RESUMEN
Glutaminyl-peptide cyclotransferases (QCs) convert the N-terminal glutamine or glutamate residues of protein and peptide substrates into pyroglutamate (pE) by releasing ammonia or a water molecule. The N-terminal pE modification protects peptides/proteins against proteolytic degradation by amino- or exopeptidases, increasing their stability. Mammalian QC is abundant in the brain and a large amount of evidence indicates that pE peptides are involved in the onset of neural human pathologies such as Alzheimer's and Huntington's disease and synucleinopathies. Hence, human QC (hQC) has become an intensively studied target for drug development against these diseases. Soon after its characterization, hQC was identified as a Zn-dependent enzyme, but a partial restoration of the enzyme activity in the presence of the Co(II) ion was also reported, suggesting a possible role of this metal ion in catalysis. The present work aims to investigate the structure of demetallated hQC and of the reconstituted enzyme with Zn(II) and Co(II) and their behavior in the presence of known inhibitors. Furthermore, our structural determinations provide a possible explanation for the presence of the mononuclear metal binding site of hQC, despite the presence of the same conserved metal binding motifs present in distantly related dinuclear aminopeptidase enzymes.
Asunto(s)
Aminoaciltransferasas , Zinc , Humanos , Aminoaciltransferasas/metabolismo , Aminoaciltransferasas/química , Zinc/metabolismo , Zinc/química , Sitios de Unión , Cobalto/metabolismo , Cobalto/química , Unión Proteica , Modelos MolecularesRESUMEN
Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.
Asunto(s)
Antagonistas del Ácido Fólico , Tetrahidrofolato Deshidrogenasa , Triazinas , Trypanosoma brucei brucei , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , Humanos , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Antagonistas del Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/química , Triazinas/farmacología , Triazinas/química , Tripanocidas/farmacología , Tripanocidas/química , Proguanil/farmacología , Proguanil/química , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Leishmania infantum/efectos de los fármacos , Leishmania infantum/enzimología , Bencimidazoles/farmacología , Bencimidazoles/química , Relación Estructura-Actividad , Antiprotozoarios/farmacología , Antiprotozoarios/química , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , OxidorreductasasRESUMEN
Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diamino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class of phenyl-, heteroaryl- and benzyloxy-nitro derivatives based on the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against TbPTR1, the X-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective compounds against T. brucei were then tested against the amastigote cellular stage of T. cruzi, searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of hERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-ß-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Compound 1p, showed the best pharmacodynamic and pharmacokinetic properties, can be considered a good candidate for further bioavailability and efficacy studies.
Asunto(s)
Antiprotozoarios , Enfermedad de Chagas , Trypanosoma brucei brucei , Trypanosoma cruzi , Humanos , Relación Estructura-Actividad , Antiprotozoarios/química , Modelos Moleculares , Antiparasitarios/farmacología , Enfermedad de Chagas/tratamiento farmacológicoRESUMEN
α-Synuclein (αSyn) constitutes the main protein component of Lewy bodies, which are the pathologic hallmark in Parkinson's disease. αSyn is unstructured in solution but the interaction of αSyn with lipid membrane modulates its conformation by inducing an α-helical structure of the N-terminal region. In addition, the interaction with metal ions can trigger αSyn conformation upon binding and/or through the metal-promoted generation of reactive oxygen species which lead to a cascade of structural alterations. For these reasons, the ternary interaction between αSyn, copper, and membranes needs to be elucidated in detail. Here, we investigated the structural properties of copper-αSyn binding through NMR, EPR, and XAS analyses, with particular emphasis on copper(I) coordination since the reduced state is particularly relevant for oxygen activation chemistry. The analysis was performed in different membrane model systems, such as micellar sodium dodecyl sulfate (SDS) and unilamellar vesicles, comparing the binding of full-length αSyn and N-terminal peptide fragments. The presence of membrane-like environments induced the formation of a copper:αSyn = 1:2 complex where Cu+ was bound to the Met1 and Met5 residues of two helical peptide chains. In this coordination, Cu+ is stabilized and is unreactive in the presence of O2 in catechol substrate oxidation.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Cobre/química , Enfermedad de Parkinson/metabolismo , Péptidos/metabolismo , Oxidación-ReducciónRESUMEN
Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.
