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1.
Chemistry ; : e202401606, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38801240

RESUMO

The development of novel antivirals is crucial not only for managing current COVID-19 infections but for addressing potential future zoonotic outbreaks. SARS-CoV-2 main protease (Mpro) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS-CoV-2 Mpro to enhance its cell permeability and in vitro antiviral activity. By identifying mutation-tolerant amino acid residues within the peptide sequence, we describe the development of a second-generation Mpro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS-CoV-2 Mpro.

2.
ACS Chem Biol ; 19(1): 141-152, 2024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38085789

RESUMO

The development of effective antiviral compounds is essential for mitigating the effects of the COVID-19 pandemic. Entry of SARS-CoV-2 virions into host cells is mediated by the interaction between the viral spike (S) protein and membrane-bound angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells. Inhibition of this viral protein-host protein interaction is an attractive avenue for the development of antiviral molecules with numerous spike-binding molecules generated to date. Herein, we describe an alternative approach to inhibit the spike-ACE2 interaction by targeting the spike-binding interface of human ACE2 via mRNA display. Two consecutive display selections were performed to direct cyclic peptide ligand binding toward the spike binding interface of ACE2. Through this process, potent cyclic peptide binders of human ACE2 (with affinities in the picomolar to nanomolar range) were identified, two of which neutralized SARS-CoV-2 entry. This work demonstrates the potential of targeting ACE2 for the generation of anti-SARS-CoV-2 therapeutics as well as broad spectrum antivirals for the treatment of SARS-like betacoronavirus infection.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Enzima de Conversão de Angiotensina 2/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/metabolismo , Pandemias , Ligantes , Ligação Proteica , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Antivirais/farmacologia , Antivirais/química
3.
J Biol Chem ; 299(12): 105482, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37992806

RESUMO

Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD-the N-terminal BD of BRD3-using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution.


Assuntos
Proteoma , Fatores de Transcrição , Humanos , Proteoma/metabolismo , Fatores de Transcrição/metabolismo , Domínios Proteicos , Motivos de Aminoácidos , Peptídeos/metabolismo , Ligação Proteica , Acetilação
4.
Angew Chem Int Ed Engl ; 62(50): e202313037, 2023 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-37818778

RESUMO

Mild strategies for the selective modification of peptides and proteins are in demand for applications in therapeutic peptide and protein discovery, and in the study of fundamental biomolecular processes. Herein, we describe the development of an electrochemical selenoetherification (e-SE) platform for the efficient site-selective functionalization of polypeptides. This methodology utilizes the unique reactivity of the 21st amino acid, selenocysteine, to effect formation of valuable bioconjugates through stable selenoether linkages under mild electrochemical conditions. The power of e-SE is highlighted through late-stage C-terminal modification of the FDA-approved cancer drug leuprolide and assembly of a library of anti-HER2 affibody conjugates bearing complex cargoes. Following assembly by e-SE, the utility of functionalized affibodies for in vitro imaging and targeting of HER2 positive breast and lung cancer cell lines is also demonstrated.


Assuntos
Antineoplásicos , Selenocisteína , Selenocisteína/química , Peptídeos/química , Proteínas , Linhagem Celular
5.
Acc Chem Res ; 56(19): 2688-2699, 2023 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-37708351

