Chemical modulation of peptoids: synthesis and conformational studies on partially constrained derivatives.

The high conformational flexibility of peptoids can generate problems in biomolecular selectivity as a result of undesired off-target interactions. This drawback can be counterbalanced by restricting the original flexibility to a certain extent, thus leading to new peptidomimetics. By starting from the structure of an active peptoid as an apoptosis inhibitor, we designed two families of peptidomimetics that bear either 7-substituted perhydro-1,4-diazepine-2,5-dione 2 or 3-substituted 1,4-piperazine-2,5-dione 3 moieties. We report an efficient, solid-phase-based synthesis for both peptidomimetic families 2 and 3 from a common intermediate. An NMR spectroscopic study of 2a,b and 3a,b showed two species in solution in different solvents that interconvert slowly on the NMR timescale. The cis/trans isomerization around the exocyclic tertiary amide bond is responsible for this conformational behavior. The cis isomers are more favored in nonpolar environments, and this preference is higher for the six-membered-ring derivative 3a,b. We propose that the hydrogen-bonding pattern could play an important role in the cis/trans equilibrium process. These hydrogen bonds were characterized in solution, in the solid state (i.e., by using X-ray studies), and by molecular modeling of simplified systems. A comparative study of a model peptoid 10 containing the isolated tertiary amide bond under study outlined the importance of the heterocyclic moiety for the prevalence of the cis configuration in 2a and 3a. The kinetics of the cis/trans interconversion in 2a, 3a, and 10 was also studied by variable-temperature NMR spectroscopic analysis. The full line-shape analysis of the NMR spectra of 10 revealed negligible entropic contribution to the energetic barrier in this conformational process. A theoretical analysis of 10 supported the results observed by NMR spectroscopic analysis. Overall, these results are relevant for the study of the peptidomimetic/biological-target interactions.

Protein-protein interaction antagonists as novel inhibitors of non-canonical polyubiquitylation.

BACKGROUND: Several pathways that control cell survival under stress, namely RNF8-dependent DNA damage recognition and repair, PCNA-dependent DNA damage tolerance and activation of NF-kappaB by extrinsic signals, are regulated by the tagging of key proteins with lysine 63-based polyubiquitylated chains, catalyzed by the conserved ubiquitin conjugating heterodimeric enzyme Ubc13-Uev. METHODOLOGY/PRINCIPAL FINDINGS: By applying a selection based on in vivo protein-protein interaction assays of compounds from a combinatorial chemical library followed by virtual screening, we have developed small molecules that efficiently antagonize the Ubc13-Uev1 protein-protein interaction, inhibiting the enzymatic activity of the heterodimer. In mammalian cells, they inhibit lysine 63-type polyubiquitylation of PCNA, inhibit activation of NF-kappaB by TNF-alpha and sensitize tumor cells to chemotherapeutic agents. One of these compounds significantly inhibited invasiveness, clonogenicity and tumor growth of prostate cancer cells. CONCLUSIONS/SIGNIFICANCE: This is the first development of pharmacological inhibitors of non-canonical polyubiquitylation that show that these compounds produce selective biological effects with potential therapeutic applications.

A semaphorin 3A inhibitor blocks axonal chemorepulsion and enhances axon regeneration.

Secreted semaphorins are a large group of extracellular proteins involved in a variety of processes during development, including neuronal migration and axon guidance. We screened a peptoid combinatorial library to search for semaphorin 3A inhibitors, and identified a peptoid (SICHI: semaphorin Induced chemorepulsion inhibitor) that blocks semaphorin 3A-chemorepulsion and growth-cone collapse in axons at millimolar concentrations. SICHI inhibits the binding of semaphorin 3A to its receptor complex (neuropilin 1/plexin A1) and semaphorin 3A-induced phosphorylation of GSK3. Chemorepulsion induced by semaphorin 3F or netrin 1 is not blocked by SICHI. We also show that SICHI promotes neural regeneration of damaged axons. We suggest that SICHI, a selective inhibitor of semaphorin 3A, is of therapeutic interest for approaches aimed at promoting axonal regeneration and brain repair.

