RESUMO
ATP-binding cassette (ABC) transport systems are crucial for bacteria to ensure sufficient uptake of nutrients that are not produced de novo or improve the energy balance. The cell surface of the pathobiont Streptococcus pneumoniae (pneumococcus) is decorated with a substantial array of ABC transporters, critically influencing nasopharyngeal colonization and invasive infections. Given the auxotrophic nature of pneumococci for certain amino acids, the Ami ABC transporter system, orchestrating oligopeptide uptake, becomes indispensable in host compartments lacking amino acids. The system comprises five exposed Oligopeptide Binding Proteins (OBPs) and four proteins building the ABC transporter channel. Here, we present a structural analysis of all the OBPs in this system. Multiple crystallographic structures, capturing both open and closed conformations along with complexes involving chemically synthesized peptides, have been solved at high resolution providing insights into the molecular basis of their diverse peptide specificities. Mass spectrometry analysis of oligopeptides demonstrates the unexpected remarkable promiscuity of some of these proteins when expressed in Escherichia coli, displaying affinity for a wide range of peptides. Finally, a model is proposed for the complete Ami transport system in complex with its various OBPs. We further disclosed, through in silico modelling, some essential structural changes facilitating oligopeptide transport into the cellular cytoplasm. Thus, the structural analysis of the Ami system provides valuable insights into the mechanism and specificity of oligopeptide binding by the different OBPs, shedding light on the intricacies of the uptake mechanism and the in vivo implications for this human pathogen.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias , Oligopeptídeos , Streptococcus pneumoniae , Streptococcus pneumoniae/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Oligopeptídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Cristalografia por Raios X , Modelos Moleculares , LipoproteínasRESUMO
AL-471, the leading exponent of a class of potent HIV and enterovirus A71 (EV-A71) entry inhibitors discovered in our research group, contains four l-tryptophan (Trp) units bearing an aromatic isophthalic acid directly attached to the C2 position of each indole ring. Starting from AL-471, we (i) replaced l-Trp with d-Trp, (ii) inserted a flexible linker between C2 and the isophthalic acid, and (iii) substituted a nonaromatic carboxylic acid for the terminal isophthalic acid. Truncated analogues lacking the Trp motif were also synthesized. Our findings indicate that the antiviral activity seems to be largely independent of the stereochemistry (l- or d-) of the Trp fragment and also that both the Trp unit and the distal isophthalic moiety are essential for antiviral activity. The most potent derivative, 23 (AL-534), with the C2 shortest alkyl urea linkage (three methylenes), showed subnanomolar potency against different EV-71 clinical isolates. This finding was only observed before with the early dendrimer prototype AL-385 (12 l-Trp units) but remained unprecedented for the reduced-size prototype AL-471. Molecular modeling showed the feasibility of high-affinity binding of the novel l-Trp-decorated branches of 23 (AL-534) to an alternative site on the VP1 protein that harbors significant sequence variation among EV-71 strains.
Assuntos
Enterovirus Humano A , Infecções por Enterovirus , Enterovirus , Inibidores da Fusão de HIV , Humanos , Triptofano/metabolismo , Antivirais/farmacologiaRESUMO
Unprecedented 3D hexa-adducts of [60]fullerene peripherally decorated with twelve tryptophan (Trp) or tyrosine (Tyr) residues have been synthesized. Studies on the antiviral activity of these novel compounds against HIV and EV71 reveal that they are much more potent against HIV and equally active against EV71 than the previously described dendrimer prototypes AL-385 and AL-463, which possess the same number of Trp/Tyr residues on the periphery but attached to a smaller and more flexible pentaerythritol core. These results demonstrate the relevance of the globular 3D presentation of the peripheral groups (Trp/Tyr) as well as the length of the spacer connecting them to the central core to interact with the viral envelopes, particularly in the case of HIV, and support the hypothesis that [60]fullerene can be an alternative and attractive biocompatible carbon-based scaffold for this type of highly symmetrical dendrimers. In addition, the functionalized fullerenes here described, which display twelve peripheral negatively charged indole moieties on their globular surface, define a new and versatile class of compounds with a promising potential in biomedical applications.