RESUMO

Tyrosine sulfation is a post-translational modification (PTM) that modulates function by mediating key protein-protein interactions. One of the early proteins shown to possess this PTM was hirudin, produced in the salivary glands of the medicinal leech Hirudo medicinalis, whereby tyrosine sulfation led to a ∼10-fold improvement in α-thrombin inhibitory activity. Outside of this pioneering discovery, the involvement of tyrosine sulfation in modulating the activity of salivary proteins from other hematophagous organisms was unknown. We hypothesized that the intrinsic instability of the tyrosine sulfate functionality, particularly under the acidic conditions used to isolate and analyze peptides and proteins, has led to poor detection during the isolation and/or expression of these molecules.Herein, we summarize our efforts to interrogate the functional role of tyrosine sulfation in the thrombin inhibitory and anticoagulant activity of salivary peptides and proteins from a range of different blood feeding organisms, including leeches, ticks, mosquitoes, and flies. Specifically, we have harnessed synthetic chemistry to efficiently generate homogeneously sulfated peptides and proteins for detailed structure-function studies both in vitro and in vivo.Our studies began with the leech protein hirudin P6 (from Hirudinaria manillensis), which is both sulfated on tyrosine and O-glycosylated at a nearby threonine residue. Synthetically, this was achieved through solid-phase peptide synthesis (SPPS) with a late-stage on-resin sulfation, followed by native chemical ligation and a folding step to generate six differentially modified variants of hirudin P6 to assess the functional interplay between O-glycosylation and tyrosine sulfation. A one-pot, kinetically controlled ligation of three peptide fragments was used to assemble homogeneously sulfoforms of madanin-1 and chimadanin from the tick Haemaphysalis longicornis. Dual tyrosine sulfation at two distinct sites was shown to increase the thrombin inhibitory activity by up to 3 orders of magnitude through a novel interaction with exosite II of thrombin. The diselenide-selenoester ligation developed by our lab provided us with a means to rapidly assemble a library of different sulfated tick anticoagulant proteins: the andersonins, hyalomins, madanin-like proteins, and hemeathrins, thus enabling the generation of key structure-activity data on this family of proteins. We have also confirmed the presence of tyrosine sulfation in the anticoagulant proteins of Anopheles mosquitoes (anophelins) and the Tsetse fly (TTI) via insect expression and mass spectrometric analysis. These molecules were subsequently synthesized and assessed for thrombin inhibitory and anticoagulant activity. Activity was significantly improved by the addition of tyrosine sulfate modifications and led to molecules with potent antithrombotic activity in an in vivo murine thrombosis model.The Account concludes with our most recent work on the design of trivalent hybrids that tandemly occupy the active site and both exosites (I and II) of α-thrombin, with a TTI-anophelin hybrid (Ki = 20 fM against α-thrombin) being one of the most potent protease inhibitors and anticoagulants ever generated. Taken together, this Account highlights the importance of the tyrosine sulfate post-translational modification within salivary proteins from blood feeding organisms for enhancing anticoagulant activity. This work lays the foundation for exploiting native or engineered variants as therapeutic leads for thrombotic disorders in the future.


Assuntos
Anticoagulantes , Trombina , Animais , Camundongos , Anticoagulantes/farmacologia , Sequência de Aminoácidos , Trombina/metabolismo , Hirudinas/farmacologia , Hirudinas/química , Hirudinas/metabolismo , Tirosina/química , Proteínas e Peptídeos Salivares
6.
Nat Rev Chem ; 7(6): 383-404, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37173596

RESUMO

The homochirality of biomolecules in nature, such as DNA, RNA, peptides and proteins, has played a critical role in establishing and sustaining life on Earth. This chiral bias has also given synthetic chemists the opportunity to generate molecules with inverted chirality, unlocking valuable new properties and applications. Advances in the field of chemical protein synthesis have underpinned the generation of numerous 'mirror-image' proteins (those comprised entirely of D-amino acids instead of canonical L-amino acids), which cannot be accessed using recombinant expression technologies. This Review seeks to highlight recent work on synthetic mirror-image proteins, with a focus on modern synthetic strategies that have been leveraged to access these complex biomolecules as well as their applications in protein crystallography, drug discovery and the creation of mirror-image life.


Assuntos
Aminoácidos , Proteínas , Aminoácidos/química , Peptídeos
7.
Nat Commun ; 13(1): 6972, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36379950

RESUMO

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (Pam2Cys), delivered to mice parenterally or mucosally. Both routes of vaccination induce substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generates anti-Spike IgA, increases nAb in the serum and airways, and increases lung CD4+ T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determine that TLR2 expression in either compartment facilitates early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells is essential for optimal lung-localised, antigen-specific responses. In K18-hACE2 mice, vaccination provides complete protection against disease and sterilising lung immunity against SARS-CoV-2, with a short-term non-specific protective effect from mucosal Pam2Cys alone. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses.


Assuntos
COVID-19 , Vacinas Virais , Camundongos , Humanos , Animais , SARS-CoV-2 , Receptor 2 Toll-Like , Vacinas contra COVID-19 , COVID-19/prevenção & controle , Glicoproteína da Espícula de Coronavírus , Vacinação , Pulmão , Anticorpos Antivirais , Imunidade nas Mucosas , Anticorpos Neutralizantes
8.
Chem Commun (Camb) ; 58(49): 6890-6893, 2022 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-35638863

RESUMO

The covalent fusion of immunostimulatory adjuvants to immunogenic antigens is a promising strategy for the development of effective synthetic vaccines for infectious diseases. Herein, we describe the conjugation of a mycobacterial peptide antigen from the 6 kDa early secretory antigenic target (ESAT6) to a suitably functionalised trehalose dibehenate (TDB), a potent glycolipid adjuvant targeting macrophage inducible C-type lectin (Mincle).


