Design and synthesis of novel azapeptide activators of apoptosis mediated by caspase-9 in cancer cells.

A set of azapeptides was designed based on the Ala-Val-Pro-Ile peptide (derived from Smac protein) to activate caspase-9 and induce apoptosis in breast cancer cells. The diversity-oriented synthesis of the aza-peptides 5-9 was accomplished by alkylation of the aza-residue of aza-Gly-Pro dipeptide 15 using potassium tert-butoxide and a range of different alkyl halides. The resulting protected aza-dipeptide building blocks were then introduced into mimics 5-9 using standard coupling conditions. Biological evaluation of 5-9 was performed in MDA-MB-231 breast cancer cells, and indicated that the aza-Gly and aza-Phe analogs 5 and 7 were most efficient in inducing cell death by a caspase-9 mediated apoptotic pathway. Revealing a relationship between azabicycloalkanone and aza peptide mimics, novel AVPI mimics were synthesized which exhibit utility for studying structure-activity relationships to develop leads for activating apoptosis in cancer cells.

A novel biased allosteric compound inhibitor of parturition selectively impedes the prostaglandin F2alpha-mediated Rho/ROCK signaling pathway.

The prostaglandin F2alpha (PGF2alpha) receptor (FP) is a key regulator of parturition and a target for pharmacological management of preterm labor. However, an incomplete understanding of signaling pathways regulating myometrial contraction hinders the development of improved therapeutics. Here we used a peptidomimetic inhibitor of parturition in mice, PDC113.824, whose structure was based on the NH(2)-terminal region of the second extracellular loop of FP receptor, to gain mechanistic insight underlying FP receptor-mediated cell responses in the context of parturition. We show that PDC113.824 not only delayed normal parturition in mice but also that it inhibited both PGF2alpha- and lipopolysaccharide-induced preterm labor. PDC113.824 inhibited PGF2alpha-mediated, G(alpha)(12)-dependent activation of the Rho/ROCK signaling pathways, actin remodeling, and contraction of human myometrial cells likely by acting as a non-competitive, allosteric modulator of PGF2alpha binding. In contrast to its negative allosteric modulating effects on Rho/ROCK signaling, PDC113.824 acted as a positive allosteric modulator on PGF2alpha-mediated protein kinase C and ERK1/2 signaling. This bias in receptor-dependent signaling was explained by an increase in FP receptor coupling to G(alpha)(q), at the expense of coupling to G(alpha)(12). Our findings regarding the allosteric and biased nature of PDC113.824 offer new mechanistic insights into FP receptor signaling relevant to parturition and suggest novel therapeutic opportunities for the development of new tocolytic drugs.

Solution-phase submonomer diversification of aza-dipeptide building blocks and their application in aza-peptide and aza-DKP synthesis.

Aza-peptides have been used as tools for studying SARs in programs aimed at drug discovery and chemical biology. Protected aza-dipeptides were synthesized by a solution-phase submonomer approach featuring alkylation of N-terminal benzophenone semicarbazone aza-Gly-Xaa dipeptides using different alkyl halides in the presence of potassium tert-butoxide as base. Benzophenone protected aza-dipeptide tert-butyl ester 31c was selectively deprotected at the C-terminal ester or N-terminal hydrazone to afford, respectively, aza-dipeptide acid and amine building blocks 36c and 40c, which were introduced into longer aza-peptides. Alternatively, removal of the benzophenone semicarbazone protection from aza-dipeptide methyl esters 29a-c led to intramolecular cyclization to produce aza-DKPs 39a-c. In light of the importance of aza-peptides and DKPs as therapeutic agents and probes of biological processes, this diversity-oriented solution-phase approach may provide useful tools for studying peptide science.

Paired Utility of Aza-Amino Acyl Proline and Indolizidinone Amino Acid Residues for Peptide Mimicry: Conception of Prostaglandin F2α Receptor Allosteric Modulators That Delay Preterm Birth.

Peptide mimicry employing a combination of aza-amino acyl proline and indolizidinone residues has been used to develop allosteric modulators of the prostaglandin F2α receptor. The systematic study of the N-terminal phenylacetyl moiety and the conformation and side chain functions of the central turn dipeptide residue has demonstrated the sensitive relationships between modulator activity and topology. Examination of aza-Gly-Pro and aza-Phe-Pro analogs 2a and 2b in a murine preterm labor model featuring treatment with lipopolysaccharide demonstrated their capacity to extend significantly (>20 h) the average time of delivery offering new prototypes for delaying premature birth.