Anti-Tat and anti-HIV activities of trimers of n-alkylglycines.

Transcription of human immunodeficiency virus (HIV-1) is activated by viral Tat protein which regulates HIV-LTR transcription and elongation. In the present report, the evaluation of the anti-Tat activity of a combinatorial library composed of 5120 N-trialkylglycines is reported. The antiviral activity was studied through luciferase-based assays targeting the HIV-1 promoter activation induced by the HIV-1 Tat protein. We identified five peptoids with specific anti-HIV-1 Tat activity; none of these peptoids affected the binding of HIV-1 Tat protein to the viral TAR RNA. Using a recombinant-virus assay in which luciferase activity correlates with the rate of HIV-1 transcription we have detected that one of the five selected peptoids, NC37-37-15C, is a potent inhibitor of HIV-1-LTR transcription in both primary T lymphocytes and transformed cell lines. The inhibitory effect of NC37-37-15C, which is additive with azidothymidine (AZT), correlates with its ability to inhibit CTD phosphorylation and shows a suitable profile for development of novel anti-HIV-1 drugs. Likewise, the structural simplicity of N-alkylglycine oligomers makes these peptidomimetics amenable to structural manipulation, thus facilitating the optimisation of lead molecules for drug-like properties.

Peptoids as source of compounds eliciting antibacterial activity.

N-Alkylglycine oligomers (peptoids) constitute a family of non-natural peptidomimetics attractive for the early drug discovery process because of their physicochemical features, easy of adaptation to combinatorial chemistry approaches and their proteolytic stability. Consequently, peptoid libraries have found application for discovering hits against a wide diversity of pharmaceutical targets, among which different examples of antibacterials are found. In the present work, research efforts addressed towards the identification of peptoids as antibacterial agents are discussed.

Design and synthesis of an optimized positional scanning library of peptoids: identification of novel multidrug resistance reversal agents.

Herein is reported the optimized solid-phase synthesis of a library of 5,120 trimeric N-alkylglycines (peptoids) using the positional scanning format and the submonomer strategy. Diversity at the N-terminal position was generated from 20 commercially available primary amines, whereas 16 primary amines were employed for the middle and C-terminal positions of the trimers. Formation of undesirable side-products observed in a previous library synthesis (Humet, M. et al. J. Comb. Chem. 2003, 5, 597-605) was averted by restricting the use of primary amines functionalized with tertiary amino groups to the third amination step. Screening of the new library for the identification of chemosensitizers yielded two peptoids, compounds 1 and 2, with potent in vitro activity as multidrug resistance (MDR) reversal agents. The structures of the lead peptoids are consistent with a pharmacophore model generated from the interaction of various known inhibitors with the MDR-implicated transmembrane glycoprotein P-gp.

Identification from a positional scanning peptoid library of in vivo active compounds that neutralize bacterial endotoxins.

Two peptoids that neutralize the Gram-negative lipopolysaccharide (LPS) were identified from the screening of a positional scanning library. The evaluation of the in vivo activity of these compounds in an endoxemia murine model is also reported. These peptoids did not neutralize lipid A, i.e., the hydrophobic toxic component of LPS. This fact suggests that they do not have access to the micellar core and that they should bind to the hydrophilic carbohydrate portion of LPS.

Trimers of N-alkylglycines are potent modulators of the multidrug resistance phenotype.