Assuntos
Enterovirus , Fulerenos , Infecções por HIV , Infecções por HIV/tratamento farmacológico , Hexosaminidase A , Humanos , Triptofano , TirosinaRESUMO
Here, we report a class of tryptophan trimers and tetramers that inhibit (at low micromolar range) dengue and Zika virus infection in vitro These compounds (AL family) have three or four peripheral tryptophan moieties directly linked to a central scaffold through their amino groups; thus, their carboxylic acid groups are free and exposed to the periphery. Structure-activity relationship (SAR) studies demonstrated that the presence of extra phenyl rings with substituents other than COOH at the N1 or C2 position of the indole side chain is a requisite for the antiviral activity against both viruses. The molecules showed potent antiviral activity, with low cytotoxicity, when evaluated on different cell lines. Moreover, they were active against laboratory and clinical strains of all four serotypes of dengue virus as well as a selected group of Zika virus strains. Additional mechanistic studies performed with the two most potent compounds (AL439 and AL440) demonstrated an interaction with the viral envelope glycoprotein (domain III) of dengue 2 virus, preventing virus attachment to the host cell membrane. Since no antiviral agent is approved at the moment against these two flaviviruses, further pharmacokinetic studies with these molecules are needed for their development as future therapeutic/prophylactic drugs.
Assuntos
Antivirais/química , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Triptofano/química , Triptofano/farmacologia , Zika virus/efeitos dos fármacos , Animais , Chlorocebus aethiops , Vírus da Dengue/patogenicidade , Células Endoteliais da Veia Umbilical Humana , Humanos , Sorogrupo , Relação Estrutura-Atividade , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Zika virus/patogenicidadeRESUMO
Foot-and-mouth disease virus (FMDV) is an RNA virus belonging to the Picornaviridae family that contains three small viral proteins (VPgs), named VPg1, VPg2 and VPg3, linked to the 5'-end of the viral genome. These VPg proteins act as primers for RNA replication, which is initiated by the consecutive binding of two UMP molecules to the hydroxyl group of Tyr3 in VPg. This process, termed uridylylation, is catalyzed by the viral RNA-dependent RNA polymerase named 3Dpol. 5-Fluorouridine triphosphate (FUTP) is a potent competitive inhibitor of VPg uridylylation. Peptide analysis showed FUMP covalently linked to the Tyr3 of VPg. This fluorouridylylation prevents further incorporation of the second UMP residue. The molecular basis of how the incorporated FUMP blocks the incorporation of the second UMP is still unknown. To investigate the mechanism of inhibition of VPg uridylylation by FUMP, we have prepared a simplified 15-mer model of VPg1 containing FUMP and studied its x-ray crystal structure in complex with 3Dpol. Unfortunately, the fluorouridylylated VPg1 was disordered and not visible in the electron density maps; however, the structure of 3Dpol in the presence of VPg1-FUMP showed an 8 Å movement of the ß9-α11 loop of the polymerase towards the active site cavity relative to the complex of 3Dpol with VPg1-UMP. The conformational rearrangement of this loop preceding the 3Dpol B motif seems to block the access of the template nucleotide to the catalytic cavity. This result may be useful in the design of new antivirals against not only FMDV but also other picornaviruses, since all members of this family require the uridylylation of their VPg proteins to initiate the viral RNA synthesis.
Assuntos
Vírus da Febre Aftosa/metabolismo , Peptídeos/química , Proteínas Virais/química , Sequência de Aminoácidos , Modelos Moleculares , Conformação Molecular , Engenharia de Proteínas , RNA Polimerase Dependente de RNA/síntese química , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-Atividade , Uridina Monofosfato/química , Proteínas Virais/síntese química , Proteínas Virais/metabolismoRESUMO
New substituted benzo[g]indazoles functionalized with a 6-nitro and 6-amino groups have been synthesized by the reaction of benzylidene tetralones with hydrazine in acetic acid. The resulting conformationally-constrained compounds were evaluated for their antiproliferative activity against selected cancer cell lines. The nitro-based indazoles 11a, 11b, 12a and 12b have shown IC50 values between 5-15 µM against the lung carcinoma cell line NCI-H460. Moreover, the nitro compounds were tested for antibacterial activity where compounds 12a and 13b have shown MIC values of 250 and 62.5 µg/mL against N. gonorrhoeae with no hemolytic activity in human red blood cells (RBC).