Synthesis and alkylation of Aza-Gly-Pro building blocks of peptidomimetic libraries for developing prostaglandin F2α receptor modulators as therapeutics to inhibit preterm labor.

Premature birth is a steadily increasing unmet medical need, for which new "tocolytic" agents are required to arrest contractions and delay labor. A peptide-based approach was developed to produce modulators of the prostaglandin F2α receptor as a novel target for tocolytic development. In this strategy, the solution-phase synthesis and alkylation of aza-glycyl-proline building blocks were key for the preparation of a series of modulators exhibiting biased signaling. An optimized method is now provided for making the aza-Gly-Pro unit with minimum side product, and alkylation of the unit is described to illustrate the library diversification step. Conditions have been reported for selectively unmasking the protecting groups at the N- and C-terminal of the aza-dipeptide unit and for its introduction into analogs that modulate the signaling of the PGF2α receptor. The merits of this protocol for azapeptide synthesis have thus been demonstrated by the synthesis of inhibitors of myometrial contraction exhibiting potential as prototypes for developing tocolytics to treat preterm labor.

Targeting the prostaglandin F2α receptor for preventing preterm labor with azapeptide tocolytics.

The prostaglandin-F2α (PGF2α) receptor (FP) was targeted to develop tocolytic agents for inhibiting preterm labor. Azabicycloalkane and azapeptide mimics 2-10 were synthesized based on the (3S,6S,9S)-indolizidin-2-one amino acid analogue PDC113.824 (1), which was shown to modulate FP by a biased allosteric mechanism, involving both Gαq- and Gα12-mediated signaling pathways, and exhibited significant tocolytic activity delaying preterm labor in a mouse model ( Goupil ; et al. J. Biol. Chem. 2010 , 285 , 25624 - 25636 ). Although changes in azabicycloalkane stereochemistry and ring size caused loss of activity, replacement of the indolizidin-2-one amino acid with azaGly-Pro and azaPhe-Pro gave azapeptides 6 and 8, which reduced PGF2α-induced myometrial contractions, potentiated the effect of PGF2α on Gαq-mediated ERK1/2 activation, and inhibited FP modulation of cell ruffling, a response dependent on the Gα12/RhoA/ROCK signaling pathway. Revealing complementarities of azabicycloalkane and azapeptide mimics, novel probes, and efficient tocolytic agents were made to study allosteric modulation of the FP receptor.

Exploring side-chain diversity by submonomer solid-phase aza-peptide synthesis.

Submonomer synthesis of aza-peptides featuring regioselective alkylation of peptide-bound aza-Gly residues provided ten aza-analogues of the Growth Hormone Releasing Peptide-6 (GHRP-6) in 15-42% yield and purity generally >or=90%. Circular dichroism demonstrated that azaPhe-peptide 7a induced a beta-turn conformation which may be responsible for its 1000-fold improvement in GHRP-6 selectivity for the CD36 receptor. This versatile method for making aza-peptides avoids solution-phase hydrazine synthesis and is well suited for studying side-chain-activity relationships of biologically active peptides.

Benzophenone semicarbazone protection strategy for synthesis of aza-glycine containing aza-peptides.

Aza-glycine has been incorporated into peptide mimics as a tool for studying the active conformation and characterizing structure-function relationships for activity. Side reactions, such as intramolecular cyclizations to form hydantoins and oxadiazalones, have, however, inhibited efforts to make activated aza-Gly residues in solution using carbamate protection. Herein, we describe efficient incorporation of aza-glycine into aza-peptides using diphenyl hydrazone protection. Hydrazone acylation with p-nitrobenzyl chloroformate provided the protected aza-Gly activated ester, which was used to acylate a set of amino ester and amino acids to provide aza-Gly-Xaa aza-dipeptide fragments for peptide synthesis. Removal of the hydrazone protection was performed under acidic conditions to provide the hydrochloride salt of the aza-Gly residue for subsequent elongation of the aza-peptide chain using standard coupling conditions. A proof of concept for the use of benzophenone protection has been established by the synthesis of an aza-peptide analog of a potent activator of caspase 9 in cancer cells.