The multidrug resistance (MDR) phenotype is considered a major cause of the failure of cancer chemotherapy. The acquisition of MDR is usually mediated by the overexpression of drug efflux pumps such as glycoprotein P (P-gp) or multidrug resistance-related protein 1 (MRP1). Thus, the identification, validation, and development of compounds that mitigate the MDR phenotype by modulating the activity of these transport proteins is an important yet elusive target. Here, we have addressed this issue and screened an N-trialkylglycine-based combinatorial library composed of 5120 compounds to search for modulators of the MDR phenotype. The screening identified 20 trimers of N-alkylglycine that increased the intracellular accumulation of daunomycin (DNM) in drug-resistant L1210R tumor cells that overexpressed the P-gp. These compounds seem to act as P-gp antagonists, as evidenced by the augmentation of DNM accumulation in the L1210(P-gp) cell line, a drug-sensitive L1210 cell stably expressing the murine P-gp protein. Similarly, several of the active N-trialkylglycines also produced an increment in DNM uptake in human HL60R cells, which primarily express the MRP1 protein. Trialkylglycines notably sensitized L1210R and HL60R tumor cells to DNM with a potency that rivaled that of verapamil. These findings provide new molecular scaffolds for the development of effective chemosensitizers against the MDR phenotype that, in due turn, could be used as adjuvant drugs in cancer chemotherapy.

Oxidative stress-induced apoptosis in retinal photoreceptor cells is mediated by calpains and caspases and blocked by the oxygen radical scavenger CR-6.

A critical role for reactive oxygen species (ROS) in photoreceptor apoptosis has been established. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. This study delineates the molecular events that occur after treatment of the photoreceptor cell line 661W with the nitric oxide donor sodium nitroprusside (SNP). Cytosolic calcium levels increased during photoreceptor apoptosis, leading to activation of the calcium-dependent proteases calpains. Furthermore, caspase activation also occurred following SNP insult. However, although treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone inhibited caspase activity per se in SNP-treated 661W cells, it did not prevent apoptosis. On the other hand, CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acted as a scavenger of ROS and reduced 661W photoreceptor apoptosis induced by SNP by preventing the activation of a pathway in which calpains have a key role. In summary, we report for the first time that both caspases and calpains are involved in 661W photoreceptor apoptosis and that calpain activation can be prevented by the ROS scavenger CR-6.

Synthesis of a library of 3-oxopiperazinium and perhydro-3-oxo-1,4-diazepinium derivatives and identification of bioactive compounds.

The design and synthesis of a library of novel families of 3-oxopiperazinium and perhydro-3-oxo-1,4-diazepinium derivatives is reported. The library was composed of 44 3-oxopiperazinium derivatives (11 of these compounds had a spiranic skeleton) and 22 perhydro-3-oxo-1,4-diazepinium compounds. The synthetic procedure involved a 6-step sequence carried out in solution, along with the use of solid-phase linked scavengers and microwave activation for the rapid removal of the excess of amine reagents. A final cyclization step performed under mild conditions led to the charged heterocyclic moiety. Screening of this library in two biological assays identified active compounds that inhibit the activity of the vanilloid receptor TRPV1 and modulators of the multidrug resistance phenomenon. Thus, this synthetic sequence represents a facile and convenient entry to unprecedented libraries of this sort of tetraalkylammonium derivatives that may be of use for identification of novel scaffolds of diverse biological activity.

A positional scanning combinatorial library of peptoids as a source of biological active molecules: identification of antimicrobials.

A positional scanning library of N-alkylglycine trimers (peptoids) containing over 10 000 compounds has been synthesized on solid phase. The synthetic pathway involved the use of the submonomer strategy and a set of 22 commercially available primary amines as a chemical diversity source. The unbiased nature of the library allowed its screening against a variety of biological targets, leading to the identification of individual peptoids exhibiting remarkable biological activities (García-Martínez, C. et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2374. Montoliu, et al. J. Pharm. Exp. Therap. 2002, 302, 29. Planells-Cases, R., et al. J. Pharm. Exp. Therap. 2002, 302, 163). In the present work, the screening of this library against a panel of Gram-positive and Gram-negative bacteria led to the identification of different compounds exhibiting antimicrobial activity.