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Bactérias/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Indazóis/química , Neoplasias/tratamento farmacológico , Antibacterianos/síntese química , Antineoplásicos/síntese química , Hemólise/efeitos dos fármacos , Humanos , Neoplasias/patologia , Células Tumorais CultivadasRESUMO
Cationic and non-peptide small molecules containing a total of six positive charges arranged on one side and a long aliphatic tail on the other have been synthesized and tested against Gram-positive and Gram-negative bacteria. The positive charges have been contributed by two aminophenol residues. These molecules have showed remarkable antimicrobial activity against Gram-positive bacteria including multidrug-resistant strains. Our structureâ»activity relationship studies demonstrated the importance of the length and flexibility of the hydrophobic tail for the antimicrobial activity. Importantly, these compounds are non-toxic to eukaryotic cells at the concentration affecting growth in bacteria, reflecting an acceptable margin of safety. The small size and easy synthetic accessibility of our molecules can be of interest for the further development of novel antimicrobials against Gram-positive bacterial pathogens, including multidrug-resistant strains.
Assuntos
Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Aminofenóis/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cátions , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/crescimento & desenvolvimento , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade da Espécie , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Tryptophan dendrimers that inhibit HIV replication by binding to the HIV envelope glycoproteins gp120 and gp41 have unexpectedly also proven to be potent, specific, and selective inhibitors of the replication of the unrelated enterovirus A71. Dendrimer 12, a consensus compound that was synthesized on the basis of the structure-activity relationship analysis of this series, is 3-fold more potent against the BrCr lab strain and, surprisingly, inhibits a large panel of clinical isolates in the low-nanomolar/high-picomolar range.
Assuntos
Fármacos Anti-HIV/farmacologia , Enterovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Relação Estrutura-AtividadeRESUMO
The synthesis and the assessment of the anti-HIV activity of a set of molecules inspired by the multivalent structures of some naturally-occurring polyphenols (tannins) are reported. Different multibranched scaffolds have been derived from pentaerythritol as the central core which distribute spatially synthetic polyphenolic subunits based on 1-substituted 2,3,4-trihydroxyphenyl moieties. A tetrapodal compound () bearing four N-(2,3,4-trihydroxyphenyl)amide groups, exhibits remarkable selective activity against HIV-1 with EC50 values in the micromolar scale, in the same range as those reported for the most representative anti-HIV tannins. Preliminary SAR studies emphasize the importance of the 1-substituted 2,3,4-trihydroxyphenyl moiety, the presence of an amide as the linker and the multivalent architecture of these molecules, since the anti-HIV activity increases with the number of polyphenolic moieties. The data support the interest in synthetic polyphenols and represent a promising starting point for further design and development of selective HIV-1 inhibitors.
Assuntos
Fármacos Anti-HIV/síntese química , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Polifenóis/síntese química , Propilenoglicóis/química , Taninos/química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Desenho de Fármacos , HIV-1/crescimento & desenvolvimento , HIV-2/crescimento & desenvolvimento , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Polifenóis/química , Polifenóis/farmacologia , Relação Estrutura-AtividadeRESUMO
Cationic ultrashort lipopeptides (USLPs) are promising antimicrobial candidates to combat multidrug-resistant bacteria. Using DICAMs, a newly synthesized family of tripeptides with net charges from -2 to +1 and a fatty amine conjugated to the C-terminus, we demonstrate that anionic and neutral zwitterionic USLPs can possess potent antimicrobial and membrane-disrupting activities against prevalent human pathogens such as Streptococcus pneumoniae and Streptococcus pyogenes. The strongest antimicrobials completely halt bacterial growth at low micromolar concentrations, reduce bacterial survival by several orders of magnitude, and may kill planktonic cells and biofilms. All of them comprise either an anionic or neutral zwitterionic peptide attached to a long fatty amine (16-18 carbon atoms) and show a preference for anionic lipid membranes enriched in phosphatidylglycerol (PG), which excludes electrostatic interactions as the main driving force for DICAM action. Hence, the hydrophobic contacts provided by the long aliphatic chains of their fatty amines are needed for DICAM's membrane insertion, while negative-charge shielding by salt counterions would reduce electrostatic repulsions. Additionally, we show that other components of the bacterial envelope, including the capsular polysaccharide, can influence the microbicidal activity of DICAMs. Several promising candidates with good-to-tolerable therapeutic ratios are identified as potential agents against S. pneumoniae and S. pyogenes. Structural characteristics that determine the preference for a specific pathogen or decrease DICAM toxicity have also been investigated.
