Probing the Dimerization Interface of <i>Leishmania infantum</i> Trypanothione Reductase with Site‐Directed Mutagenesis and Short Peptides

Toro, Miguel Ángel; Sánchez‐Murcia, Pedro A.; Moreno, David F.; Ruiz-Santaquiteria, Marta; Alzate, Juan F.; Negri, Ana; Camarasa, Marı́a-José; Gago, Federico; Velázquez, Sonsoles; Jiménez-Ruı́z, Antonio

doi:10.1002/cbic.201200744

Cited by 25 publications

(75 citation statements)

References 32 publications

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“…Nevertheless, two of the 12 targets identified in this study have been successfully inhibited by interface-derived peptides. An interface-derived peptide helix has been demonstrated to inhibit Li TryR through a mechanism that disrupts the PPI [34,40]. This helix had also been identified by HippDB as potentially contributing 15% of the interface energy, while the loop region identified in this analysis is predicted to contribute 22%.…”

Section: Resultsmentioning

confidence: 74%

“…cruzi , and L . infantum TryR containing three hotspots, Ile-72, Phe-78, and Leu-82, which matches a loop in Li TryR identified by Loopfinder (heat score: 3), overlaps a segment predicted by Peptiderive (22% interface energy), and contains one hotspot (Trp-80) that has been verified experimentally (Fig 1) [34]. This proposed inhibitory peptide is predicted to contribute more to the interface energy than the established helix-based inhibitor.…”

Section: Resultsmentioning

confidence: 75%

“…Recent computational and experimental studies have identified a hot-spot-containing helix in L . infantum TryR that inhibits TS 2 reduction by disrupting dimerization of the enzyme, as demonstrated by kinetics and ELISA [34,40]. This helix overlaps, but shares little sequence homology with, a helix that disrupts dimerization of human glutathione reductase (hGR), although it presents a strikingly similar helical face (S1 Fig).…”

Section: Resultsmentioning

confidence: 99%

“…This contrast is most apparent when examining specific PPIs in this study. In the case of TryR (Fig 1), an experimentally verified hot spot (Trp-80) [34] was not identified by the computational alanine scan, despite being buried in the opposite chain of the protein. TryR Trp-80 is conserved across kinetoplastids (S5 Table), as are two of the three calculated hot spots, Ile/Leu-71 and Phe-78.…”

Section: Resultsmentioning

confidence: 99%

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Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions

2017

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Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

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Section: Resultsmentioning

confidence: 74%

Section: Resultsmentioning

confidence: 75%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions

2017

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“…Then, the enzymatic reaction was quenched (50 µL) at the desired time point (0,5,15,30,60,180 and 300 min for linear peptide 2 and 0, 15,30,60,120,240,360,480 Stock solutions of the linear peptide 2 and amide-bridge cyclic analogue 11 (100 µM) were prepared using 10% DMSO in TBS buffer (pH = 7.6, Aldrich).…”

Section: Protease Susceptibility Assaysmentioning

confidence: 99%

Comparison of hydrocarbon-and lactam-bridged cyclic peptides as dimerization inhibitors of Leishmania infantum trypanothione reductase

et al. 2015

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A table of contents entryHelical peptides stabilized via all-hydrocarbon or lactam side-chain bridging were investigated as disruptors of Leishmania infantum trypanothione reductase and the biological results were rationalized by NMR spectroscopy studies and molecular dynamics simulations.Abstract-All-hydrocarbon and lactam-bridged staples linking amino acid side-chains have been used to stabilize the α-helical motif in short 13-mer peptides that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR). The design of the best positions for covalent hydrocarbon closure relied on a theoretical prediction of the degree of helicity of the corresponding cyclic peptides in water. Selected (i, i+4) and (i, i+7) hydrocarbon-stapled peptides were prepared by using solid-phase synthesis protocols and optimized ring-closing metathesis reactions under microwave conditions. Structural analysis by NMR spectroscopy confirmed high helical contents in aqueous TFE solutions for both types of helix-constrained cyclic peptides. Remarkably, the ability to prevent Li-TryR dimerization was reduced in both (i, i+4) and (i, i+7) hydrocarbon stapled peptides but was retained in the corresponding (i, i+4) Glu-Lys lactam-bridged analogue, which also showed a higher resistance to proteolytic degradation by proteinase K relative to the linear peptide prototype. In silico studies indicated that the introduction of a hydrocarbon staple vs a lactam bridge likely perturbs critical interactions required for proper binding of the peptide to the Li-TryR monomer.corresponding N-Fmoc-protected amino acid (1.2 equiv), HCTU (1.2 equiv) and DIEA (2.4 equiv) in anhydrous DMF (0.5-1.0 mL) was added over the swollen peptidil-resin (1.0 equiv) in anhydrous DMF in a microwave vial (5-10 mL). The coupling reaction was heated at 40 ºC using microwave radiation for 10 min. Each coupling step was repeated 3 times using freshly prepared Fmoc-amino acid/coupling reagent mixtures. After complete couplings, the resin was drained and washed with DMF/CH 2 Cl 2 /DMF/CH 2 Cl 2 (4 x 0.5 min).Coupling reactions to primary and secondary amines were monitored by the ninhydrin and the chloranil tests, respectively. Ring closing metathesis (RCM) reaction.To the peptidyl-resins 12 and 13 (0.05-0.07 mmol) swollen in anhydrous CH 2 Cl 2 (5 mL) in a microwave vial (5-10 mL) second generation Grubb's catalyst (0.2 equiv) was added and the vial was sealed and gently bubbled with argon. Then, the reaction was heated at 40 ºC using microwave radiation for 30-150 min. The resin was then filtered and washed successively with CH 2 Cl 2 /MeOH.Residual ruthenium impurities were removed by stirring the resin bound peptide with a solution of DMSO (50 equiv relative to the catalyst) in DMF for 12 h. 37

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A recent update on new synthetic chiral compounds with antileishmanial activity

2022

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Parasitic diseases, including malaria, leishmaniasis, and trypanosomiasis, affect billions of people and are responsible for almost 500,000 deaths/year. In particular, leishmaniasis, a neglected tropical disease, is considered a global public health problem because current drugs have several drawbacks including to toxicity, high cost, and drug resistance, which result in a lack of effective and readily available therapies. Therefore, the synthesis of new, safe, and effective molecules still requires the attention of the scientific community. Moreover, it is well known that chirality plays a crucial role in the antiparasitic activity of molecules, driving the design of their synthesis. Therefore, in this review we report a recent update on new chiral compounds with promising antileishmanial activity, focusing on synthetic approaches. Where reported, in most cases the enantiopure compound has shown better potency against the protozoa than its enantiomer or corresponding racemic mixture.

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Probing the Dimerization Interface of Leishmania infantum Trypanothione Reductase with Site‐Directed Mutagenesis and Short Peptides

Abstract: 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.

Cited by 25 publications

References 32 publications

Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions

Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions

Comparison of hydrocarbon-and lactam-bridged cyclic peptides as dimerization inhibitors of Leishmania infantum trypanothione reductase

A recent update on new synthetic chiral compounds with antileishmanial activity

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