RESUMO
The dimeric architecture of tandem-repeat type galectins, such as galectin-4 (Gal-4), modulates their biological activities, although the underlying molecular mechanisms have remained elusive. Emerging evidence show that tandem-repeat galectins play an important role in innate immunity by recognizing carbohydrate antigens present on the surface of certain pathogens, which very often mimic the structures of the human self-glycan antigens. Herein, we have analyzed the binding preferences of the C-domain of Gal-4 (Gal-4C) toward the ABH-carbohydrate histo-blood antigens with different core presentations and their recognition features have been rationalized by using a combined experimental approach including NMR, solid-phase and hemagglutination assays, and molecular modeling. The data show that Gal-4C prefers A over B antigens (two-fold in affinity), contrary to the N-domain (Gal-4N), although both domains share the same preference for the type-6 presentations. The behavior of the full-length Gal-4 (Gal-4FL) tandem-repeat form has been additionally scrutinized. Isothermal titration calorimetry and NMR data demonstrate that both domains within full-length Gal-4 bind to the histo-blood antigens independently of each other, with no communication between them. In this context, the heterodimeric architecture does not play any major role, apart from the complementary A and B antigen binding preferences. However, upon binding to a bacterial lipopolysaccharide containing a multivalent version of an H-antigen mimetic as O-antigen, the significance of the galectin architecture was revealed. Indeed, our data point to the linker peptide domain and the F-face of the C-domain as key elements that provide Gal-4 with the ability to cross-link multivalent ligands, beyond the glycan binding capacity of the dimer.
RESUMO
The Gram-negative selective antibiotic darobactin A has attracted interest owing to its intriguing fused bicyclic structure and unique targeting of the outer membrane protein BamA. Darobactin, a ribosomally synthesized and post-translationally modified peptide (RiPP), is produced by a radical S-adenosyl methionine (rSAM)-dependent enzyme (DarE) and contains one ether and one C-C cross-link. Herein, we analyze the substrate tolerance of DarE and describe an underlying catalytic principle of the enzyme. These efforts produced 51 enzymatically modified darobactin variants, revealing that DarE can install the ether and C-C cross-links independently and in different locations on the substrate. Notable variants with fused bicyclic structures were characterized, including darobactin W3Y, with a non-Trp residue at the twice-modified central position, and darobactin K5F, which displays a fused diether ring pattern. While lacking antibiotic activity, quantum mechanical modeling of darobactins W3Y and K5F aided in the elucidation of the requisite features for high-affinity BamA engagement. We also provide experimental evidence for ß-oxo modification, which adds support for a proposed DarE mechanism. Based on these results, ether and C-C cross-link formation was investigated computationally, and it was determined that more stable and longer-lived aromatic Cß radicals correlated with ether formation. Further, molecular docking and transition state structures based on high-level quantum mechanical calculations support the different indole connectivity observed for ether (Trp-C7) and C-C (Trp-C6) cross-links. Finally, mutational analysis and protein structural predictions identified substrate residues that govern engagement to DarE. Our work informs on darobactin scaffold engineering and further unveils the underlying principles of rSAM catalysis.
Assuntos
Antibacterianos , Antibacterianos/química , Antibacterianos/farmacologia , Modelos MolecularesRESUMO
Selective cleavage of amide bonds holds prominent significance by facilitating precise manipulation of biomolecules, with implications spanning from basic research to therapeutic interventions. However, achieving selective cleavage of amide bonds via mild synthetic chemistry routes poses a critical challenge. Here, we report a novel amide bond-cleavage reaction triggered by Na[AuCl4] in mild aqueous conditions, where a crucial cyclization step leads to the formation of a 5-membered ring intermediate that rapidly hydrolyses to release the free amine in high yields. Notably, the reaction exhibits remarkable site-specificity to cleave peptide bonds at the C-terminus of allyl-glycine. The strategic introduction of a leaving group at the allyl position facilitated a dual-release approach through π-acid catalyzed substitution. This reaction was employed for the targeted release of the cytotoxic drug monomethyl auristatin E in combination with an antibody-drug conjugate in cancer cells. Finally, Au-mediated prodrug activation was shown in a colorectal zebrafish xenograft model, leading to a significant increase in apoptosis and tumor shrinkage. Our findings reveal a novel metal-based cleavable reaction expanding the utility of Au complexes beyond catalysis to encompass bond-cleavage reactions for cancer therapy.
