF/00624/2015)
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Protein-Protein Conjugates: Tyrosine Delivers.
The residues cysteine and lysine are the undisputed champions of bioconjugation chemistry. Targeting other amino acids has been touted as a potential way to improve the synthesis of protein–peptide and protein–protein conjugates that are widely studied for their potential therapeutic ability and used as tools for understanding biological function. Now, a team of researchers at the University of California, Berkeley, have targeted solvent-exposed tyrosine residues to develop a method of preparing such conjugates
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Urban Endocrine Disruptors Targeting Breast Cancer Proteins.
Humans are exposed to a huge amount of environmental pollutants called endocrine disrupting chemicals (EDCs). These molecules interfere with the homeostasis of the body, usually through mimicking natural hormones leading to activation or blocking of their receptors. Many of these compounds have been associated with a broad range of diseases including the development or increased susceptibility to breast cancer, the most prevalent cancer in women worldwide, according to the World Health Organization. Thus, this article presents a virtual high-throughput screening (vHTS) to evaluate the affinity of proteins related to breast cancer, such as ESR1, ERBB2, PGR, BCRA1, and SHBG, among others, with EDCs from urban sources. A blind docking strategy was employed to screen each protein-ligand pair in triplicate in AutoDock Vina 2.0, using the computed binding affinities as ranking criteria. The three-dimensional structures were previously obtained from EDCs DataBank and Protein Data Bank, prepared and optimized by SYBYL X-2.0. Some of the chemicals that exhibited the best affinity scores for breast cancer proteins in each category were 1,3,7,8-tetrachlorodibenzo-p-dioxin, bisphenol A derivatives, perfluorooctanesulfonic acid, and benzo(a)pyrene, for catalase, several proteins, sex hormone-binding globulin, and cytochrome P450 1A2, respectively. An experimental validation of this approach was performed with a complex that gave a moderate binding affinity in silico, the sex hormone binding globulin (SHBG), and bisphenol A (BPA) complex. The protein was obtained using DNA recombinant technology and the physical interaction with BPA assessed through spectroscopic techniques. BPA binds on the recombinant SHBG, and this results in an increase of its α helix content. In short, this work shows the potential of several EDCs to bind breast cancer associated proteins as a tool to prioritize compounds to perform in vitro analysis to benefit the regulation or exposure prevention by the general population.This work was supported by Colciencias (567-2012 to D.M-G., 1107-519-29058, 1107-459- 21616), the University of Cartagena (0342010) and the EPSRC. G.J.L.B. is a Royal Society University Research Fellow.This is the author accepted manuscript. The final version is available from ACS via http://dx.doi.org/10.1021/acs.chemrestox.5b0034
Tetrazine Carbon Nanotubes for Pretargeted In Vivo "Click-to-Release" Bioorthogonal Tumour Imaging.
The bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) cleavage reaction between tetrazine and trans-cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single-walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO-caged molecule was used to deliver active effector molecules. To optimize a turn-on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near-infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real-time, non-invasive tumour visualization with a high target-to-background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine-functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off-site activation of fluorophore/drug
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Computational advances in combating colloidal aggregation in drug discovery.
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.D.R. is a Swiss National Science Foundation Fellow (Grants P2EZP3_168827 and P300P2_177833). G.J.L.B. is a Royal Society URF (UF110046 and URF/R/180019), an iFCT Investigator (IF/00624/2015), and the recipient of an ERC StG (TagIt, Grant Agreement 676832). T.R. and G.J.L.B. acknowledge Marie Sklodowska-Curie ITN Protein Conjugates (Grant Agreement 675007) for funding. T.R. is a Marie Curie Fellow (Grant Agreement 743640). T.R. acknowledges the H2020 (TWINN-2017 ACORN, Grant Agreement 807281) and POR Lisboa 2020/FEDER (02/SAICT/2017, Grant Agreement Lisboa-01-0145-FEDER-028333) for funding. D.R. acknowledges the MIT-IBM Watson AI Lab and the MIT SenseTime coalition for funding
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Natural product modulators of transient receptor potential (TRP) channels as potential anti-cancer agents.
Treatment of cancer is a significant challenge in clinical medicine, and its research is a top priority in chemical biology and drug discovery. Consequently, there is an urgent need for identifying innovative chemotypes capable of modulating unexploited drug targets. The transient receptor potential (TRPs) channels persist scarcely explored as targets, despite intervening in a plethora of pathophysiological events in numerous diseases, including cancer. Both agonists and antagonists have proven capable of evoking phenotype changes leading to either cell death or reduced cell migration. Among these, natural products entail biologically pre-validated and privileged architectures for TRP recognition. Furthermore, several natural products have significantly contributed to our current knowledge on TRP biology. In this Tutorial Review we focus on selected natural products, e.g. capsaicinoids, cannabinoids and terpenes, by highlighting challenges and opportunities in their use as starting points for designing natural product-inspired TRP channel modulators. Importantly, the de-orphanization of natural products as TRP channel ligands may leverage their exploration as viable strategy for developing anticancer therapies. Finally, we foresee that TRP channels may be explored for the selective pharmacodelivery of cytotoxic payloads to diseased tissues, providing an innovative platform in chemical biology and molecular medicine.We thank FCT Portugal (FCT Investigator to G. J. L. B.), the EU
(Marie-Curie CIG and Marie-Curie ITN Protein Conjugates to
G. J. L. B.), Deutsche Forschungsgemeinschaft (Postdoctoral
Fellowship to F. S.), the EPSRC and MRC for funding. G. J. L. B.
is a Royal Society University Research Fellow and the recipient
of an European Research Council Starting Grant (TagIt)
Sequential dual site-selective protein labelling enabled by lysine modification.
Methods that allow for chemical site-selective dual protein modification are scarce. Here, we provide proof-of-concept for the orthogonality and compatibility of a method for regioselective lysine modification with strategies for protein modification at cysteine and genetically encoded ketone-tagged amino acids. This sequential, orthogonal approach was applied to albumin and a therapeutic antibody to create functional dual site-selectively labelled proteins
A Microfluidic Co-Flow Route for Human Serum Albumin-Drug-Nanoparticle Assembly.
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
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