Asunto(s)
Neoplasias Ováricas , Timidilato Sintasa , Femenino , Animales , Ratones , Humanos , Sitios de Unión , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Fluorouracilo/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Inhibidores Enzimáticos/farmacologíaRESUMEN
Metallo-ß-lactamases (MBLs) contribute to the resistance of Gram-negative bacteria to carbapenems, last-resort antibiotics at hospital, and MBL inhibitors are urgently needed to preserve these important antibacterial drugs. Here, we describe a series of 1,2,4-triazole-3-thione-based inhibitors displaying an α-amino acid substituent, which amine was mono- or disubstituted by (hetero)aryl groups. Compounds disubstituted by certain nitrogen-containing heterocycles showed submicromolar activities against VIM-type enzymes and strong NDM-1 inhibition (Ki = 10-30 nM). Equilibrium dialysis, native mass spectrometry, isothermal calorimetry (ITC), and X-ray crystallography showed that the compounds inhibited both VIM-2 and NDM-1 at least partially by stripping the catalytic zinc ions. These inhibitors also displayed a very potent synergistic activity with meropenem (16- to 1000-fold minimum inhibitory concentration (MIC) reduction) against VIM-type- and NDM-1-producing ultraresistant clinical isolates, including Enterobacterales and Pseudomonas aeruginosa. Furthermore, selected compounds exhibited no or moderate toxicity toward HeLa cells, favorable absorption, distribution, metabolism, excretion (ADME) properties, and no or modest inhibition of several mammalian metalloenzymes.
Asunto(s)
Tionas , Inhibidores de beta-Lactamasas , Humanos , Inhibidores de beta-Lactamasas/farmacología , Inhibidores de beta-Lactamasas/química , Tionas/farmacología , Células HeLa , Antibacterianos/farmacología , Antibacterianos/química , beta-Lactamasas/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that stabilizes client proteins in a folded and functional state. It is composed of two identical and symmetrical subunits and each monomer consists of three domains, the N-terminal (NTD), the middle (MD), and the C-terminal domain (CTD). Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP-binding pocket in the NTD act as Hsp90 inhibitors, leading to client protein degradation and cell death. Therefore, human Hsp90 represents a validated target for developing new anticancer drugs. Since protozoan parasites use their Hsp90 to trigger important transitions between different stages of their life cycle, this protein also represents a profitable target in anti-parasite drug discovery. Nevertheless, the development of molecules able to selectively target the ATP-binding site of protozoan Hsp90 is challenging due to the high homology with the human Hsp90 NTD (hHsp90-NTD). In a previous work, a series of potent Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold was developed. The most promising inhibitor of the series, JMC31, showed potent Hsp90 binding and antiproliferative activity in NCI-H460 cells in the low-nanomolar range. In this work, we present the structural characterization of hHsp90-NTD in complex with JMC31 through X-ray crystallography. In addition, to elucidate the role of residue 112 on the ligand binding and its exploitability for the development of selective inhibitors, we investigated the crystal structures of hHsp90-NTD variants (K112R and K112A) in complex with JMC31.
Asunto(s)
Proteínas HSP90 de Choque Térmico , Triazoles , Adenosina Trifosfato/metabolismo , Sitios de Unión , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Unión Proteica , Triazoles/farmacologíaRESUMEN
The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective antiparasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure-activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei while retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.
