RESUMO
Chemical post-translational protein-protein conjugation is an important technique with growing applications in biotechnology and pharmaceutical research. Maleimides represent one of the most widely employed bioconjugation reagents. However, challenges associated with the instability of first- and second-generation maleimide technologies are yet to be fully addressed. We report the development of a novel class of maleimide reagents that can undergo on-demand ring-opening hydrolysis of the resulting thio-succinimide. This strategy enables rapid post-translational assembly of protein-protein conjugates. Thio-succinimide hydrolysis, triggered upon application of chemical, photochemical, or enzymatic stimuli, allowed homobifunctional bis-maleimide reagents to be applied in the production of stable protein-protein conjugates, with complete temporal control. Bivalent and bispecific protein-protein dimers constructed from small binders targeting antigens of oncological importance, PD-L1 and HER2, were generated with high purity, stability, and improved functionality compared to monomeric building blocks. The modularity of the approach was demonstrated through elaboration of the linker moiety through a bioorthogonal propargyl handle to produce protein-protein-fluorophore conjugates. Furthermore, extending the functionality of the homobifunctional reagents by temporarily masking reactive thiols included in the linker allowed the assembly of higher order trimeric and tetrameric single-domain antibody conjugates. The potential for the approach to be extended to proteins of greater biochemical complexity was demonstrated in the production of immunoglobulin single-domain antibody conjugates. On-demand control of thio-succinimide hydrolysis combined with the facile assembly of chemically defined homo- and heterodimers constitutes an important expansion of the chemical methods available for generating stable protein-protein conjugates.
Assuntos
Maleimidas , Succinimidas , Hidrólise , Succinimidas/química , Maleimidas/química , Proteínas/química , Receptor ErbB-2/metabolismo , Receptor ErbB-2/química , Humanos , Estrutura MolecularRESUMO
Targeted drug delivery approaches that selectively and preferentially deliver therapeutic agents to specific tissues are of great interest for safer and more effective pharmaceutical treatments. We investigated whether cathepsin B cleavage of a valine-citrulline [VC(S)]-containing linker is required for the release of monomethyl auristatin E (MMAE) from albumin-drug conjugates. In this study, we used an engineered version of human serum albumin, Veltis High Binder II (HBII), which has enhanced binding to the neonatal Fc (fragment crystallizable) receptor (FcRn) to improve drug release upon binding and FcRn-mediated recycling. The linker-payload was conjugated to cysteine 34 of albumin using a carbonylacrylic (caa) reagent which produced homogeneous and plasma stable conjugates that retained FcRn binding. Two caa-linker-MMAE reagents were synthesizedâone with a cleavable [VC(S)] linker and one with a noncleavable [VC(R)] linkerâto question whether protease-mediated cleavage is needed for MMAE release. Our findings demonstrate that cathepsin B is required to achieve efficient and selective antitumor activity. The conjugates equipped with the cleavable [VC(S)] linker had potent antitumor activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalization. In addition to the pronounced antitumor activity of the albumin conjugates in vivo, we also demonstrated their preferable tumor biodistribution and biocompatibility with no associated toxicity or side effects. These results suggest that the use of engineered albumins with high FcRn binding combined with protease cleavable linkers is an efficient strategy to target delivery of drugs to solid tumors.
Assuntos
Antineoplásicos , Imunoconjugados , Neoplasias , Humanos , Recém-Nascido , Albuminas/metabolismo , Catepsina B/metabolismo , Linhagem Celular Tumoral , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Imunoconjugados/metabolismo , Neoplasias/tratamento farmacológico , Peptídeo Hidrolases , Distribuição TecidualRESUMO
The development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α-galactosyl ceramide (α-GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B-cell activation. Herein, we introduce a novel derivatization hotspot at the α-GalCer skeleton, namely the N-substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self-adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen-specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α-GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α-GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.
