New antitumor leads from a peptidomimetic library

A parallel combinatorial library of over 1600 compounds has been designed and synthesized for the development of new potential peptidomimetic protein tyrosine kinase (PTK) inhibitor leads. These peptidomimetic molecules are aimed at intervening with the substrate binding site of the pp60^c-src enzyme. The new structures were based on known PTK inhibitors with at least two variously substituted aromatic moieties attached by spacer groups of different length and flexibility. Eleven bis-aryl-type inhibitory compounds were found in the range of 18–100 M IC₅₀ concentrations from combinations of 12 different substituents. Molecular modeling of the active compounds showed a characteristic distance of 12–14 Å between the farthest sp² carbon atoms of the two aromatic rings. Conformational analysis of several peptide substrates recently found for pp60^c-src PTK showed that the energy-minimized conformers had the same distance between the two aromatic moieties. Several compounds in the library not only showed remarkable PTK inhibitory activity but also a significant apoptosis-inducing effect on HT-29 human colon tumor cells.     

Identification of FAK substrate peptides via colorimetric screening of a one‐bead one‐peptide combinatorial library

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is associated with regulating cellular functions such as cell adhesion and migration and has emerged as an important target for cancer research. Short peptide substrates that are selectively and efficiently phosphorylated by FAK have not been previously identified and tested. Here we report the synthesis and screening of a one‐bead one‐peptide combinatorial library to identify novel substrates for FAK. Using a solid‐phase colorimetric antibody tagging detection platform, the peptide beads phosphorylated by FAK were sequenced via Edman degradation and then validated through radioisotope kinetic studies with [γ‐³²P] ATP to derive Michaelis–Menton constants. The combination of results gathered from both colorimetric and radioisotope kinase assays led to the rational design of a second generation of FAK peptide substrates. Out of all the potential peptide substrates evaluated, the most active was GDYVEFKKK with a K_M = 92 μM and a V_max = 1920 nmol/min/mg. Peptide substrates discovered within this study may be useful diagnostic tools for future kinase investigations and may lead to novel therapeutic agents. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of novel peptide ligands for the cancer‐specific receptor mutation EFGRvIII using a mixture‐based synthetic combinatorial library

We report here, the design and synthesis of a positional scanning synthetic combinatorial library for the identification of novel peptide ligands targeted against the cancer‐specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in several kinds of cancer, in particular, ovarian, glioblastomas, and breast cancer, but not in normal tissue. The library consisted of six individual positional sublibraries in the format, H‐O_1–6XXXXX‐NH₂, O being one of the 19 proteinogenic amino acids (cysteine omitted) and X an equimolar mixture of these. The library consisted of 114 mixtures in total. Using a biotin‐streptavidin assay, the binding of each sublibrary to NR6M, NR6W‐A, and NR6 cells was tested. These cells express EGFRvIII, EGFR, and neither of the receptors, respectively. The result from each sublibrary was examined to identify the most active amino acid residue at each position. On the basis of this knowledge, eight peptides were synthesized and tested for binding to EGFRvIII. We identified one peptide, H‐FALGEA‐NH₂, that showed more selective binding to the mutated receptor than the EGFRvIII specific peptide PEPHC1. This study demonstrates the value of using mixture‐based combinatorial positional scanning libraries for the identification of novel peptide ligands targeted against the cancer‐specific EGFRvIII. Our best candidate H‐FALGEA‐NH₂ will be radioactively labeled and evaluated as an imaging agent for positron emission tomography investigation for diagnosis, staging, and monitoring of therapy of various types of cancer. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 201–206, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Stably folded de novo proteins from a designed combinatorial library

Binary patterning of polar and nonpolar amino acids has been used as the key design feature for constructing large combinatorial libraries of de novo proteins. Each position in a binary patterned sequence is designed explicitly to be either polar or nonpolar; however, the precise identities of these amino acids are varied extensively. The combinatorial underpinnings of the "binary code" strategy preclude explicit design of particular side chains at specified positions. Therefore, packing interactions cannot be specified a priori. To assess whether the binary code strategy can nonetheless produce well-folded de novo proteins, we constructed a second-generation library based upon a new structural scaffold designed to fold into 102-residue four-helix bundles. Characterization of five proteins chosen arbitrarily from this new library revealed that (1) all are alpha-helical and quite stable; (2) four of the five contain an abundance of tertiary interactions indicative of well-ordered structures; and (3) one protein forms a well-folded structure with native-like features. The proteins from this new 102-residue library are substantially more stable and dramatically more native-like than those from an earlier binary patterned library of 74-residue sequences. These findings demonstrate that chain length is a crucial determinant of structural order in libraries of de novo four-helix bundles. Moreover, these results show that the binary code strategy--if applied to an appropriately designed structural scaffold--can generate large collections of stably folded and/or native-like proteins.     