Asunto(s)
Leishmania major , Oxidorreductasas , Tetrahidrofolato Deshidrogenasa , Trypanosoma brucei brucei , Leishmania major/efectos de los fármacos , Leishmania major/enzimología , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Pteridinas/química , Pteridinas/farmacología , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimologíaRESUMEN
Pteridine reductase 1 (PTR1) is a key enzyme of the folate pathway in protozoan parasites of the genera Leishmania and Trypanosoma and is a valuable drug target for tropical diseases. This enzyme is able to catalyze the NADPH-dependent reduction of both conjugated (folate) and unconjugated (biopterin) pterins to their tetrahydro forms, starting from oxidized- or dihydro-state substrates. The currently available X-ray structures of Leishmania major PTR1 (LmPTR1) show the enzyme in its unbound, unconjugated substrate-bound (with biopterin derivatives) and inhibitor-bound forms. However, no structure has yet been determined of LmPTR1 bound to a conjugated substrate. Here, the high-resolution crystal structure of LmPTR1 in complex with folic acid is presented and the intermolecular forces that drive the binding of the substrate in the catalytic pocket are described. By expanding the collection of LmPTR1 structures in complex with process intermediates, additional insights into the active-site rearrangements that occur during the catalytic process are provided. In contrast to previous structures with biopterin derivatives, a small but significant difference in the orientation of Asp181 and Tyr194 of the catalytic triad is found. This feature is shared by PTR1 from T. brucei (TbPTR1) in complex with the same substrate molecule and may be informative in deciphering the importance of such residues at the beginning of the catalytic process.
Asunto(s)
Leishmania major , Biopterinas/metabolismo , Cristalografía por Rayos X , Ácido Fólico/química , Ácido Fólico/metabolismo , Leishmania major/metabolismo , NADP/metabolismo , OxidorreductasasRESUMEN
Metallo-ß-lactamases (MBLs) are increasingly involved as a major mechanism of resistance to carbapenems in relevant opportunistic Gram-negative pathogens. Unfortunately, clinically efficient MBL inhibitors still represent an unmet medical need. We previously reported several series of compounds based on the 1,2,4-triazole-3-thione scaffold. In particular, Schiff bases formed between diversely 5-substituted-4-amino compounds and 2-carboxybenzaldehyde were broad-spectrum inhibitors of VIM-type, NDM-1 and IMP-1 MBLs. Unfortunately, these compounds were unable to restore antibiotic susceptibility of MBL-producing bacteria, probably because of poor penetration and/or susceptibility to hydrolysis. To improve their microbiological activity, we synthesized and characterized compounds where the hydrazone-like bond of the Schiff base analogues was replaced by a stable ethyl link. This small change resulted in a narrower inhibition spectrum, as all compounds were poorly or not inhibiting NDM-1 and IMP-1, but showed a significantly better activity on VIM-type enzymes, with Ki values in the µM to sub-µM range. The resolution of the crystallographic structure of VIM-2 in complex with one of the best inhibitors yielded valuable information about their binding mode. Interestingly, several compounds were shown to restore the ß-lactam susceptibility of VIM-type-producing E. coli laboratory strains and also of K. pneumoniae clinical isolates. In addition, selected compounds were found to be devoid of toxicity toward human cancer cells at high concentration, thus showing promising safety.
Asunto(s)
Tionas , Inhibidores de beta-Lactamasas , Antibacterianos/química , Antibacterianos/farmacología , Escherichia coli , Humanos , Pruebas de Sensibilidad Microbiana , Tionas/farmacología , Triazoles/química , Inhibidores de beta-Lactamasas/química , beta-Lactamasas/metabolismoRESUMEN
The use of light-responsive proteins to control both living or synthetic cells, is at the core of the expanding fields of optogenetics and synthetic biology. It is thus apparent that a richer reaction toolbox for the preparation of such systems is of fundamental importance. Here, we provide a proof-of-principle demonstration that Morita-Baylis-Hillman adducts can be employed to perform a facile site-specific, irreversible and diastereoselective click-functionalization of a lysine residue buried into a lipophilic binding pocket and yielding an unnatural chromophore with an extended π-system. In doing so we effectively open the path to the inâ vitro preparation of a library of synthetic proteins structurally reminiscent of xanthopsin eubacterial photoreceptors. We argue that such a library, made of variable unnatural chromophores inserted in an easy-to-mutate and crystallize retinoic acid transporter, significantly expand the scope of the recently introduced rhodopsin mimics as both optogenetic and "lab-on-a-molecule" tools.