RESUMO
Binding at the interface: We tested the inhibitory activity of a set of peptide sequences derived from an α-helix of the dimeric trypanothione reductase from Leishmania infantum. Replacement of a glutamic acid residue with a lysine promoted monomer dissociation and enzyme inhibition.
Assuntos
Leishmania infantum/enzimologia , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Leishmania infantum/genética , Leishmania infantum/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NADH NADPH Oxirredutases/genética , Peptídeos/genética , Multimerização ProteicaRESUMO
Expediting drug discovery to fight antibacterial resistance requires holistic approaches at system levels. In this study, we focused on the human-adapted pathogen Haemophilus influenzae, and by constructing a high-quality genome-scale metabolic model, we rationally identified new metabolic drug targets in this organism. Contextualization of available gene essentiality data within in silico predictions identified most genes involved in lipid metabolism as promising targets. We focused on the ß-ketoacyl-acyl carrier protein synthase III FabH, responsible for catalyzing the first step in the FASII fatty acid synthesis pathway and feedback inhibition. Docking studies provided a plausible three-dimensional model of FabH in complex with the synthetic inhibitor 1-(5-(2-fluoro-5-(hydroxymethyl)phenyl)pyridin-2-yl)piperidine-4-acetic acid (FabHi). Validating our in silico predictions, FabHi reduced H. influenzae viability in a dose- and strain-dependent manner, and this inhibitory effect was independent of fabH gene expression levels. fabH allelic variation was observed among H. influenzae clinical isolates. Many of these polymorphisms, relevant for stabilization of the dimeric active form of FabH and/or activity, may modulate the inhibitory effect as part of a complex multifactorial process with the overall metabolic context emerging as a key factor tuning FabHi activity. Synergies with antibiotics were not observed and bacteria were not prone to develop resistance. Inhibitor administration during H. influenzae infection on a zebrafish septicemia infection model cleared bacteria without signs of host toxicity. Overall, we highlight the potential of H. influenzae metabolism as a source of drug targets, metabolic models as target-screening tools, and FASII targeting suitability to counteract this bacterial infection. IMPORTANCE Antimicrobial resistance drives the need of synergistically combined powerful computational tools and experimental work to accelerate target identification and drug development. Here, we present a high-quality metabolic model of H. influenzae and show its usefulness both as a computational framework for large experimental data set contextualization and as a tool to discover condition-independent drug targets. We focus on ß-ketoacyl-acyl carrier protein synthase III FabH chemical inhibition by using a synthetic molecule with good synthetic and antimicrobial profiles that specifically binds to the active site. The mechanistic complexity of FabH inhibition may go beyond allelic variation, and the strain-dependent effect of the inhibitor tested supports the impact of metabolic context as a key factor driving bacterial cell behavior. Therefore, this study highlights the systematic metabolic evaluation of individual strains through computational frameworks to identify secondary metabolic hubs modulating drug response, which will facilitate establishing synergistic and/or more precise and robust antibacterial treatments.
Assuntos
Haemophilus influenzae , Metabolismo dos Lipídeos , Humanos , Animais , Peixe-Zebra , Antibacterianos/farmacologia , Bactérias , Redes e Vias MetabólicasRESUMO
There is a clear need for novel antiviral concepts to control SARS-CoV-2 infection. Based on the promising anti-coronavirus activity observed for a class of 1,4,4-trisubstituted piperidines, we here conducted a detailed analysis of the structure-activity relationship of these structurally unique inhibitors. Despite the presence of five points of diversity, the synthesis of an extensive series of analogues was readily achieved by Ugi four-component reaction from commercially available reagents. After evaluating 63 analogues against human coronavirus 229E, four of the best molecules were selected and shown to have micromolar activity against SARS-CoV-2. Since the action point was situated post virus entry and lying at the stage of viral polyprotein processing and the start of RNA synthesis, enzymatic assays were performed with CoV proteins involved in these processes. While no inhibition was observed for SARS-CoV-2 nsp12-nsp7-nsp8 polymerase, nsp14 N7-methyltransferase and nsp16/nsp10 2'-O-methyltransferase, nor the nsp3 papain-like protease, the compounds clearly inhibited the nsp5 main protease (Mpro). Although the inhibitory activity was quite modest, the plausibility of binding to the catalytic site of Mpro was established by in silico studies. Therefore, the 1,4,4-trisubstituted piperidines appear to represent a novel class of non-covalent CoV Mpro inhibitors that warrants further optimization and development.
