Rosario González-Muñiz

Modulation of Protein-Protein Interactions by Stabilizing/Mimicking Protein Secondary Structure Elements

In view of the crucial role of protein-protein intercommunication both in biological and pathological processes, the search of modulators of protein-protein interactions (PPIs) is currently a challenging issue. The development of rational strategies to imitate key secondary structure elements of protein interfaces is complementary to other approaches based on the screening of synthetic or virtual libraries. In this sense, the present review provides representative examples of compounds that are able to disturb PPIs of therapeutic relevance, through the stabilization or the imitation of peptide hot-spots detected in contact areas of the interacting proteins. The review is divided into three sections, covering mimetics of the three main secondary structural elements found in proteins, in general, and in protein-protein interfaces, in particular (alpha-helices, beta-sheets, and reverse turns). Once the secondary element has been identified, the first approach typically involves the translation of the primary peptide structure into different cyclic analogues. This is normally followed by gradual decrease of the peptide nature through combination of peptide and non-peptide fragments in the same molecule. The final step usually consists in the development of pertinent organic scaffolds for appending key functional groups in the right spatial disposition, as a means towards totally non-peptide small molecule PPI modulators.

Advances in modulating thermosensory TRP channels

Introduction: Thermosensory channels are a subfamily of the transient receptor potential (TRP) channel family that are activated by changes in the environmental temperature. These channels, known as thermoTRPs, cover the entire spectrum of temperatures, from noxious cold (< 15°C) to injurious heat (> 42°C). In addition, dysfunction of these channels contributes to the thermal hypersensitivity that accompanies painful conditions. Moreover, because of their wide tissue and cellular distribution, thermoTRPs are also involved in the pathophysiology of several diseases, from inflammation to cancer. Areas covered: Although the number of thermoTRPs is increasing with the identification of novel members such as TRPM3, we will cover the recent advances in the pharmacology of the classical thermosensory channels, namely TRPV1, TRPV2, TRPV3, TRPV4, TRPM8 and TRPA1. This review will focus on the therapeutic progress carried out for all these channels and will highlight the tenet that TRPV1, TRPM8 and TRPA1 are the most exploited channels, and that the interest on TRPV3 and TRPV4 is growing with the first TRPV3 antagonist that moves into Phase-II clinical trials. In contrast, the pharmacology of TRPV2 is yet in its infancy. Expert opinion: Despite the tremendous academic and industrial investment to develop therapeutic modulators of thermoTRPs, it apparently seems that we are still far from the first successful product, although hope is maintained high for all compounds currently in clinical trials. A major concern has been the appearance of side effects. A better knowledge of the thermosensory protein networks (signal-plexes), along with the application of system biology approaches may provide novel strategies to modulate thermoTRPs activity with improved therapeutic index. A case in point is TRPV1, where acting on interacting proteins is providing new therapeutic opportunities.

A role for ring-closing metathesis in medicinal chemistry: mimicking secondary architectures in bioactive peptides.

Synthetically versatile and easy to carry out, Ring‐Closing Metathesis (RCM) constitutes an attractive chemical tool, easily amenable for multiple substrates in mild conditions. In medicinal chemistry, the use of RCM has been especially prolific during the last few years. An important application that has benefited from this reaction is the stabilization of spatial conformations in bioactive peptides, since their 3D arrangements play relevant roles in biomolecular recognition processes. RCM reaction is being widely used to introduce conformational constraints into small peptides, through the generation of cyclic structures from appropriate linear precursors. As an alternative to strategies like disulfide or lactam‐bridged cyclizations, RCM shows the additional advantage of generating hydrocarbon bridges, less prone to metabolic degradation, and metabolically more stable, which could benefit their pharmacokinetic properties. Particularly remarkable is the application of RCM to the preparation of small peptide modulators able to mimic epitopes identified as hotspots within the surface contact areas in protein‐protein interactions (PPIs). This review deals with the replacement of S‐S and thioether linkages of cyclic peptides by C‐C‐bridges and with the stabilization of peptide secondary architectures (α‐helix, β‐hairpins, β‐turns) through RCM, as a useful strategy for the modulation of therapeutically relevant signaling pathways.   © 2010 Wiley Periodicals, Inc. Med Res Rev 31: 677‐715, 2011

