Hirokazu Tamamura

Mid-size Drugs Based on Peptides and Peptidomimetics

In the middle-size region, between small and macromolecules, there is an indispensable drug-like chemical space. These drugs with middle-size molecules are designated as mid-size drugs, which have the advantages that small and macromolecules possess and reduce the drawbacks. Peptide and peptide derivatives are mainly located in the above-mentioned middle-size region, and target not only the active centers of enzymes and pockets of receptors but also the protein–protein interactions because the mid-size drugs can cover target molecules broadly. Thus, the mid-size drugs might be expected as the next generation drugs. However, in therapeutical use of peptides, there is some limitation that involves several factors: low metabolic stability toward proteolysis, undesired activity resulting from interactions with several receptors, etc. Therefore, modifications of their structures for maintenance of biological activity have been considered and tried to develop peptidomimetics. Generally, peptidomimetics point peptide bond isosteres that mimic primary structures of peptides, such as transition-state mimics and ground-state mimics. As ground-state mimics, we have focused on the development of several alkene-type dipeptide isosteres (ADIs) such as chloroalkene dipeptide isosteres (CADIs). In the broad sense, peptidomimetics include mimetics of secondary and tertiary structures of peptides, which are useful for the development of inhibitors of protein–protein interactions.

Peptide-derived mid-sized anti-HIV agents

To date, several anti-human immunodeficiency virus (HIV) drugs such as reverse transcriptase inhibitors, protease inhibitors and integrase inhibitors have been developed, and the use in combination of these drugs has brought great success in the treatment of HIV-infected and acquired immunodeficiency syndrome (AIDS) patients. We have produced several anti-HIV agents including fusion inhibitors, coreceptor antagonists, integrase inhibitors, CD4 mimics and matrix peptides, and vaccines. These have been developed from the corresponding peptides and proteins. The number of available potent drugs is limited and entry inhibitors such as CCR5/CXCR4 antagonists and CD4 mimics, fusion inhibitors, vaccines and allosteric type integrase inhibitors might be useful for an expansion of the drug repertoire. This chapter is an update of our contribution on the topic of peptide-derived anti-HIV agents with a focus on mid-size drugs.

Tag-Probe System for Imaging of Intracellular Proteins

Fluorescent imaging of proteins in living cells is a useful technique in chemical biology. A tag-probe system can label target proteins by fluorescent probes that bind specifically to tag peptides fused to the proteins. ZIP tag-probe pairs based on three α-helical antiparallel leucine zipper peptides have been developed. A probe peptide is designed as an α-helical peptide, which possesses an environmental sensitive fluorescent dye such as 4-nitrobenzo-2-oxa-1,3-diazole (NBD). Tag peptides are designed as two α-helical antiparallel peptides. The binding of the probe to its complement tag causes a remarkable change of fluorescent properties, blue shift of fluorescent spectra and an increase in fluorescent intensity, because the environment surrounding the fluorescent dye in the probe is changed to more hydrophobic. Since the labeled protein with strong fluorescence can be distinguished from the free probe with weak fluorescence, washing steps for excessed probes is not required for detection of target proteins. As the first application, the fluorescent imaging of a cell surface protein, chemokine receptor CXCR4, which was transiently expressed as tag-fusion on CHO-K1 cells was performed. In this study, as an example of intracellular proteins, protein kinase C (PKC) was successfully labeled by probe peptides having octaarginine as a cell penetrating peptide (CPP) sequence. Taken together, the ZIP tag-probe system involving three α-helical antiparallel leucine zipper peptides is a useful tool for fluorescent imaging of proteins.

Biological Effects of Bivalent-Type CXCR4 Ligands with Rigid Linkers

A chemokine receptor CXCR4 belongs to the G-protein coupled receptor (GPCR) family. Interaction with its endogenous ligand, stromal-cell derived factor-1α (SDF-1)/CXCL12, induces various physiological functions in an embryonic stage. Recent studies have indicated a pivotal role of homoand hetero-oligomerization of CXCR4 in cancer metastasis. In the previous study, we have designed and synthesized novel CXCR4 bivalent ligands utilizing two FC131 analogues [cyclo(-D-Tyr-Arg-Arg-Nal-D-Cys-)](Nal = L-3-(2-naphthyl)alanine) as ligand units. The units are connected by a polyproline or a PEGylated polyproline linker. A ligand with an optimum linker-length showed the strongest binding affinity. Thus the dimer-state of CXCR4 on the cell surface was estimated by the linker length. FACS analyses showed that the bivalent ligand can distinguish the amount of CXCR4 expression. As the next challenge, migration of cells was targeted by the bivalent ligands because it was unclear whether the binding of CXCR4 bivalent ligands can promote or suppress the migration induced by the chemotaxis depending on the concentration of SDF-1α. The bivalent ligand with a T140 analog showed anti-chemotaxis activity in a chamber assay. Activity of SDF-1α/CXCR4/PI-3K/Akt signaling was also investigated. The results indicate that our ligand design approach utilizing rigid polyproline linker would be useful for development of more effective anti-CXCR4 ligands.