RESUMO
Natural proteins are highly optimized for function but are often difficult to produce at a scale suitable for biotechnological applications due to poor expression in heterologous systems, limited solubility, and sensitivity to temperature. Thus, a general method that improves the physical properties of native proteins while maintaining function could have wide utility for protein-based technologies. Here, we show that the deep neural network ProteinMPNN, together with evolutionary and structural information, provides a route to increasing protein expression, stability, and function. For both myoglobin and tobacco etch virus (TEV) protease, we generated designs with improved expression, elevated melting temperatures, and improved function. For TEV protease, we identified multiple designs with improved catalytic activity as compared to the parent sequence and previously reported TEV variants. Our approach should be broadly useful for improving the expression, stability, and function of biotechnologically important proteins.
Assuntos
Endopeptidases , Temperatura , Endopeptidases/metabolismo , Proteínas Recombinantes de FusãoRESUMO
The ability to control the activation of prodrugs by transition metals has been shown to have great potential for controlled drug release in cancer cells. However, the strategies developed so far promote the cleavage of C-O or C-N bonds, which limits the scope of drugs to only those that present amino or hydroxyl groups. Here, we report the decaging of an ortho-quinone prodrug, a propargylated ß-lapachone derivative, through a palladium-mediated C-C bond cleavage. The reaction's kinetic and mechanistic behavior was studied under biological conditions along with computer modeling. The results indicate that palladium (II) is the active species for the depropargylation reaction, activating the triple bond for nucleophilic attack by a water molecule before the C-C bond cleavage takes place. Palladium iodide nanoparticles were found to efficiently trigger the C-C bond cleavage reaction under biocompatible conditions. In drug activation assays in cells, the protected analogue of ß-lapachone was activated by nontoxic amounts of nanoparticles, which restored drug toxicity. The palladium-mediated ortho-quinone prodrug activation was further demonstrated in zebrafish tumor xenografts, which resulted in a significant anti-tumoral effect. This work expands the transition-metal-mediated bioorthogonal decaging toolbox to include cleavage of C-C bonds and payloads that were previously not accessible by conventional strategies.
Assuntos
Naftoquinonas , Neoplasias , Pró-Fármacos , Animais , Humanos , Pró-Fármacos/farmacologia , Pró-Fármacos/química , Paládio/química , Peixe-ZebraRESUMO
Invited for the cover of this issue are the groups of Gonzalo Jiménez-Osés and Fernando López-Gallego at CIC bioGUNE and CIC biomaGUNE, respectively. The image depicts the substrate scope of an engineered acyl transferases for the synthesis of statin derivatives. Read the full text of the article at 10.1002/chem.202300911.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , AciltransferasesRESUMO
This study identifies new acyl donors for manufacturing statin analogues through the acylation of monacolin J acid by the laboratory evolved acyltransferase LovD9. Vinyl and p-nitrophenyl esters have emerged as alternate substrates for LovD9-catalyzed acylation. While vinyl esters can reach product yields as high as the ones obtained by α-dimethyl butyryl-S-methyl-3-mercaptopropionate (DMB-SMMP), the thioester for which LovD9 was evolved, p-nitrophenyl esters display a reactivity even higher than DMB-SMMP for the first acylation step yet the acylation product yield is lower. The reaction mechanisms were elucidated through quantum mechanics (QM) calculations.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Aciltransferases/metabolismo , Biocatálise , Acilação , Ésteres , Especificidade por SubstratoRESUMO
Fluorine (19 F) incorporation into glycan-binding proteins (lectins) has been achieved and exploited to monitor the binding to carbohydrate ligands by nuclear magnetic resonance (NMR) spectroscopy. Galectins are a family of lectins that bind carbohydrates, generally with weak affinities, through a combination of intermolecular interactions including a key CH-π stacking involving a conserved tryptophan residue. Herein, Galectin-3 (Gal3) and Galectin-8 (Gal8) with one and two carbohydrate recognition domains (CRDs), respectively, were selected. Gal3 contains one Trp, whereas Gal8 contains three, one at each binding site and a third one not involved in sugar binding; these were substituted by the corresponding F-Trp analogues. The presence of fluorine did not significantly modify the affinity for glycan binding, which was in slow exchange on the 19 F NMR chemical-shift timescale, even for weak ligands, and allowed binding events taking place at two different binding sites within the same lectin to be individualized.