Asunto(s)
Receptores de Ácido Retinoico/metabolismo , Rodopsina/metabolismo , Química Clic , Cristalografía por Rayos X , Modelos Moleculares , Estructura Molecular , Receptores de Ácido Retinoico/química , Rodopsina/química , EstereoisomerismoRESUMEN
Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screening by GlaxoSmithKline (Tres Cantos Anti-Kinetoplastid Screening, TCAKS) were exploited for the discovery of a novel core structure inspiring new treatments of parasitic diseases targeting the trypansosmatidic pteridine reductase 1 (PTR1) and dihydrofolate reductase (DHFR) enzymes. In total, 592 compounds were tested through medium-throughput screening assays. A subset of 14 compounds successfully inhibited the enzyme activity in the low micromolar range of at least one of the enzymes from both Trypanosoma brucei and Lesihmania major parasites (pan-inhibitors), or from both PTR1 and DHFR-TS of the same parasite (dual inhibitors). Molecular docking studies of the protein-ligand interaction focused on new scaffolds not reproducing the well-known antifolate core clearly explaining the experimental data. TCMDC-143249, classified as a benzenesulfonamide derivative by the QikProp descriptor tool, showed selective inhibition of PTR1 and growth inhibition of the kinetoplastid parasites in the 5 µM range. In our work, we enlarged the biological profile of the GSK Kinetobox and identified new core structures inhibiting selectively PTR1, effective against the kinetoplastid infectious protozoans. In perspective, we foresee the development of selective PTR1 and DHFR inhibitors for studies of drug combinations.
RESUMEN
Metallo-ß-lactamases (MBLs) are important contributors of Gram-negative bacteria resistance to ß-lactam antibiotics. MBLs are highly worrying because of their carbapenemase activity, their rapid spread in major human opportunistic pathogens while no clinically useful inhibitor is available yet. In this context, we are exploring the potential of compounds based on the 1,2,4-triazole-3-thione scaffold as an original ligand of the di-zinc active sites of MBLs, and diversely substituted at its positions 4 and 5. Here, we present a new series of compounds substituted at the 4-position by a thioether-containing alkyl chain with a carboxylic and/or an aryl group at its extremity. Several compounds showed broad-spectrum inhibition with Ki values in the µM to sub-µM range against VIM-type enzymes, NDM-1 and IMP-1. The presence of the sulfur and of the aryl group was important for the inhibitory activity and the binding mode of a few compounds in VIM-2 was revealed by X-ray crystallography. Importantly, in vitro antibacterial susceptibility assays showed that several inhibitors were able to potentiate the activity of meropenem on Klebsiella pneumoniae clinical isolates producing VIM-1 or VIM-4, with a potentiation effect of up to 16-fold. Finally, a selected compound was found to only moderately inhibit the di-zinc human glyoxalase II, and several showed no or only moderate toxicity toward several human cells, thus favourably completing a promising behaviour.
