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

Abstract

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

Design and structural characterisation of monomeric water-soluble α-helix and β-hairpin peptides: State-of-the-art

“…More recently, hydrocarbonstapled peptides have emerged providing flexible linkers that not only confer α-helical stability, but also improve their drug-like properties. But, a lactam-stapled peptide showed better dimerization inhibitor capacity than its hydrocarbon-stapled counterpart [179]. Diverse stapled peptides have been successfully designed offering promising therapeutic avenues [169,172,180].…”

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

“…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 ).…”

Stapled Peptides—A Useful Improvement for Peptide-Based Drugs

“…For what is concerning the proteolytic stability, there are no evidence that the use of different stapling techniques determines relevant differences in the half-life of the peptide in the cellular environment. In fact, degradative proteolytic enzymes recognize only extended peptide chains and the side brace of the stapled peptides prevent them from entering in the active site of these enzymes [80,81]. However, even if in many cases the use of a staple enhances the resistance towards hydrolysis [17,25,80,81], there is the possibility that the stapled peptides show a life-time shorter than the non-stapled one—for example, in 2017 Chou et al found that the amino acidic chains outside the staple degrade faster than the unstapled peptide, probably due to a conformational change determined by the staple [82].…”