Assuntos
Células T Matadoras Naturais , Vacinas , Adjuvantes Imunológicos/farmacologia , Galactosilceramidas/farmacologia , Galactosilceramidas/químicaRESUMO
Macrocyclization of peptides is typically used to fix specific bioactive conformations and improve their pharmacological properties. Recently, macrobicyclic peptides have received special attention owing to their capacity to mimic protein structures or be key components of peptide-drug conjugates. Here, we describe the development of novel synthetic strategies for two distinctive types of peptide macrobicycles. A multicomponent macrocyclo-dimerization approach is introduced for the production of interconnected ß-turns, allowing two macrocyclic rings to be formed and dimerized in one pot. Also, an on-resin double stapling strategy is described for the assembly of lactam-bridged macrobicycles with stable tertiary folds.
Assuntos
Peptídeos Cíclicos , Peptídeos , Peptídeos Cíclicos/química , Ciclização , Peptídeos/química , Lactamas , Conformação MolecularRESUMO
Multicomponent reactions are of utmost importance at generating a unique, wide, and complex chemical space. Herein we describe a novel multicomponent approach based on the combination of the isonitrile-tetrazine (4+1) cycloaddition and the Ugi four-component reaction to generate pyrazole amide derivatives. The scope of the reaction as well as mechanistic insights governing the 4H-pyrazol-4-imine tautomerization are provided. This multicomponent process provides access to a new chemical space of pyrazole amide derivatives and offers a tool for peptide modification and stapling.
RESUMO
Conjugate vaccines against encapsulated pathogens like Streptococcus pneumoniae face many challenges, including the existence of multiple serotypes with a diverse global distribution that constantly requires new formulations and higher coverage. Multivalency is usually achieved by combining capsular polysaccharide-protein conjugates from invasive serotypes, and for S. pneumoniae, this has evolved from 7- up to 20-valent vaccines. These glycoconjugate formulations often contain high concentrations of carrier proteins, which may negatively affect glycoconjugate immune response. This work broadens the scope of an efficient multicomponent strategy, leading to multivalent pneumococcal glycoconjugates assembled in a single synthetic operation. The bioconjugation method, based on the Ugi four-component reaction, enables the one-pot incorporation of two different polysaccharide antigens to a tetanus toxoid carrier, thus representing the fastest approach to achieve multivalency. The reported glycoconjugates incorporate three combinations of capsular polysaccharides 1, 6B, 14, and 18C from S. pneumoniae. The glycoconjugates were able to elicit functional specific antibodies against pneumococcal strains comparable to those shown by mixtures of the two monovalent glycoconjugates.
Assuntos
Glicoconjugados/química , Vacinas Pneumocócicas/química , Vacinas Conjugadas/química , Animais , Técnicas de Química Sintética , Glicoconjugados/síntese química , Glicoconjugados/imunologia , Glicoconjugados/uso terapêutico , Humanos , Camundongos , Modelos Moleculares , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/síntese química , Vacinas Pneumocócicas/imunologia , Vacinas Pneumocócicas/uso terapêutico , Coelhos , Streptococcus pneumoniae/imunologia , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/uso terapêuticoRESUMO
Stapled peptides derived from the Ugi macrocyclization comprise a special class of cyclopeptides with an N-substituted lactam bridge cross-linking two amino acid side chains. Herein we report a comprehensive analysis of the structural factors influencing the secondary structure of these cyclic peptides in solution. Novel insights into the s-cis/s-trans isomerism and the effect of N-functionalization on the conformation are revealed.
Assuntos
Lactamas/química , Peptídeos/química , Ciclização , Peptídeos/síntese química , Estrutura Secundária de ProteínaRESUMO
Antimicrobial resistance to conventional antibiotics and the limited alternatives to combat plant-threatening pathogens are worldwide problems. Antibiotic lipopeptides exert remarkable membrane activity, which usually is not prone to fast resistance formation, and often show organism-type selectivity. Additional modes of action commonly complement the bioactivity profiles of such compounds. The present work describes a multicomponent-based methodology for the synthesis of cyclic polycationic lipopeptides with stabilized helical structures. The protocol comprises an on solid support Ugi-4-component macrocyclization in the presence of a lipidic isocyanide. Circular dichroism was employed to study the influence of both macrocyclization and lipidation on the amphiphilic helical structure in water and micellar media. First bioactivity studies against model phytopathogens demonstrated a positive effect of the lipidation on the antimicrobial activity.
