De Novo Designed Library of Linear Helical Peptides: An Exploratory Tool in the Discovery of Protein–Protein Interaction Modulators

Abstract

Protein-protein interactions (PPIs) have emerged as important targets for pharmaceutical intervention because of their essential role in numerous physiological and pathological processes, but screening efforts using small-molecules have led to very low hit rates. Linear peptides could represent a quick and effective approach to discover initial PPI hits, particularly if they have inherent ability to adopt specific peptide secondary structures. Here, we address this hypothesis through a linear helical peptide library, composed of four sublibraries, which was designed by theoretical predictions of helicity (Agadir software). The 13-mer peptides of this collection fixes either a combination of three aromatic or two aromatic and one aliphatic residues on one face of the helix (Ac-SSEEX(5)ARNX(9)AAX(12)N-NH2), since these are structural features quite common at PPIs interfaces. The 81 designed peptides were conveniently synthesized by parallel solid-phase methodologies, and the tendency of some representative library components to adopt the intended secondary structure was corroborated through CD and NMR experiments. As proof of concept in the search for PPI modulators, the usefulness of this library was verified on the widely studied p53-MDM2 interaction and on the communication between VEGF and its receptor Flt-1, two PPIs for which a hydrophobic α-helix is essential for the interaction. We have demonstrated here that, in both cases, selected peptides from the library, containing the right hydrophobic sequence of the hot-spot in one of the protein partners, are able to interact with the complementary protein. Moreover, we have discover some new, quite potent inhibitors of the VEGF-Flt-1 interaction, just by replacing one of the aromatic residues of the initial F(5)Y(9)Y(12) peptide by W, in agreement with previous results on related antiangiogenic peptides. Finally, the HTS evaluation of the full collection on thermoTRPs has led to a few antagonists of TRPV1 and TRPA1 channels, which open new avenues on the way to innovative modulators of these channels.

Transient Receptor Potential Melastatin 8 Channel (TRPM8) Modulation: Cool Entryway for Treating Pain and Cancer

“…220,240 Notably, peptides patterned after this domain in TRPV1 channels inhibit receptor function, 228 indicating that this domain may also be a novel site for drug intervention in TRPM8 channels. Up to date, the destabilization of protein-protein interactions (PPI) within ion channels has barely been studied, 241 but most probably the application of strategies followed for other PPIs could also be relevant for TRPM8 drug discovery.…”

Disrupting VEGF–VEGFR1 Interaction: De Novo Designed Linear Helical Peptides to Mimic the VEGF13-25 Fragment

“…We had previously described a collection of linear 13-mer peptide library, de novo designed for adopting helical conformations. Our hypothesis was based on the fact that α-helices are the secondary structure element most frequently involved in PPIs [ 15 ]. They were designed to fix a combination of either three aromatic—or two aromatic and one aliphatic—residues on one face of the helix (Ac-SSEE X 5 ARN X 9 AA X 12 N-NH 2 ), taking into account that frequently only the hydrophobic face of the helix is involved in binding, and also that key residues for affinity are located at relative positions i , i + 3( i + 4), i + 7.…”

“…In both cases, the protein–protein contact takes place through interfaces in which the mediation of a hydrophobic α-helix is essential. The results of the appropriate binding experiments showed that some of the tested peptides were able to mimic p53 and VEGF ‘hot-spots’, binding with their complementary proteins MDM2 (murine form of the HDM2 suppressor factor) and VEGFR1, respectively, and finally hampering their interaction with the respective partners, p53 and VEGF [ 15 ].…”

“…To validate our helical library in the VEGF/VEGFR system [ 15 ], seven peptides of the whole collection were initially selected, including the FYY containing derivative as the most related to VEGF 17-25 fragment. Six of them had in their sequence three aromatic residues located at relative positions i , i + 4, i + 7, corresponding to positions 5, 9, 12 of the sequence (Ac-SSEE X 5 ARN X 9 AA X 12 N-NH 2 ) [ 15 ].…”

Effect of acyclic monoterpene alcohols and their derivatives on TRP channels

“…9,11 It is well known that the TRP domain of the different transient potential receptor channels contains various sets of hydrophobic residues, involved in channel assembly and activation. 13 Esters 1a-i, 2a-j, 3a-e were prepared by acylation of the corresponding alcohols with either acetic anhydride (esters 1a, 2a, 3a) or the appropriate carboxylic acids, using N-ethyl-N 0 -(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as the carboxylate activator and 4-dimethylaminopyridine (DMAP) as the nucleophilic catalyst (esters 1b-i, 2b-j, 3b-e) (Scheme 1). Amides 1j,k and 2k,l were prepared by condensation of geranylamine (4a) and nerylamine (4b), respectively, with the appropriate carboxylic acids, using 1-hydroxybenzotriazole (HOBt)/EDC as the carboxylate activator (Scheme 2).…”