RESUMO
We have recently described a novel family of compounds of reduced size and dual anti-HIV and anti-EV71 activity that encompasses tripodal and tetrapodal derivatives. The tripodal prototype, AL-470, has a nitro group at the focal point of the central scaffold and three attached tryptophan residues, each of which bearing an isophthaloyl moiety at the C2 position of the indole ring. A nitro to amino substitution has allowed us now to introduce a chemically addressable functionality to perform further structural modifications consisting of both direct and linker-mediated attachment of several aromatic groups, including the fluorescent dye Alexa Fluor 647 and the antibody-recruiting 2,4-dinitrophenyl motif. Some of the derivatives turned out to be more potent and selective than AL-470 against HIV-1, HIV-2 and EV-A71. The fluorescent probe demonstrated a specific tropism for intestines and lungs, two important niches for the human microbiome in health and disease.
Assuntos
Dendrímeros , Enterovirus Humano A , Infecções por Enterovirus , Inibidores da Fusão de HIV , HIV-1 , Dendrímeros/química , Inibidores da Fusão de HIV/farmacologia , HIV-2 , Humanos , Internalização do VírusRESUMO
Redox homeostasis in trypanosomatids is based on the low-molecular-weight trypanothione, an essential dithiol molecule that is synthetized by trypanothione synthetase (TryS) and maintained in its reduced state by trypanothione disulfide reductase (TryR). The fact that both enzymes are indispensable for parasite survival and absent in the mammalian hosts makes them ideal drug targets against leishmaniasis. Although many efforts have been directed to developing TryR inhibitors, much less attention has been focused on TryS. The screening of an in-house library of 144 diverse molecules using two parallel biochemical assays allowed us to detect 13 inhibitors of L. infantum TryS. Compounds 1 and 3 were characterized as competitive inhibitors with Ki values in the low micromolar range and plausible binding modes for them were identified by automated ligand docking against refined protein structures obtained through computational simulation of an entire catalytic cycle. The proposed binding site for both inhibitors overlaps the polyamine site in the enzyme and, additionally, 1 also occupies part of the ATP site. Compound 4 behaves as a mixed hyperbolic inhibitor with a Ki of 0.8 µM. The activity of 5 is clearly dependent on the concentration of the polyamine substrate, but its kinetic behavior is clearly not compatible with a competitive mode of inhibition. Analysis of the activity of the six best inhibitors against intracellular amastigotes identified 5 as the most potent leishmanicidal candidate, with an EC50 value of 0.6 µM and a selectivity index of 35.
Assuntos
Amida Sintases , Antiprotozoários , Animais , Amida Sintases/metabolismo , NADH NADPH Oxirredutases , Sítios de Ligação , Oxirredução , Antiprotozoários/farmacologia , Antiprotozoários/química , Mamíferos/metabolismoRESUMO
N-methylation of the triazole moiety present in our recently described triazole-phenyl-thiazole dimerization disruptors of Leishmania infantum trypanothione disulfide reductase (LiTryR) led to a new class of potent inhibitors that target different binding sites on this enzyme. Subtle structural changes among representative library members modified their mechanism of action, switching from models of classical competitive inhibition to time-dependent mixed noncompetitive inhibition. X-ray crystallography and molecular modeling results provided a rationale for this distinct behavior. The remarkable potency and selectivity improvements, particularly against intracellular amastigotes, of the LiTryR dimerization disruptors 4c and 4d reveal that they could be exploited as leishmanicidal agents. Of note, L. infantum promastigotes treated with 4c significantly reduced their low-molecular-weight thiol content, thus providing additional evidence that LiTryR is the main target of this novel compound.
Assuntos
Antiprotozoários , Leishmania infantum , Dissulfetos , Antiprotozoários/química , NADH NADPH Oxirredutases , Triazóis/farmacologia , Triazóis/metabolismoRESUMO
Here we highlight a sound and unique work reported by Chen and co-workers entitled "HIV-1 fusion inhibitors targeting the membrane-proximal external region of Env spikes" (Xiao etâ al., Nat. Chem. Biol. 2020, 16, 529). In this article, the authors identify, by means of a clever antibody-guided strategy, several small molecules as fusion inhibitors of HIV-1 replication acting at the membrane proximal external region (MPER) of the HIV-1 envelope (Env) spike. MPER, which was previously recognized as a vaccine target, emerges as a novel druggable target for the discovery of HIV-1 fusion inhibitors. The compounds (exemplified by dequalinium and dequalinium-inspired analogues) prevent the conformational changes of Env from the prefusion species to the intermediate states required for membrane fusion. This work not only paves the way to novel, specific and useful anti-HIV-1 inhibitors, but also discloses new therapeutic strategies against other infectious diseases.