Assuntos
Antifúngicos/química , Lactamas/química , Lipopeptídeos/química , Peptídeos Cíclicos/química , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/síntese química , Antifúngicos/farmacologia , Botrytis/efeitos dos fármacos , Lipopeptídeos/síntese química , Lipopeptídeos/farmacologia , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Phytophthora infestans/efeitos dos fármacosRESUMO
Peptide stapling is traditionally used to lock peptide conformations into α-helical structures using a variety of macrocyclization chemistries. In an endeavor to add a diversity-generating tool to this repertoire, we introduce a multicomponent stapling approach enabling the simultaneous stabilization of helical secondary structures and the exocyclic N-functionalization of the side chain-tethering lactam bridge. This is accomplished by means of a novel solid-phase methodology comprising, for the first time, the on-resin Ugi reaction-based macrocyclization of peptide side chains bearing amino and carboxylic acid groups. The exocyclic diversity elements arise from the isocyanide component used in the Ugi multicomponent stapling protocol, which allows for the incorporation of relevant fragments such as lipids, sugars, polyethylene glycol, fluorescent labels, and reactive handles. We prove the utility of such exocyclic reactive groups in the bioconjugation of a maleimide-armed lactam-bridged peptide to a carrier protein. The on-resin multicomponent stapling proved efficient for the installation of not only one, but also two consecutive lactam bridges having either identical or dissimilar N-functionalities. The easy access to helical peptides with a diverse set of exocyclic functionalities shows prospect for applications in peptide drug discovery and chemical biology.
Assuntos
Lactamas/química , Lipídeos/química , Peptídeos/química , Polietilenoglicóis/química , Açúcares/químicaRESUMO
The cyclization of peptide side chains has been traditionally used to either induce or stabilize secondary structures (ß-strands, helices, reverse turns) in short peptide sequences. So far, classic peptide coupling, nucleophilic substitution, olefin metathesis, and click reactions have been the methods of choice to fold synthetic peptides by means of macrocyclization. This article describes the utilization of the Ugi reaction for the side chain-to-side chain and side chain-to-termini macrocyclization of peptides, thus enabling not only access to stable folded structures but also the incorporation of exocyclic functionalities as N-substituents. Analysis of the NMR-derived structures revealed the formation of helical turns, ß-bulges, and α-turns in cyclic peptides cross-linked at i, i + 3 and i, i + 4 positions, proving the folding effect of the multicomponent Ugi macrocyclization. Molecular dynamics simulation provided further insights on the stability and molecular motion of the side chain cross-linked peptides.
Assuntos
Peptídeos Cíclicos/síntese química , Peptídeos/química , Sequência de Aminoácidos , Cristalografia por Raios X , Ciclização , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Peptídeos Cíclicos/químicaRESUMO
Constraining small peptides into specific secondary structures has been a major challenge in peptide ligand design. So far, the major solution for decreasing the conformational flexibility in small peptides has been cyclization. An alternative is the use of topological templates, which are able to induce and/or stabilize peptide secondary structures by means of covalent attachment to the peptide. Herein a multicomponent strategy and structural analysis of a new type of peptidosteroid architecture having the steroid as N-substituent of an internal amide bond is reported. The approach comprises the one-pot conjugation of two peptide chains (or amino acid derivatives) to aminosteroids by means of the Ugi reaction to give a unique family of N-steroidal peptides. The conjugation efficiency of a variety of peptide sequences and steroidal amines, as well as their consecutive head-to-tail cyclization to produce chimeric cyclopeptide-steroid conjugates, that is, macrocyclic lipopeptides, was assessed. Determination of the three-dimensional structure of an acyclic N-steroidal peptide in solution proved that the bulky, rigid steroidal template is capable of both increasing significantly the conformational rigidity, even in a peptide sequence as short as five amino acid residues, and inducing a ß-turn secondary structure even in the all-s-trans isomer. This report provides the first evidence of the steroid skeleton as ß-turn inducer in linear peptide sequences.