Assuntos
Lectinas Tipo C , Trealose , Adjuvantes Imunológicos/farmacologia , Antígenos , Glicolipídeos/farmacologia , Trealose/farmacologia , Vacinas Sintéticas
9.
Angew Chem Int Ed Engl ; 61(20): e202200163, 2022 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-35194928

RESUMO

Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one-pot semi-synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi-synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane-associated GTPase YPT6, and site-specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.


Assuntos
Peptídeos , Proteínas
10.
Biochem Soc Trans ; 50(1): 387-401, 2022 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-34994377

RESUMO

Tyrosine-O-sulfation is a common post-translational modification (PTM) of proteins following the cellular secretory pathway. First described in human fibrinogen, tyrosine-O-sulfation has long been associated with the modulation of protein-protein interactions in several physiological processes. A number of relevant interactions for hemostasis are largely dictated by this PTM, many of which involving the serine proteinase thrombin (FIIa), a central player in the blood-clotting cascade. Tyrosine sulfation is not limited to endogenous FIIa ligands and has also been found in hirudin, a well-known and potent thrombin inhibitor from the medicinal leech, Hirudo medicinalis. The discovery of hirudin led to successful clinical application of analogs of leech-inspired molecules, but also unveiled several other natural thrombin-directed anticoagulant molecules, many of which undergo tyrosine-O-sulfation. The presence of this PTM has been shown to enhance the anticoagulant properties of these peptides from a range of blood-feeding organisms, including ticks, mosquitos and flies. Interestingly, some of these molecules display mechanisms of action that mimic those of thrombin's bona fide substrates.


Assuntos
Hirudinas , Trombina , Sequência de Aminoácidos , Animais , Anticoagulantes , Hirudinas/química , Hirudinas/metabolismo , Hirudinas/farmacologia , Trombina/metabolismo , Tirosina/metabolismo
11.
Angew Chem Weinheim Bergstr Ger ; 134(20): e202200163, 2022 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38505698

RESUMO

Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one-pot semi-synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi-synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane-associated GTPase YPT6, and site-specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.

12.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33468674

RESUMO

The global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Mycobacterium tuberculosis Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants Pam2Cys-SK4 or Pam3Cys-SK4 These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4+ T cell responses against ESAT6 and provided significant protection in the lungs from virulent M. tuberculosis aerosol challenge when administered to the pulmonary mucosa of mice. The flexible synthetic platform we describe, which allows incorporation of adjuvants to multiantigenic vaccines, represents a general approach that can be applied to rapidly assess vaccination strategies in preclinical models for a range of diseases, including against novel pandemic pathogens such as SARS-CoV-2.


Assuntos
Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/farmacologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinas Conjugadas/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Vacina BCG/farmacologia , Proteínas de Bactérias , Linfócitos T CD4-Positivos/imunologia , COVID-19/prevenção & controle , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , SARS-CoV-2/imunologia , Receptor 2 Toll-Like/imunologia , Vacinas contra a Tuberculose/imunologia , Vacinas Conjugadas/imunologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologia
13.
Curr Opin Chem Biol ; 58: 72-85, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32777686

RESUMO

The decoration of proteins with post-translational modifications (PTMs) serves as a mechanism to expand the functional repertoire of the proteome. Tyrosine sulfation is a PTM that has been shown to be a key regulator of extracellular protein-protein interactions in a select number of examples. However, the challenges associated with identifying and characterising the functional consequences of tyrosine sulfation have hindered our ability to understand the full scope of its role in the wider proteome when compared with that of other PTMs, for example, phosphorylation and glycosylation. In this account, we highlight recent advances in the prediction and detection of tyrosine sulfation and outline the need for continued innovation in this area. We also discuss the utility of emerging solid-phase synthesis and peptide ligation strategies for accessing libraries of homogeneously sulfated peptides and proteins to help reveal functional aspects of the sulfoproteome.


Assuntos
Peptídeos/química , Proteínas/química , Sulfatos/química , Tirosina/química , Humanos , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Sulfatos/metabolismo , Tirosina/metabolismo
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