David Sabatino

Azapeptides and their therapeutic potential

Azapeptides are peptide analogs in which one or more of the amino residues is replaced by a semicarbazide. This substitution of a nitrogen for the α-carbon center results in conformational restrictions, which bend the peptide about the aza-amino acid residue away from a linear geometry. The resulting azapeptide turn conformations have been observed by x-ray crystallography and spectroscopy, as well as predicted based on computational models. In biologically active peptide analogs, the aza-substitution has led to enhanced activity and selectivity as well as improved properties, such as prolonged duration of action and metabolic stability. In light of these characteristics, azapeptides have found important uses as receptor ligands, enzyme inhibitors, drugs, pro-drugs, probes and imaging agents. Recent improvements in synthetic methods for their procurement have ushered in a new era of azapeptide chemistry. This review aims to provide a historical look at the development of azapeptide science along with a focus on recent developments and perspectives on the future of this useful tool for medicinal chemistry.

Peptide Scanning for Studying Structure‐Activity Relationships in Drug Discovery

Peptide‐based therapeutics have grown in importance over the last few decades. Furthermore, peptides have been extensively used as lead compounds in the drug discovery process to investigate the nature of chemical space required for molecular recognition and activity at a variety of targets. This critical commentary reviews scanning techniques, which employ natural and non‐proteinogenic amino acids to facilitate understanding of structural requirements for peptide biological activity. The value of sequence analysis by such methods is highlighted by examples, in which the elements for peptide affinity and activity have been elucidated and employed to prepare peptidomimetic leads for drug development.

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.

Structure–Activity Relationships of GHRP-6 Azapeptide Ligands of the CD36 Scavenger Receptor by Solid-Phase Submonomer Azapeptide Synthesis

The cluster of differentiation 36 (CD36) class B scavenger receptor binds a variety of biologically endogenous ligands in addition to synthetic peptides (i.e., growth hormone-releasing peptides, GHRPs), which modulate biological function related to anti-angiogenic and anti-atherosclerotic activities. Affinity labeling had previously shown that GHRP-6 analogues such as hexarelin, [2-Me-W(2)]GHRP-6 (1), bind to the lysine-rich domain of the CD36 receptor. Moreover, the azapeptide analogue [aza-F(4)]GHRP-6, 2, exhibited a characteristic β-turn conformation as described by CD and NMR spectroscopy and a slightly higher CD36 binding affinity relative to hexarelin (1.34 and 2.37 μM, respectively), suggesting receptor binding was mediated by the conformation and the aromatic residues of these peptide sequences. Ligand-receptor binding interactions were thus explored using azapeptides to examine influences of side-chain diversity and backbone conformation. In particular, considering that aromatic cation interactions may contribute to binding affinity, we have explored the potential of introducing salt bridges to furnish GHRP-6 azapeptide ligands of the CD36 receptor. Fifteen aza-glutamic acid analogues related to 2 were prepared by submonomer solid-phase synthesis. The azapeptide side chains were installed by novel approaches featuring alkylation of resin-bound semicarbazone with Michael acceptors and activated allylic acetates in the presence of phosphazene base (BTPP). Moreover, certain Michael adducts underwent intramolecular cyclization during semicarbazone deprotection, leading to novel pyrrazoline and aza-pyroglutamate N-terminal residues. Structural studies indicated that contingent on sequence the [aza-Glu]GHRP-6 analogues exhibited CD spectra characteristic of random coil, polyproline type II and β-turn secondary structures in aqueous media. In covalent competition binding studies with the GHRP-6 prototype hexarelin bearing a radiotracer, certain [aza-Glu]GHRP-6 azapeptides retained relatively high (2-27 μM) affinity for the CD36 scavenger receptor.

Synthesis, characterization, and biological activity of poly(arginine)-derived cancer-targeting peptides in HepG2 liver cancer cells

The solid‐phase synthesis, structural characterization, and biological evaluation of a small library of cancer‐targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose‐regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3–12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9‐fluorenylmethoxycarbonyl‐based solid‐phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14–46%) and crude purities >95% as analyzed by liquid chromatography–mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure–activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose‐regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose‐response (0–1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer‐targeting delivery agents. Copyright

Synthesis, DNA binding and anti-leukemic activity of an aminoacyl nucleolipid

The synthesis and characterization of a new class of DNA binding molecule exhibiting potent and selective anti-leukemic activity is described. The synthesis of an aminoacyl nucleolipid was developed from an efficient EEDQ coupling strategy, in which a series of seven bioconjugates were synthesized in yields of 53-78%. Guanosine bioconjugate 7, was used as building block for the synthesis of a target aminoacyl nucleolipid 14. Its GRP78 DNA binding affinity was confirmed by gel shift assay, CD spectroscopy, Tm measurements and dynamic light scattering experiments. Moreover, in a single dose (10 μM) screen against a panel of 60 cancer cell lines, aminoacyl nucleolipid 14 was found to selectively trigger greater than 90% cell death in a SR human leukemia cancer cell line. The reported aminoacyl nucleolipid represents a useful model for a new class of DNA binding molecules for the development of potent and selective anti-cancer agents.

