Tetsuo Narumi

CD4 mimics targeting the mechanism of HIV entry.

A structure-activity relationship study was conducted of several CD4 mimicking small molecules which block the interaction between HIV-1 gp120 and CD4. These CD4 mimics induce a conformational change in gp120, exposing its co-receptor-binding site. This induces a highly synergistic interaction in the use in combination with a co-receptor CXCR4 antagonist and reveals a pronounced effect on the dynamic supramolecular mechanism of HIV-1 entry.

Cell-Permeable Stapled Peptides Based on HIV-1 Integrase Inhibitors Derived from HIV-1 Gene Products

HIV-1 integrase (IN) is an enzyme which is indispensable for the stable infection of host cells because it catalyzes the insertion of viral DNA into the genome and thus is an attractive target for the development of anti-HIV agents. Earlier, we found Vpr-derived peptides with inhibitory activity against HIV-1 IN. These Vpr-derived peptides are originally located in an α-helical region of the parent Vpr protein. Addition of an octa-arginyl group to the inhibitory peptides caused significant inhibition against HIV replication associated with an increase in cell permeability but also relatively high cytotoxicity. In the current study, stapled peptides, a new class of stabilized α-helical peptidomimetics were adopted to enhance the cell permeability of the above lead peptides. A series of stapled peptides, which have a hydrocarbon link formed by a ruthenium-catalyzed ring-closing metathesis reaction between successive turns of α-helix, were designed, synthesized, and evaluated for biological activity. In cell-based assays some of the stapled peptides showed potent anti-HIV activity comparable with that of the original octa-arginine-containing peptide (2) but with lower cytotoxicity. Fluorescent imaging experiments revealed that these stapled peptides are significantly cell permeable, and CD analysis showed they form α-helical structures, whereas the unstapled congeners form β-sheet structures. The application of this stapling strategy to Vpr-derived IN inhibitory peptides led to a remarkable increase in their potency in cells and a significant reduction of their cytotoxicity.

CD4 mimics targeting the HIV entry mechanism and their hybrid molecules with a CXCR4 antagonist

Small molecules behaving as CD4 mimics were previously reported as HIV-1 entry inhibitors that block the gp120-CD4 interaction and induce a conformational change in gp120, exposing its co-receptor-binding site. A structure-activity relationship (SAR) study of a series of CD4 mimic analogs was conducted to investigate the contribution from the piperidine moiety of CD4 mimic 1 to anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. In addition, several hybrid molecules based on conjugation of a CD4 mimic analog with a selective CXCR4 antagonist were also synthesized and their utility evaluated.

Small molecular CD4 mimics as HIV entry inhibitors

Derivatives of CD4 mimics were designed and synthesized to interact with the conserved residues of the Phe43 cavity in gp120 to investigate their anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. Significant potency gains were made by installation of bulky hydrophobic groups into the piperidine moiety, resulting in discovery of a potent compound with a higher selective index and CD4 mimicry. The current study identified a novel lead compound 11 with significant anti-HIV activity and lower cytotoxicity than those of known CD4 mimics.

CD4 mimics as HIV entry inhibitors: Lead optimization studies of the aromatic substituents

Several CD4 mimics have been reported as HIV-1 entry inhibitors that can intervene in the interaction between a viral envelope glycoprotein gp120 and a cell surface protein CD4. Our previous SAR studies led to a finding of a highly potent analogue 3 with bulky hydrophobic groups on a piperidine moiety. In the present study, the aromatic ring of 3 was modified systematically in an attempt to improve its antiviral activity and CD4 mimicry which induces the conformational changes in gp120 that can render the envelope more sensitive to neutralizing antibodies. Biological assays of the synthetic compounds revealed that the introduction of a fluorine group as a meta-substituent of the aromatic ring caused an increase of anti-HIV activity and an enhancement of a CD4 mimicry, and led to a novel compound 13a that showed twice as potent anti-HIV activity compared to 3 and a substantial increase in a CD4 mimicry even at lower concentrations.