Asunto(s)
Sulfuros/farmacología , Tionas/farmacología , Triazoles/farmacología , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-Actividad , Sulfuros/química , Tionas/síntesis química , Tionas/química , Triazoles/síntesis química , Triazoles/química , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/químicaRESUMEN
Ferritins are nanocage proteins that store iron ions in their central cavity as hydrated ferric oxide biominerals. In mammals, further the L (light) and H (heavy) chains constituting cytoplasmic maxi-ferritins, an additional type of ferritin has been identified, the mitochondrial ferritin (MTF). Human MTF (hMTF) is a functional homopolymeric H-like ferritin performing the ferroxidase activity in its ferroxidase site (FS), in which Fe(II) is oxidized to Fe(III) in the presence of dioxygen. To better investigate its ferroxidase properties, here we performed time-lapse X-ray crystallography analysis of hMTF, providing structural evidence of how iron ions interact with hMTF and of their binding to the FS. Transient iron binding sites, populating the pathway along the cage from the iron entry channel to the catalytic center, were also identified. Furthermore, our kinetic data at variable iron loads indicate that the catalytic iron oxidation reaction occurs via a diferric peroxo intermediate followed by the formation of ferric-oxo species, with significant differences with respect to human H-type ferritin.
Asunto(s)
Ceruloplasmina , Compuestos Férricos , Animales , Apoferritinas/metabolismo , Sitios de Unión , Ceruloplasmina/metabolismo , Ferritinas/metabolismo , Humanos , Hierro/metabolismo , Oxidación-ReducciónRESUMEN
Trypanosoma and Leishmania parasites are the etiological agents of various threatening neglected tropical diseases (NTDs), including human African trypanosomiasis (HAT), Chagas disease, and various types of leishmaniasis. Recently, meaningful progresses in the treatment of HAT, due to Trypanosoma brucei (Tb), have been achieved by the introduction of fexinidazole and the combination therapy eflornithine-nifurtimox. Nevertheless, due to drug resistance issues and the exitance of animal reservoirs, the development of new NTD treatments is still required. For this purpose, we explored the combined targeting of two key folate enzymes, dihydrofolate reductase (DHFR) and pteridine reductase 1 (PTR1). We formerly showed that the TbDHFR inhibitor cycloguanil (CYC) also targets TbPTR1, although with reduced affinity. Here, we explored a small library of CYC analogues to understand how their substitution pattern affects the inhibition of both TbPTR1 and TbDHFR. Some novel structural features responsible for an improved, but preferential, ability of CYC analogues to target TbPTR1 were disclosed. Furthermore, we showed that the known drug pyrimethamine (PYR) effectively targets both enzymes, also unveiling its binding mode to TbPTR1. The structural comparison between PYR and CYC binding modes to TbPTR1 and TbDHFR provided key insights for the future design of dual inhibitors for HAT therapy.
RESUMEN
Combining drugs represent an approach to efficiently prevent and overcome drug resistance and to reduce toxicity; yet it is a highly challenging task, particularly if combinations of inhibitors of the same enzyme target are considered. To show that crystallographic and inhibition kinetic information can provide indicators of cancer cell growth inhibition by combinations of two anti-human thymidylate synthase (hTS) drugs, we obtained the X-ray crystal structure of the hTS:raltitrexed:5-fluorodeoxyuridine monophosphate (FdUMP) complex. Its analysis showed a ternary complex with both molecules strongly bound inside the enzyme catalytic cavity. The synergistic inhibition of hTS and its mechanistic rationale were consistent with the structural analysis. When administered in combination to A2780 and A2780/CP ovarian cancer cells, the two drugs inhibited ovarian cancer cell growth additively/synergistically. Together, these results support the idea that X-ray crystallography can provide structural indicators for designing combinations of hTS (or any other target)-directed drugs to accelerate preclinical research for therapeutic application.
RESUMEN
The diazabicyclooctanes (DBOs) are a class of serine ß-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of ß-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of ß-lactam antibiotics against carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO "critical priority pathogen" producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure-activity relationship, leading to the discovery of a novel DBO, ANT3310, which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.