Assuntos
Peptídeos/química , Esteroides/química , Sequência de Aminoácidos , Ciclização , Isomerismo , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Secundária de ProteínaRESUMO
Multicomponent reactions (MCRs) are recently expanding the plethora of solid-phase protocols for the synthesis and derivatization of peptides. Herein, we describe a solid-phase-compatible strategy based on MCRs as a powerful strategy for peptide cyclization and ligation . We illustrate, using Gramicidin S as a model peptide, how the execution of on-resin Ugi reactions enables the simultaneous backbone N-functionalization and cyclization, which are important types of derivatizations in peptide-based drug development or for incorporation of conjugation handles, or labels.
Assuntos
Peptídeos Cíclicos/química , Ciclização , GramicidinaRESUMO
Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular polysaccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical challenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the polysaccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR results were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.
Assuntos
Glicoconjugados , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Polissacarídeos , Polissacarídeos Bacterianos/química , Vacinas Conjugadas/químicaRESUMO
Solid-phase synthesis represents the methodological showcase for technological advances such as split-and-pool combinatorial chemistry and the automated synthesis of peptides, nucleic acids and polysaccharides. These strategies involve iterative coupling cycles that do not generate functional diversity besides that incorporated by the amino acids, nucleosides and monosaccharide building blocks. In sharp contrast, multicomponent reactions (MCRs) are traditionally used to generate both skeletal and appendage diversity in short, batchwise procedures. On-resin MCRs have traditionally been employed for the construction of heterocycle and peptidomimetic libraries, but that scenario has changed recently, and today the focus is more on the solid-phase derivatization of peptides and oligonucleotides. This review presents relevant experimental details and addresses the synthetic scope of such on-resin multicomponent protocols employed to accomplish specific biopolymer covalent modifications that are practically inviable by traditional solution-phase methodologies. Recommendations are provided to facilitate the implementation of solid-supported protocols and avoid possible pitfalls associated with the selection of the polymeric resin, the solvent and the order and amount of the reagents employed. We describe procedures comprising the multicomponent lipidation, biotinylation and labeling of both termini and the side chains, as well as the use of MCRs in the traceless on-resin synthesis of ligated and cyclic peptides. Solid-phase protocols for the assembly of α-helical and parallel ß-sheet peptides as well as hybrid peptide-peptoid and peptide-peptide nucleic acid architectures are described. Finally, the solid-supported multicomponent derivatization of DNA oligonucleotides is illustrated as part of the DNA-encoded library technology relying on MCR-derived heterocyclic compounds.
Assuntos
Biopolímeros/química , Técnicas de Química Combinatória/métodos , Técnicas de Síntese em Fase Sólida/métodos , Aminas , Aminoácidos , Biopolímeros/biossíntese , Biotinilação , DNA , Compostos Heterocíclicos , Oligonucleotídeos , Peptídeos/síntese química , Peptídeos Cíclicos , Resinas Sintéticas/químicaRESUMO
The property of the isonitrile group to enable the simultaneous α-addition of a strong electrophile and a nucleophile has always attracted the attention of organic chemists. Its versatility is augmented when recognizing that its high structural compactness, the inertia to most of the naturally occurring functional groups, and relatively prolonged physiological and metabolical stability, convert it into the smallest bioorthogonal group. The discovery and optimization of the isonitrile-tetrazine [4+1] cycloaddition as an alternative tool for the development of ligation and decaging strategies and the recently reported reaction of isonitriles with chlorooximes bring new opportunities for the utilization of this functional group in biological systems. Although several approaches have been reported for the synthesis of isonitrile-modified carbohydrates and polysaccharides, its incorporation in proteins has been barely explored. Besides compiling the reported methods for the assembly of isonitrile-modified proteins, this Mini-Review aims at calling attention to the real potential of this modification for protein ligation, decaging, immobilization, imaging, and many other applications at a low structural and functional cost.