Solid-phase synthesis, characterization and RNAi activity of branch and hyperbranch siRNAs

Linear, branch and hyperbranch siRNAs were effectively prepared for down-regulating GRP78 expression and inducing cell death in HepG2 liver cancer cells. Branch and hyperbranch GRP78 siRNAs were synthesized by automated solid-phase synthesis in good yields (44-78%) and isolated in excellent purities (>99%) following HPLC purification. Moreover, siRNAs adopted stable intramolecular hybrids as discerned by native PAGE and thermal denaturation studies. These sequences also exhibited the pre-requisite A-type helical trajectory for triggering RNAi activity as determined by CD spectroscopy. Biological studies confirmed potent suppression of GRP78 expression (50-60%) while compromising cancer cell viability by ~20%. Thus, branch and hyperbranch siRNAs may serve as potent siRNA candidates in cancer gene therapy applications.

RNAi Screening of the Glucose-Regulated Chaperones in Cancer with Self-Assembled siRNA Nanostructures

The emerging field of RNA nanotechnology has been used to design well-programmed, self-assembled nanostructures for applications in chemistry, biology, and medicine. At the forefront of its utility in cancer is the unrestricted ability to self-assemble multiple siRNAs within a single nanostructure formulation for the RNAi screening of a wide range of oncogenes while potentiating the gene therapy of malignant tumors. In our RNAi nanotechnology approach, V- and Y-shape RNA templates were designed and constructed for the self-assembly of discrete, higher-ordered siRNA nanostructures targeting the oncogenic glucose regulated chaperones. The GRP78-targeting siRNAs self-assembled into genetically encoded spheres, triangles, squares, pentagons and hexagons of discrete sizes and shapes according to TEM imaging. Furthermore, gel electrophoresis, thermal denaturation, and CD spectroscopy validated the prerequisite siRNA hybrids for their RNAi application. In a 24 sample siRNA screen conducted within the AN3CA endometrial cancer cells known to overexpress oncogenic GRP78 activity, the self-assembled siRNAs targeting multiple sites of GRP78 expression demonstrated more potent and long-lasting anticancer activity relative to their linear controls. Extending the scope of our RNAi screening approach, the self-assembled siRNA hybrids (5 nM) targeting of GRP-75, 78, and 94 resulted in significant (50-95%) knockdown of the glucose regulated chaperones, which led to synergistic effects in tumor cell cycle arrest (50-80%) and death (50-60%) within endometrial (AN3CA), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines. Interestingly, a nontumorigenic lung (MRC5) cell line displaying normal glucose regulated chaperone levels was found to tolerate siRNA treatment and demonstrated less toxicity (5-20%) relative to the cancer cells that were found to be addicted to glucose regulated chaperones. These remarkable self-assembled siRNA nanostructures may thus encompass a new class of potent siRNAs that may be useful in screening important oncogene targets while improving siRNA therapeutic efficacy and specificity in cancer.

Synthesis and Photophysical and Photocatalytic Properties of a Highly Fluorinated and Durable Phthalocyanine–Peptide Bioconjugate for Potential Theranostic Applications

The functionalized, asymmetric fluoro-fluoroalkyl scaffold F48H7COOHPcZn (3) was used to prepare F48H7COOPcZn-6-amino-hexanoate-CTVALPGGYVRVC (5), a Pep42 peptide bioconjugate envisioned for photodynamic therapy, which can specifically target the GRP78 chaperone protein overexpressed and exclusively localized on some cancer cell surfaces. The analogous F48H7COOHPcCu (4) has also been prepared, and its single-crystal X-ray structure was elucidated. Despite reduced steric hindrance relative to the nonfunctionalized, single-site complexes of the F64Pc scaffold, no aggregation was detected in solution via UV-vis spectroscopy, for either 3, 4, or 5, consistent with the lack of π stacking observed for the crystalline 4. The 6-aminohexanoic acid-Pep42 moiety diminishes the fluorescence efficiency of 5, relative to 3, but for singlet oxygen (1O2) generation, photochemical hydroperoxidation of β-(-)-citronellol using 5 and 3 occurs with comparable substrate turnover efficiency, albeit at a slower initial triplet oxygen uptake for 5. The bioconjugate 5 is durable; it does not decompose under 1O2 photoreaction conditions. These results suggest a synthetic coupling pathway for obtaining diverse biotargeting polypeptide-fluorinated phthalocyanine bioconjugates of potential utility as both fluorescence reporters and photocatalysts and highlight the importance of fluorinated scaffolds for generating chemically resilient, catalytic, theranostic materials.

Nucleic Acid Bioconjugates in Cancer Detection and Therapy.

Nucleoside‐ and nucleotide‐based chemotherapeutics have been used to treat cancer for more than 50 years. However, their inherent cytotoxicities and the emergent resistance of tumors against treatment has inspired a new wave of compounds in which the overall pharmacological profile of the bioactive nucleic acid component is improved by conjugation with delivery vectors, small‐molecule drugs, and/or imaging modalities. In this manner, nucleic acid bioconjugates have the potential for targeting and effecting multiple biological processes in tumors, leading to synergistic antitumor effects. Consequently, tumor resistance and recurrence is mitigated, leading to more effective forms of cancer therapy. Bioorthogonal chemistry has led to the development of new nucleoside bioconjugates, which have served to improve treatment efficacy en route towards FDA approval. Similarly, oligonucleotide bioconjugates have shown encouraging preclinical and clinical results. The modified oligonucleotides and their pharmaceutically active formulations have addressed many weaknesses of oligonucleotide‐based drugs. They have also paved the way for important advancements in cancer diagnosis and treatment. Cancer‐targeting ligands such as small‐molecules, peptides, and monoclonal antibody fragments have all been successfully applied in oligonucleotide bioconjugation and have shown promising anticancer effects in vitro and in vivo. Thus, the application of bioorthogonal chemistry will, in all likelihood, continue to supply a promising pipeline of nucleic acid bioconjugates for applications in cancer detection and therapy.