Asunto(s)
Acinetobacter/efectos de los fármacos , Antibacterianos/farmacología , Enterobacteriaceae/efectos de los fármacos , Octanos/química , beta-Lactamasas/química , Animales , Antibacterianos/química , Antibacterianos/metabolismo , Sitios de Unión , Carbapenémicos/farmacología , Evaluación Preclínica de Medicamentos , Farmacorresistencia Bacteriana/efectos de los fármacos , Semivida , Ratones , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Octanos/metabolismo , Octanos/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/metabolismo , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/metabolismoRESUMEN
Resistance to ß-lactam antibiotics in Gram-negatives producing metallo-ß-lactamases (MBLs) represents a major medical threat and there is an extremely urgent need to develop clinically useful inhibitors. We previously reported the original binding mode of 5-substituted-4-amino/H-1,2,4-triazole-3-thione compounds in the catalytic site of an MBL. Moreover, we showed that, although moderately potent, they represented a promising basis for the development of broad-spectrum MBL inhibitors. Here, we synthesized and characterized a large number of 4-amino-1,2,4-triazole-3-thione-derived Schiff bases. Compared to the previous series, the presence of an aryl moiety at position 4 afforded an average 10-fold increase in potency. Among 90 synthetic compounds, more than half inhibited at least one of the six tested MBLs (L1, VIM-4, VIM-2, NDM-1, IMP-1, CphA) with Ki values in the µM to sub-µM range. Several were broad-spectrum inhibitors, also inhibiting the most clinically relevant VIM-2 and NDM-1. Active compounds generally contained halogenated, bicyclic aryl or phenolic moieties at position 5, and one substituent among o-benzoic, 2,4-dihydroxyphenyl, p-benzyloxyphenyl or 3-(m-benzoyl)-phenyl at position 4. The crystallographic structure of VIM-2 in complex with an inhibitor showed the expected binding between the triazole-thione moiety and the dinuclear centre and also revealed a network of interactions involving Phe61, Tyr67, Trp87 and the conserved Asn233. Microbiological analysis suggested that the potentiation activity of the compounds was limited by poor outer membrane penetration or efflux. This was supported by the ability of one compound to restore the susceptibility of an NDM-1-producing E. coli clinical strain toward several ß-lactams in the presence only of a sub-inhibitory concentration of colistin, a permeabilizing agent. Finally, some compounds were tested against the structurally similar di-zinc human glyoxalase II and found weaker inhibitors of the latter enzyme, thus showing a promising selectivity towards MBLs.
Asunto(s)
Bases de Schiff/farmacología , Tionas/farmacología , Triazoles/farmacología , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/metabolismo , Cristalografía por Rayos X , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Pruebas de Sensibilidad Microbiana , Unión Proteica , Pseudomonas aeruginosa/química , Bases de Schiff/síntesis química , Bases de Schiff/metabolismo , Tionas/síntesis química , Tionas/metabolismo , Triazoles/síntesis química , Triazoles/metabolismo , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/metabolismoRESUMEN
The clinical effectiveness of the important ß-lactam class of antibiotics is under threat by the emergence of resistance, mostly due to the production of acquired serine- (SBL) and metallo-ß-lactamase (MBL) enzymes. To address this resistance issue, multiple ß-lactam/ß-lactamase inhibitor combinations have been successfully introduced into the clinic over the past several decades. However, all of those combinations contain SBL inhibitors and, as yet, there are no MBL inhibitors in clinical use. Consequently, there exists an unaddressed yet growing healthcare problem due to the rise in recent years of highly resistant strains which produce New Delhi metallo (NDM)-type metallo-carbapenemases. Previously, we reported the characterization of an advanced MBL inhibitor lead compound, ANT431. Herein, we discuss the completion of a lead optimization campaign culminating in the discovery of the preclinical candidate ANT2681, a potent NDM inhibitor with strong potential for clinical development.
Asunto(s)
Enterobacteriaceae , Inhibidores de beta-Lactamasas , Antibacterianos/farmacología , Meropenem/farmacología , Monobactamas , Inhibidores de beta-Lactamasas/farmacologíaRESUMEN
Breakthroughs in Medicinal Chemistry [...].