Azamacrocyclic Metal Complexes as CXCR4 Antagonists

The chemokine receptor CXCR4 is a member of the seven transmembrane GPCR family, which is implicated in multiple diseases, including HIV infection, cancers, and rheumatoid arthritis. Low‐molecular‐weight nonpeptidic compounds, including AMD3100 and various pyridyl macrocyclic zinc(II) complexes, have been identified as selective antagonists of CXCR4. In the present study, structure–activity relationship studies were performed by combining the common structural features of alkylamino and pyridiyl macrocyclic antagonists. Several new zinc(II) or copper(II) complexes demonstrated potent anti‐HIV activity, strong CXCR4‐binding activity, and significant inhibitory activity against Ca2+ mobilization induced by CXCL12 stimulation. These results may prove useful in the design of novel CXCR4 antagonists, and the compounds described could potentially be developed as therapeutics against CXCR4‐relevant diseases or chemical probes to study the biological activity of CXCR4.

A Synthetic C34 Trimer of HIV-1 gp41 Shows Significant Increase in Inhibition Potency

The development of new anti-HIV-1 drugs such as inhibitors of protease and integrase has been contributed to highly active anti-retroviral therapy (HAART) for the treatment of AIDS. The entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope glycoprotein (Env), a type I transmembrane protein that consists of surface subunit gp120 and noncovalently associated transmembrane subunit gp41. Sequential binding of HIV-1 gp120 to its cell receptor CD4 and a co-receptor (CCR5 or CXCR4) can trigger a series of conformational rearrangements in gp41 to mediate fusion between viral and cellular membranes. The protein gp41 is hidden beneath gp120, and its ectodomain contains helical Nand C-terminal leucine/isoleucine heptad repeat domains, NHR and C-HR. Particular regions of N-HR and C-HR are involved in membrane fusion, and 36-mer and 34-mer peptides, which are derived from N-HR and C-HR, have been designated as the N-terminal helix (N36) and C-terminal helix (C34), respectively. In the membrane fusion of HIV-1, these helices assemble to form a six-helical bundle (6-HB) consisting of a central parallel trimer of N36 surrounded by C34 in an antiparallel hairpin fashion. Synthetic peptides derived from these helices have potent antiviral activity against both laboratory-adapted strains and primary isolates of HIV-1. They inhibit the membrane fusion stage of HIV-1 infection in a dominant-negative manner by binding to the counterpart regions of gp41 (N-HR or C-HR), blocking formation of the viral gp41 core. Several potent anti-HIV-1 peptides based on the C-HR region have been discovered, 8] and T20 was subsequently developed as the clinical anti-HIV-1 drug enfuvirtide (Roche/Trimeris). 10–13] It is a 36-mer peptide derived from the gp41 C-HR sequence and can bind to the N-HR to prevent formation of the 6-HB in a dominant-negative fashion. T20 therapy has brought safety, potent antiretroviral activity, and immunological benefit to patients, but its clinical application is limited by the development of resistance. The C-terminal helix C34 is also a C-HR-derived peptide, and contains the amino acid residues required for docking into the hydrophobic pocket, termed the “deep pocket”, of the trimer of the N-HR region. This peptide potently inhibits HIV-1 fusion in vitro. To date, several gp41 mimetics, especially those of N36 regions, which assemble these helical peptides with branched peptide linkers, have been synthesized as antigens. Recently, by using a novel template with C3-symmetric linkers of equal length, we synthesized a three-helix bundle mimetic that corresponds to the trimeric form of N36. The antisera obtained from mice immunized by the peptide antigen showed strong recognition against the N36 trimer peptide with structural preference. At the same time, the trimer peptide was also investigated as a fusion inhibitor. However, the trimer N36 showed only a threefold increase in inhibition of HIV-1 fusion relative to the N36 monomer. In terms of N36 content, the trimer and monomer have nearly the same inhibitory potency. This phenomenon is consistent with the results from other studies. The multimerization of the functional unit, such as synthetic ligands against receptors, show synergistic binding and strong binding activity. Thus, we hypothesized that our strategy using C3-symmetric linkers in the design of trimer mimics of gp41 could be applied to the C34 peptide, which shows significant inhibition potency in the monomeric form. In the present study, we designed and synthesized a novel three-helical bundle structure of the trimeric form of C34. This equivalent mimic of the trimeric form of C34 was evaluated as a novel form of fusion inhibitor. The C-terminal region of gp41 is known to be an assembly site involving a trimeric coiled-coil conformation. In the design of the C34-derived peptides C34REG-thioester (Figure 1 A) and C34REG (Figure 1 B), the triplet repeat of arginine and glutamic acid (RERERE) was added to the C-terminal end of the C34 sequence (residues 628–661) to increase aqueous solubility, and for C34REG-thioester, a glycine thioester was fused to the C terminus. To form a triple helix corresponding precisely to the gp41 pre-fusion form, we designed the novel C3-symmetric template depicted in Figure 1 C. This designed template linker has three branches of equal length, a hydrophilic structure, and a ligation site for coupling with C34REG-thioester. The template was synthesized as shown in Scheme 1. This approach uses native chemical ligation for chemoselective coupling of unprotected C34REG-thioester with a three-armed cysteine scaffold to produce triC34e (Figure 2). 25] Circular dichroism (CD) spectra of C34REG and triC34e are shown in Figure 3 A. The peptides were dissolved in 50 mm sodium phosphate buffer with 150 mm NaCl, pH 7.2. Both spectra display minima at ~200 nm, indicating that these peptides form random structures. We previously reported that the [a] Dr. W. Nomura, C. Hashimoto, A. Ohya, Dr. T. Tanaka, Dr. T. Narumi, T. Nakahara, Prof. Dr. H. Tamamura Institute of Biomaterials and Bioengineering Tokyo Medical and Dental University 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062 (Japan) E-mail : tamamura.mr@tmd.ac.jp [b] Dr. K. Miyauchi, Dr. E. Urano, Dr. J. A. Komano AIDS Research Center, National Institute of Infectious Diseases 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640 (Japan) [c] Prof. Dr. N. Yamamoto Department of Microbiology, Yong Loo Lin School of Medicine National University of Singapore 5 Science Drive 2, Singapore 117597 (Singapore) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201100542.