Disulfide bonds versus Trp···Trp pairs in irregular β-hairpins: NMR structure of vammin loop 3-derived peptides as a case study

Where a noncovalent interaction is better than a covalent bond: The most stabilising cross‐strand pairs were incorporated into an irregular β‐hairpin, loop 3 of vammin. 1H and 13C NMR conformational analyses of these designed peptides indicated that an edge‐to‐face Trp⋅⋅⋅Trp interaction leads to a β‐hairpin that is more stable than a disulfide bond.

Memory of chirality in the stereoselective synthesis of β-lactams: importance of the starting amino acid derivative

Abstract The enantioselectivity of the base-promoted cyclization of N -alkyl- N -chloroacetyl amino acid derivatives to β-lactams is dependent on the substituents on the starting material. While t Bu esters are preferred over Me esters, and N -Bzl-, N -Pmb, N -Nph and N -Mom groups gave similar e.e. values, only amino acid derivatives with branched side-chains at the γ-position were able to show a good memory of chirality.

Pharmacological evaluation of IQM-95,333, a highly selective CCKA receptor antagonist with anxiolytic-like activity in animal models

The pyridopyrimidine derivative IQM‐95,333 ((4aS,5R)‐2‐benzyl‐5‐[Nα‐tert‐butoxicarbonyl)L‐tryptophyl]amino‐1,3dioxoperhydropyrido[1,2‐c]pyrimidine), a new non‐peptide antagonist of cholecystokinin type A (CCKA) receptors, has been evaluated in vitro and in vivo in comparison with typical CCKA and CCKB receptor antagonists, such as devazepide, lorglumide, L‐365,260 and PD‐135,158. IQM‐95,333 displaced [3H]‐CCK‐8S binding to CCKA receptors from rat pancreas with a high potency in the nanomolar range. Conversely, the affinity of this new compound at brain CCKB receptors was negligible (IC50>10 μM). IQM‐95,333 was a more selective CCKA receptor ligand than devazepide and other CCKA receptor antagonists. Like devazepide, IQM‐95,333 was a more potent antagonist of CCK‐8S‐ than of CCK‐4‐induced contraction of the longitudinal muscle from guinea‐pig ileum, suggesting selective antagonism at CCKA receptors. IQM‐95,333 and devazepide were also potent inhibitors of CCK‐8S‐stimulated amylase release from isolated pancreatic acini, a CCKA receptor‐mediated effect. The drug concentrations required (IC50s around 20 nM) were higher than in binding studies to pancreas homogenates. Low doses (50–100 μg kg−1, i.p.) of IQM‐95,333 and devazepide, without any intrinsic effect on food intake or locomotion, blocked the hypophagia and the hypolocomotion induced by systemic administration of CCK‐8S, two effects associated with stimulation of peripheral CCKA receptors. IQM‐95,333 showed an anxiolytic‐like profile in the light/dark exploration test in mice over a wide dose range (10–5,000 μg kg−1). Typical CCKA and CCKB antagonists, devazepide and L‐365,260 respectively, were only effective within a more limited dose range. In a classical conflict paradigm for the study of anxiolytic drugs, the punished‐drinking test, IQM‐95,333, devazepide and L‐365,260 were effective within a narrow dose range. The dose‐response curve for the three drugs was biphasic, suggesting that other mechanisms are operative at higher doses. In conclusion, IQM‐95,333 is a potent and selective CCKA receptor antagonist both in vitro and in vivo with an anxiolytic‐like activity in two different animal models, which can only be attributed to blockade of this CCK receptor subtype.

