Improved proteolytic stability and potent activity against Leishmania infantum trypanothione reductase of α/β-peptide foldamers conjugated to cell-penetrating peptides

Abstract

The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/β-peptide foldamers of different length (from 9-mers to 13-mers) and different α→β substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/β-peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,β-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/β-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes.

Chiral Cyclobutane-Containing Cell-Penetrating Peptides as Selective Vectors for Anti-Leishmania Drug Delivery Systems

“…There are few examples of the use of CPP in the treatment of Leishmania [ 38 , 39 , 40 ]. Even so, the use of CPP as DDS on Leishmania parasites has been reported for the delivery of cargoes which are noncovalently complexed with CPP [ 41 , 42 , 43 ], or as covalent conjugates [ 44 , 45 , 46 , 47 , 48 ].…”

Trypanothione reductase inhibition and anti-leishmanial activity of all-hydrocarbon stapled α-helical peptides with improved proteolytic stability

“…Finally, to deliver the peptides into the parasites for leishmanicidal in vitro evaluation, the N-terminal end of 2a was covalently linked to a polyarginine (R 9 ) chain using standard SPPS methodology (compound 5, Scheme 2), as previously reported for our CPP-conjugates of linear peptide prototypes, lactam-bridged analogues and ,-peptide foldamers [10,29,33].…”

“…Fluorescence microscopy with the fluorescein-labeled conjugate [Flu]-PEG-[peptide] (compound 4) demonstrated the low ability of the hydrocarbon-bridged analogues tocross the cell membrane (data not shown). To deliver the peptides into the parasites, 2 was covalently linked to an R 9 sequence used as a CPP (compound 5), following the successful strategy already employed for the efficient uptake of the linear peptide prototype 1 and several lactam-bridged and mixed ,-peptide foldamer analogues[29,33]. The R 9 sequence was also studied in parallel as a control.…”

Small Molecule–Peptide Conjugates as Dimerization Inhibitors of Leishmania infantum Trypanothione Disulfide Reductase

“…The C-terminal truncated peptide Ac-PKIIQSVGI-NH 2 ( TRL38 ) was identified as the shortest peptide sequence that showed inhibition of Li TryR oxidoreductase activity and also the ability to disrupt enzyme homodimerization [ 22 ]. With a view to optimizing these novel Li TryR inhibitors and developing peptidomimetics with enhanced proteolytic stability, a variety of helix-stabilized cyclic and stapled peptides [ 23 , 24 , 25 ], as well as α,β 3 -peptide foldamers [ 26 ], were prepared thereafter. All these peptides and peptidomimetics required attachment to cell-penetrating peptides to allow their cellular uptake and subsequent killing of the parasites in cell culture [ 25 , 26 ].…”

“…With a view to optimizing these novel Li TryR inhibitors and developing peptidomimetics with enhanced proteolytic stability, a variety of helix-stabilized cyclic and stapled peptides [ 23 , 24 , 25 ], as well as α,β 3 -peptide foldamers [ 26 ], were prepared thereafter. All these peptides and peptidomimetics required attachment to cell-penetrating peptides to allow their cellular uptake and subsequent killing of the parasites in cell culture [ 25 , 26 ]. To overcome the unfavorable drug-like properties of these early prototypes, our efforts were then directed towards the design of small molecules (pyrrolopyrimidine-, imidazole-, and triazole-phenyl-thiazole scaffolds) endowed with high potency and more favorable pharmacokinetic characteristics [ 27 , 28 ].…”