Pyrrolopyrimidine vs imidazole-phenyl-thiazole scaffolds in nonpeptidic dimerization inhibitors of leishmania infantum trypanothione reductase

Disruption of protein-protein interactions of essential oligomeric enzymes by small molecules represents a significant challenge. We recently reported some linear and cyclic peptides derived from an α-helical region present in the homodimeric interface of Leishmania infantum trypanothione reductase (Li-TryR) that showed potent effects on both dimerization and redox activity of this essential enzyme. Here, we describe our first steps toward the design of nonpeptidic small-molecule Li-TryR dimerization disruptors using a proteomimetic approach. The pyrrolopyrimidine and the 5-6-5 imidazole-phenyl-thiazole α-helix-mimetic scaffolds were suitably decorated with substituents that could mimic three key residues (K, Q, and I) of the linear peptide prototype (PKIIQSVGIS-Nle-K-Nle). Extensive optimization of previously described synthetic methodologies was required. A library of 15 compounds bearing different hydrophobic alkyl and aromatic substituents was synthesized. The imidazole-phenyl-thiazole-based analogues outperformed the pyrrolopyrimidine-based derivatives in both inhibiting the enzyme and killing extracellular and intracellular parasites in cell culture. The most active imidazole-phenyl-thiazole compounds 3e and 3f inhibit Li-TryR and prevent growth of the parasites at low micromolar concentrations similar to those required by the peptide prototype. The intrinsic fluorescence of these compounds inside the parasites visually demonstrates their good permeability in comparison with previous peptide-based Li-TryR dimerization disruptors.We thank the Spanish Government (MINECO/FEDER Projects SAF2015-64629-C2, BFU2017-90030-P), the Comunidad de Madrid (BIPEDD-2-CM ref S-2010/BMD-2457), and the Consejo Superior de Investigaciones Cientıfí cas (CSIC Project 201980E028) for financial support. We thank staff from ALBA Synchrotron (Barcelona, Spain) for support during data collection.Peer Reviewe

Identification of 1,2,3-triazolium salt-based inhibitors of Leishmania infantum trypanothione disulfide reductase with enhanced antileishmanial potency in cellulo and increased selectivity

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 in- hibitors 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.This work has been financially supported by the Spanish MICINN (Projects PID2019-104070RB-C21, PID2019-104070RB-C22 and PID2020-115331 GB-I00), the Spanish Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC, Projects CSIC-PIE-201980E100 and CSIC-PIE-201980E028), and the Comunidad de Madrid (PLATESA2-CM ref. S-2018/BAA-4370). The Spanish MEC is also acknowledged for FPU grants to A. R. and to J.C.G. P.A.S.M. thanks to the Division of Physio- logical Chemistry and the Otto-Loewi Research Center of the Medical University of Graz for their support with the scienfic cluster where the calculations contained in this work were run. We thank Ricardo Lau- reano-Rodríguez, Juan Antonio Rodríguez-Gutierrez, and Laura Lagar- tera for technical assistance with SPR experiments.Peer reviewe

Structural Cues for Understanding eEF1A2 Moonlighting

18 pags., 9 figs.Spontaneous mutations in the EEF1A2 gene cause epilepsy and severe neurological disabilities in children. The crystal structure of eEF1A2 protein purified from rabbit skeletal muscle reveals a post-translationally modified dimer that provides information about the sites of interaction with numerous binding partners, including itself, and maps these mutations onto the dimer and tetramer interfaces. The spatial locations of the side chain carboxylates of Glu301 and Glu374, to which phosphatidylethanolamine is uniquely attached via an amide bond, define the anchoring points of eEF1A2 to cellular membranes and interorganellar membrane contact sites. Additional bioinformatic and molecular modeling results provide novel structural insight into the demonstrated binding of eEF1A2 to SH3 domains, the common MAPK docking groove, filamentous actin, and phosphatidylinositol-4 kinase IIIβ. In this new light, the role of eEF1A2 as an ancient, multifaceted, and articulated G protein at the crossroads of autophagy, oncogenesis and viral replication appears very distant from the “canonical” one of delivering aminoacyl-tRNAs to the ribosome that has dominated the scene and much of the thinking for many decades.A.A.C.is grateful for a predoctoral grant (FPU14/03190)from the Spanish Ministry of Education. inancial support from the Spanish Ministry of Science, Innovation and Universities (BFU2017-90030-P to J.A.H. and SAF2015-64629-C2-2-R to F.G.),and ISCIII and ERDF (PT17/0019 to D.G.) is gratefully acknowledged. We thank fully acknowledge the technical support provided by staff at the ALBA synchrotron facility, the Proteomics Unit of Complutense University of Madrid (a member of Proteo Red),and Plácido Galindo (Chromatography and MS Service,IQFR,CSIC,Madrid

Pyrrolopyrimidine vs Imidazole-Phenyl-Thiazole Scaffolds in Nonpeptidic Dimerization Inhibitors of Leishmania infantum Trypanothione Reductase

Disruption of protein–protein interactions of essential oligomeric enzymes by small molecules represents a significant challenge. We recently reported some linear and cyclic peptides derived from an α-helical region present in the homodimeric interface of Leishmania infantum trypanothione reductase (Li-TryR) that showed potent effects on both dimerization and redox activity of this essential enzyme. Here, we describe our first steps toward the design of nonpeptidic small-molecule Li-TryR dimerization disruptors using a proteomimetic approach. The pyrrolopyrimidine and the 5-6-5 imidazole-phenyl-thiazole α-helix-mimetic scaffolds were suitably decorated with substituents that could mimic three key residues (K, Q, and I) of the linear peptide prototype (PKII­QSV­GIS-Nle-­K-Nle). Extensive optimization of previously described synthetic methodologies was required. A library of 15 compounds bearing different hydrophobic alkyl and aromatic substituents was synthesized. The imidazole-phenyl-thiazole-based analogues outperformed the pyrrolopyrimidine-based derivatives in both inhibiting the enzyme and killing extracellular and intracellular parasites in cell culture. The most active imidazole-phenyl-thiazole compounds 3e and 3f inhibit Li-TryR and prevent growth of the parasites at low micromolar concentrations similar to those required by the peptide prototype. The intrinsic fluorescence of these compounds inside the parasites visually demonstrates their good permeability in comparison with previous peptide-based Li-TryR dimerization disruptors