New amide linked dimeric 1,2,3-triazoles bearing aryloxy scaffolds as a potent antiproliferative agents and EGFR tyrosine kinase phosphorylation inhibitors

A search for potent antiproliferative agents has prompted to design and synthesize aryloxy bridged and amide linked dimeric 1,2,3-triazoles (7a–j) by using 1,3-dipolar cycloaddition reaction between 2-azido-N-phenylacetamides (4a–e) and bis(prop-2-yn-1-yloxy)benzenes (6a–b) via copper (I)-catalyzed click chemistry approach with good to excellent yields. All the newly synthesized compounds have been screened for their in vitro antiproliferative activities against two human cancer cell lines. The compounds 7d, 7e, 7h, 7i and 7j have revealed promising antiproliferative activity against human breast cancer cell line (MCF-7), whereas, the compounds 7a, 7b, 7c, 7i and 7j were observed as potent antiproliferative agents against human lung cancer cell line (A-549). The active compounds against MCF-7 have been also analysed for their mechanism of action by the enzymatic study, which shows that the compounds 7d, 7h and 7j were acts as active EGFR tyrosine kinase phosphorylation inhibitors. In support to this biological study, the molecular docking as well as in silico ADME properties of all the newly synthesized hybrids were predicted.     

Selection of ceramic fluorapatite‐binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography

Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA‐specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N‐terminal sequence was found in two selected peptides: F4‐2 (KPRSMLH) and F5‐4 (KPRSVSG). The peptide F5‐4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5‐4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage‐derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Analyzing the catalytic mechanism of MPtpA: a low molecular weight protein tyrosine phosphatase from Mycobacterium tuberculosis through site-directed mutagenesis

Mycobacterium tuberculosis adopts various measures to escape from the hostile environment of the host cells. A low molecular weight protein tyrosine phosphatase (LMWPTPase) MPtpA was found to be active in virulent mycobacterial forms during the phagocytosis process. To ascertain the importance of conserved residues Cys11, Arg17, and Asp126 in the catalytic mechanism of MPtpA, site-directed mutagenesis was performed, namely C11S, R17A, D126A, and D126N. Kinetic characterization of wild-type and the mutant MPtpAs using para-nitrophenyl phosphate revealed the reaction mechanism followed by this LMWPTPase and it is similar to the other PTPases. All the LMWPTPases have a common signature motif, 'C(X)(5)R(S/T)' and an Asp as the general acid residue and the mechanism followed by MPtpA can be aptly attributed to other LMWPTPases as well, considering the similar three-dimensional conformation. We have shown that the mutations caused major changes in the chemical environment surrounding the mutated residues and resulted in the decrease of catalytic activity significantly. Inhibition kinetics was performed with phosphate analogues: sodium molybdate, sodium orthovanadate, and sodium tungstate.     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

As an activator of adenylate cyclase, the neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) impacts levels of cyclic AMP, a key second messenger available in brain cells. PACAP is involved in certain adult behaviors. To elucidate PACAP interactions, a compendium of microarrays representing mRNA expression in the adult mouse whole brain was pooled from the Phenogen database for analysis. A regulatory network was computed based on mutual information between gene pairs using gene expression data across the compendium. Clusters among genes directly linked to PACAP, and probable interactions between corresponding proteins were computed. Database “experts” affirmed some of the inferred relationships. The findings suggest ADCY7 is probably the adenylate cyclase isoform most relevant to PACAP's action. They also support intervening roles for kinases including GSK3B, PI 3-kinase, SGK3 and AMPK. Other high-confidence interactions are hypothesized for future testing. This new information has implications for certain behavioral and other disorders.