Assuntos
Inibidores da Fusão de HIV/química , HIV-1/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Dequalínio/análogos & derivados , Dequalínio/metabolismo , Dequalínio/farmacologia , Inibidores da Fusão de HIV/metabolismo , Inibidores da Fusão de HIV/farmacologia , Humanos , Relação Estrutura-Atividade , Internalização do Vírus/efeitos dos fármacos , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The nuclear export receptor exportin-1 (XPO1, CRM1) mediates the nuclear export of proteins that contain a leucine-rich nuclear export signal (NES) towards the cytoplasm. XPO1 is considered a relevant target in different human diseases, particularly in hematological malignancies, tumor resistance, inflammation, neurodegeneration and viral infections. Thus, its pharmacological inhibition is of significant therapeutic interest. The best inhibitors described so far (leptomycin B and SINE compounds) interact with XPO1 through a covalent interaction with Cys528 located in the NES-binding cleft of XPO1. Based on the well-established feature of chalcone derivatives to react with thiol groups via hetero-Michael addition reactions, we have synthesized two series of chalcones. Their capacity to react with thiol groups was tested by incubation with GSH to afford the hetero-Michael adducts that evolved backwards to the initial chalcone through a retro-Michael reaction, supporting that the covalent interaction with thiols could be reversible. The chalcone derivatives were evaluated in antiproliferative assays against a panel of cancer cell lines and as XPO1 inhibitors, and a good correlation was observed with the results obtained in both assays. Moreover, no inhibition of the cargo export was observed when the two prototype chalcones 9 and 10 were tested against a XPO1-mutated Jurkat cell line (XPO1C528S), highlighting the importance of the Cys at the NES-binding cleft for inhibition. Finally, their interaction at the molecular level at the NES-binding cleft was studied by applying the computational tool CovDock.
RESUMO
Inhibition of Leishmania infantum trypanothione disulfide reductase (LiTryR) by disruption of its homodimeric interface has proved to be an alternative and unexploited strategy in the search for novel antileishmanial agents. Proof of concept was first obtained by peptides and peptidomimetics. Building on previously reported dimerization disruptors containing an imidazole-phenyl-thiazole scaffold, we now report a new 1,2,3-triazole-based chemotype that yields noncompetitive, slow-binding inhibitors of LiTryR. Several compounds bearing (poly)aromatic substituents dramatically improve the ability to disrupt LiTryR dimerization relative to reference imidazoles. Molecular modeling studies identified an almost unexplored hydrophobic region at the interfacial domain as the putative binding site for these compounds. A subsequent structure-based design led to a symmetrical triazole analogue that displayed even more potent inhibitory activity over LiTryR and enhanced leishmanicidal activity. Remarkably, several of these novel triazole-bearing compounds were able to kill both extracellular and intracellular parasites in cell cultures.
Assuntos
Desenho de Fármacos , Leishmania infantum/enzimologia , NADH NADPH Oxirredutases/química , Multimerização Proteica/efeitos dos fármacos , Tiazóis/química , Tiazóis/farmacologia , Triazóis/química , Antiprotozoários/química , Antiprotozoários/farmacologia , Linhagem Celular , Humanos , Leishmania infantum/efeitos dos fármacos , NADH NADPH Oxirredutases/metabolismo , Estrutura Quaternária de Proteína , Relação Estrutura-AtividadeRESUMO
Trypanothione disulfide reductase (TryR) is an essential homodimeric enzyme of trypanosomatid parasites that has been validated as a drug target to fight human infections. Using peptides and peptidomimetics, we previously obtained proof of concept that disrupting protein-protein interactions at the dimer interface of Leishmania infantum TryR (LiTryR) offered an innovative and so far unexploited opportunity for the development of novel antileishmanial agents. Now, we show that linking our previous peptide prototype TRL38 to selected hydrophobic moieties provides a novel series of small-molecule-peptide conjugates that behave as good inhibitors of both LiTryR activity and dimerization.