Regio- and enantioselectivity of the Candida antarctica lipase catalyzed amidations of Cbz-l- and Cbz-d-glutamic acid diesters

Abstract Candida antarctica lipase (CAL) catalyzed amidation of Cbz-glutamic acid diesters takes place in a regioselective way to give the corresponding monoamide derivatives. The regioselectivity was found to be dependent on the reacting Glu enantiomer. Thus, amidations of Cbz- l -Glu diesters regiospecifically afforded α-amide while the γ-ester is selectively substituted in the d -enantiomer. This enzymatic reaction also shows enantioselectivity when a chiral amine is used as nucleophile.

Azetidine-Derived Amino Acids versus Proline Derivatives. Alternative Trends in Reverse Turn Induction

The influence of 2-alkyl-2-carboxyazetidines (Aze) on the 3D structure of model tetrapeptides R2CO-2-R1Aze-l-Ala-NHMe has been analyzed by molecular modeling, 1H NMR, and FT-IR studies. The conformational constraints introduced by the four-membered ring resulted in an effective way to stabilize gamma-turn-like conformations in these short peptides. The conformational preferences of these Aze-containing peptides have been compared to those of the corresponding peptide analogues containing Pro or alpha-MePro in the place of 2-alkyl-Aze residue. In the model studied, both Pro and Aze derivatives are able to induce reverse turns, but the nature of the turn is different as a function of the ring size. While the five-membered ring of Pro tends to induce beta-turns, as previously suggested by different authors, the four-membered ring of Aze residues forces the peptide to preferentially adopt gamma-turn conformations. In both cases, the presence of an alkyl group at the alpha-position of Pro or the azetidine-2-carboxylate ring enhances significantly the turn-inducing ability. These results might open the opportunity of using 2-alkyl-Aze residues as versatile tools in defining the role of gamma-turn structures within the bioactive conformation of selected peptides, and represent an alternative to Pro derivatives as turn inducers.

Identification, functional gastrointestinal stability and molecular docking studies of lentil peptides with dual antioxidant and angiotensin I converting enzyme inhibitory activities

The objective was to identify peptides with dual antioxidant and angiotensin I converting enzyme (ACE) inhibitory activities released from lentil proteins by Savinase®. The influence of gastrointestinal digestion on peptide bioactivity was also assayed. Fragments from vicilin, convicilin and legumin were the most abundant peptides identified. Peptides LLSGTQNQPSFLSGF, NSLTLPILRYL, TLEPNSVFLPVLLH showed the highest antioxidant (0.013-1.432μmol Trolox eq./μmol peptide) and ACE inhibitory activities (IC50=44-120μM). Gastrointestinal digestion of peptides improved their dual activity (10-14μmol Trolox eq./μmol peptide; IC50=11-21μM). In general, C-terminal heptapeptide was crucial for their dual activity. ACE inhibition relies on the formation of hydrogen bonds between C-terminal residues of lentil peptides and residues of the ACE catalytic site. The present study helps clarifying the relationship between structure and dual antioxidant/antihypertensive activity of lentil peptides opening new opportunities to food industry such as the application of lentil protein hydrolysates as ingredients for development of functional foods.

Quaternary α,α-2-oxoazepane α-amino acids: synthesis from ornithine-derived β-lactams and incorporation into model dipeptides.

To explore further the chemistry of amino acid-derived β-lactams, their conversion to α,α-heterocyclic quaternary amino acid derivatives is investigated. The latter derivatives, containing 2-oxoazepane as the α,α-substituent, are synthesized by a simple Pd-C-catalyzed hydrogenolysis of Orn(Z)-derived 2-azetidinones. The rearrangement from four- to seven-membered lactam ring is driven by the key intramolecular opening of the 1-Boc-β-lactam, initiated by 7-exotrig ring closure from the NH(2) of the Orn side chain. The synthetic route is applied to the stereoselective preparation of enantiomerically pure 4-amino-3-methyl-2-oxoazepane-4-carboxylate derivatives, for which the structure and configuration is confirmed by X-ray diffraction. Molecular modeling and NMR experiments indicate that these quaternary amino acids are able to drive the adoption of β-turn secondary structures when incorporated in model dipeptide derivatives.