The successes and failures of HIV drug discovery.

Introduction: To date, several anti-human immunodeficiency virus (HIV) drugs, including reverse transcriptase inhibitors and protease inhibitors, have been developed and used clinically for the treatment of patients infected with HIV. Recently, novel drugs have been discovered which have different mechanisms of action from those of the above inhibitors, including entry inhibitors and integrase (IN) inhibitors; the clinical use of three of these inhibitors has been approved. Other inhibitors are still in development. Areas covered: This review article summarizes the history of the development of anti-HIV drugs and also focuses on successes in the development of these entry and IN inhibitors, along with looking at exploratory approaches for the development of other inhibitors. Expert opinion: Currently used highly active antiretroviral therapy can be subject to a loss of efficacy, due to the emergence of multi-drug resistant (MDR) strains; a change of regimens of the drug combination is required to combat this, along with careful monitoring of the virus and CD4 in the blood, by methods such as cellular tropism testing. In such a situation, entry inhibitors such as CCR5/CXCR4 antagonists, CD4 mimics, fusion inhibitors and IN inhibitors might be optional agents for an expansion of the drug repertoire available to patients at all stages of HIV infection.

Remodeling of Dynamic Structures of HIV-1 Envelope Proteins Leads to Synthetic Antigen Molecules Inducing Neutralizing Antibodies

A synthetic antigen targeting membrane-fusion mechanism of HIV-1 has a newly designed template with C3-symmetric linkers mimicking N36 trimeric form. The antiserum produced by immunization of the N36 trimeric form antigen showed structural preference in binding to N36 trimer and stronger inhibitory activity against HIV-1 infection than the N36 monomer. Our results suggest an effective strategy of HIV vaccine design based on a relationship to the native structure of proteins involved in HIV fusion mechanisms.

Peptidic HIV integrase inhibitors derived from HIV gene products: structure-activity relationship studies.

Structure-activity relationship studies were conducted on HIV integrase (IN) inhibitory peptides which were found by the screening of an overlapping peptide library derived from HIV-1 gene products. Since these peptides located in the second helix of Vpr are considered to have an alpha-helical conformation, Glu-Lys pairs were introduced into the i and i+4 positions to increase the helicity of the lead compound possessing an octa-arginyl group. Ala-scan was also performed on the lead compound for the identification of the amino acid residues responsible for the inhibitory activity. The results indicated the importance of an alpha-helical structure for the expression of inhibitory activity, and presented a binding model of integrase and the lead compound.