Assuntos
Flúor , Galectinas , Galectinas/metabolismo , Carboidratos , Polissacarídeos/química , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Galectina 3/metabolismoRESUMO
Peptides containing variations of the ß-amyloid hydrophobic core and five-membered sulfamidates derived from ß-amino acid α-methylisoserine have been synthesized and fully characterized in the gas phase, solid state and in aqueous solution by a combination of experimental and computational techniques. The cyclic sulfamidate group effectively locks the secondary structure at the N-terminus of such hybrid peptides imposing a conformational restriction and stabilizing non-extended structures. This conformational bias, which is maintained in the gas phase, solid state and aqueous solution, is shown to be resistant to structure templating through assays of inâ vitro ß-amyloid aggregation, acting as ß-sheet breaker peptides with moderate activity.
Assuntos
Aminoácidos , Peptídeos beta-Amiloides , Conformação Proteica em Folha beta , Peptídeos beta-Amiloides/química , Estrutura Secundária de ProteínaRESUMO
Poly-aromatic systems that contain quinodimethyl (QDM) units are appealing for several photonic and spintronic applications owing to the unique electronic structure, aromaticity, and spin state(s) of the QDM ring. Herein, we report the synthesis and characterization of novel QDM-based chromophores 1-3, which exhibit unique photo-excited behavior and aromaticity. Extending the aromatic core with a biphenyl/phenanthryl- and a pyrrolo-fragment led to reducing the optoelectronic bandgap and modulating the photophysics QDM 1-3. Yet, QDM 2 and 3 suffer from "aromaticity imbalance" and become relatively unstable compared to the parent compound QDM 1. Further assessment of local aromaticity using computational tools revealed that the pseudo-quinoidal ring B is the main driving force allowing to easily populate the excited triplet state of these chromophores. The present study provides complementary guidelines for designing novel non-classical poly-aromatic systems.
RESUMO
Thermostability enhancement is a fundamental aspect of protein engineering as a biocatalyst's half-life is key for its industrial and biotechnological application, particularly at high temperatures and under harsh conditions. Thermostability changes upon mutation originate from modifications of the free energy of unfolding (ΔGu), making thermostabilization extremely challenging to predict with computational methods. In this contribution, we combine global conformational sampling with energy prediction using AlphaFold and Rosetta to develop a new computational protocol for the quantitative prediction of thermostability changes upon laboratory evolution of acyltransferase LovD and lipase LipA. We highlight how using an ensemble of protein conformations rather than a single three-dimensional model is mandatory for accurate thermostability predictions. By comparing our approaches with existing ones, we show that ensembles based on AlphaFold models provide more accurate and robust calculated thermostability trends than ensembles based solely on crystallographic structures as the latter introduce a strong distortion (scaffold bias) in computed thermostabilities. Eliminating this bias is critical for computer-guided enzyme design and evaluating the effect of multiple mutations on protein stability.
Assuntos
Engenharia de Proteínas , Estabilidade Enzimática , Mutação , Estabilidade Proteica , Conformação ProteicaRESUMO
Iridium-catalyzed borylations of aromatic C-H bonds are highly attractive transformations because of the diversification possibilities offered by the resulting boronates. These transformations are best carried out using bidentate bipyridine or phenanthroline ligands, and tend to be governed by steric factors, therefore resulting in the competitive functionalization of meta and/or para positions. We have now discovered that a subtle change in the bipyridine ligand, namely, the introduction of a CF3 substituent at positionâ 5, enables a complete change of regioselectivity in the borylation of aromatic amides, allowing the synthesis of a wide variety of ortho-borylated derivatives. Importantly, thorough computational studies suggest that the exquisite regio- and chemoselectivity stems from unusual outer-sphere interactions between the amide group of the substrate and the CF3 -substituted aryl ring of the bipyridine ligand.
RESUMO
Trifluoromethyl-substituted cyclopropanes (CF3 -CPAs) constitute an important class of compounds for drug discovery. While several methods have been developed for synthesis of trans-CF3 -CPAs, stereoselective production of corresponding cis-diastereomers remains a formidable challenge. We report a biocatalyst for diastereo- and enantio-selective synthesis of cis-CF3 -CPAs with activity on a variety of alkenes. We found that an engineered protoglobin from Aeropyrnum pernix (ApePgb) can catalyze this unusual reaction at preparative scale with low-to-excellent yield (6-55 %) and enantioselectivity (17-99 % ee), depending on the substrate. Computational studies revealed that the steric environment in the active site of the protoglobin forced iron-carbenoid and substrates to adopt a pro-cis near-attack conformation. This work demonstrates the capability of enzyme catalysts to tackle challenging chemistry problems and provides a powerful means to expand the structural diversity of CF3 -CPAs for drug discovery.