RESUMO
A solid-phase approach including on-resin Ugi reactions was developed for the construction of ß-hairpins. Various N-alkylated dipeptide fragments proved capable of aligning antiparallel ß-sheets in a macrocyclic scaffold, thus serving as ß-hairpin templates. Gramicidin S was used as the model ß-hairpin to compare the Ugi-derived ß-turns with the type-II' ß-turn. The results show that the multicomponent incorporation of such N-alkylated residues allows for the simultaneous stabilization and exo-cyclic functionalization of cyclic ß-hairpins.
Assuntos
Dipeptídeos/química , Gramicidina/síntese química , Alquilação , Dipeptídeos/síntese química , Gramicidina/química , Conformação Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Técnicas de Síntese em Fase SólidaRESUMO
The Escherichia coli neutral M1-aminopeptidase (ePepN) is a novel target identified for the development of antimicrobials. Here we describe a solid-phase multicomponent approach which enabled the discovery of potent ePepN inhibitors. The on-resin protocol, developed in the frame of the Distributed Drug Discovery (D3) program, comprises the implementation of parallel Ugi-azide four-component reactions with resin-bound amino acids, thus leading to the rapid preparation of a focused library of tetrazole-peptidomimetics (TPMs) suitable for biological screening. By dose-response studies, three compounds were identified as potent and selective ePepN inhibitors, as little inhibitory effect was exhibited for the porcine ortholog aminopeptidase. The study allowed for the identification of the key structural features required for a high ePepN inhibitory activity. The most potent and selective inhibitor (TPM 11) showed a non-competitive inhibition profile of ePepN. We predicted that both diastereomers of compound TPM 11 bind to a site distinct from that occupied by the substrate. Theoretical models suggested that TPM 11 has an alternative inhibition mechanism that doesn't involve Zn coordination. On the other hand, the activity landscape analysis provided a rationale for our findings. Of note, compound TMP 2 showed in vitro antibacterial activity against Escherichia coli. Furthermore, none of the three identified inhibitors is a potent haemolytic agent, and only two compounds showed moderate cytotoxic activity toward the murine myeloma P3X63Ag cells. These results point to promising compounds for the future development of rationally designed TPMs as antibacterial agents.
Assuntos
Aminopeptidases/antagonistas & inibidores , Antibacterianos/síntese química , Descoberta de Drogas , Escherichia coli/enzimologia , Peptidomiméticos/síntese química , Tetrazóis/síntese química , Animais , Antibacterianos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Escherichia coli/efeitos dos fármacos , Humanos , Camundongos , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Técnicas de Síntese em Fase SólidaRESUMO
A new synthetic strategy for the development of multivalent antibacterial glycoconjugate vaccines is described. The approach comprises the utilization of an isocyanide-based multicomponent process for the conjugation of functionalized capsular polysaccharides of S. pneumoniae and S. Typhi to carrier proteins such as diphtheria and tetanus toxoids. For the first time, oxo- and carboxylic acid-functionalized polysaccharides could be either independently or simultaneously conjugated to immunogenic proteins by means of the Ugi-multicomponent reaction, thus leading to mono- or multivalent unimolecular glycoconjugates as vaccine candidates. Despite the high molecular weight of the two or three reacting biomolecules, the multicomponent bioconjugation proved highly efficient and reproducible. The Ugi-derived glycoconjugates showed notable antigenicity and elicited good titers of functional specific antibodies. To our knowledge, this is the only bioconjugation method that enables the incorporation of two different polysaccharidic antigens to a carrier protein in a single step. Applications in the field of self-adjuvanting, eventually anticancer, multicomponent vaccines are foreseeable.