Assuntos
Ciclopropanos , Metano , Ciclopropanos/química , Estereoisomerismo , Metano/química , CatáliseRESUMO
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
Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody-drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the ß-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.
Assuntos
Cisteína , Proteínas , Ciclopropanos , Cisteína/química , Indicadores e Reagentes , Proteínas/químicaRESUMO
Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties.
Assuntos
Antineoplásicos , Imunoconjugados , Acetais , Antineoplásicos/química , Antineoplásicos/farmacologia , Imunoconjugados/química , Maleimidas/química , Mutação , Compostos de Sulfidrila/química , Trastuzumab/químicaRESUMO
Chemo- and diastereoselective 1,4-conjugate additions of anionic and radical C-nucleophiles to a chiral bicyclic dehydroalanine (Dha) are described. Of particular importance, radical carbon photolysis by a catalytic photoredox process using a simple method with a metal-free photocatalyst provides exceptional yields and selectivities at room temperature. Moreover, these 1,4-conjugate additions offer an excellent starting point for synthesizing enantiomerically pure carbon-ß-substituted unnatural α-amino acids (UAAs), which could have a high potential for applications in chemical biology.
Assuntos
Aminoácidos , Carbono , Estereoisomerismo , CatáliseRESUMO
Chiral bicyclic N,O-acetal isoserine derivatives have been synthesized by an acid-catalyzed tandem N,O-acetalization/intramolecular transcarbamoylation reaction between conveniently protected l-isoserine and 2,2,3,3-tetramethoxybutane. The delicate balance of the steric interactions between the different functional groups on each possible diastereoisomer controls their thermodynamic stability and hence the experimental product distribution. These chiral isoserine derivatives undergo diastereoselective alkylation at the α position, proceeding with either retention or inversion of the configuration depending on the relative configuration of the stereocenters. Quantum mechanical calculations revealed that a concave-face alkylation is favored due to smaller torsional and steric interactions at the bicyclic scaffold. This synthetic methodology gives access to chiral ß2,2-amino acids, attractive compounds bearing a quaternary stereocenter at the α position with applications in peptidomimetic and medicinal chemistry. Thus, enantiopure α-alkylisoserine derivatives were produced upon acidic hydrolysis of these alkylated scaffolds. In addition, α-benzylisoserine was readily transformed into a five-membered ring cyclic sulfamidate, which was ring opened regioselectively with representative nucleophiles to yield other types of enantiopure ß2,2-amino acids such as α-benzyl-α-heterofunctionalized-ß-alanines and α-benzylnorlanthionine derivatives.
Assuntos
Aminoácidos , Serina , Alquilação , Aminas , Aminoácidos/química , Serina/análogos & derivados , EstereoisomerismoRESUMO
Aminyl radicals are nitrogen-centered radicals of interest in synthetic strategies involving C-N bond formation due to their high reactivity. These intermediate radicals are generated by the reaction of an organic azide with tributyltin hydride (Bu3SnH) in the presence of substoichiometric amounts of azobisisobutyronitrile (AIBN). In this work, we report the regioselective rearrangement of azanorbornanic ([2.2.1]azabicyclic) aminyl radicals into 2,8-diazabicyclo[3.2.1]oct-2-ene systems. With the aim to establish the structural requirements for this ring expansion, we have studied the effect of different bridgehead atoms of the [2.2.1]bicyclic system and the presence of an alkyl substituent at C4. Attempts to perform this ring expansion on a monocyclic analogue have been also explored to evaluate the influence of the bicyclic skeleton on the rearrangement. A detailed mechanistic proposal supported by computational studies is reported.
Assuntos
NitrogênioRESUMO
A multidisciplinary study is presented to shed light on how pyrylium frameworks, as π-hole donors, establish π-π interactions. The combination of CSD analysis, computational modelling (ab intitio, DFT and MD simulations) and experimental NMR spectroscopy data provides essential information on the key parameters that characterize these intereactions, opening new avenues for further applications of this versatile heterocycle.