Haruo Aikawa

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.

Stereoselective formation of trisubstituted (Z)-chloroalkenes adjacent to a tertiary carbon stereogenic center by organocuprate-mediated reduction/alkylation.

A robust and efficient method for the synthesis of trisubstituted (Z)-chloroalkenes is described. A one-pot reaction of γ,γ-dichloro-α,β-enoyl sultams involving organocuprate-mediated reduction/asymmetric alkylation affords α-chiral (Z)-chloroalkene derivatives in moderate to high yields with excellent diastereoselectivity, and allylic alkylation of internal allylic gem-dichlorides is also demonstrated. This study provides the first examples of the use of allylic gem-dichlorides adjacent to the chiral center for novel 1,4-asymmetric induction.

Pharmacophore-based small molecule CXCR4 ligands

Low molecular weight CXCR4 ligands were developed based on the peptide T140, which has previously been identified as a potent CXCR4 antagonist. Some compounds with naphthyl, fluorobenzyl and pyridyl moieties as pharmacophore groups in the molecule showed significant CXCR4-binding activity and anti-HIV activity. Structure-activity relationships were studied and characteristics of each of these three moieties necessary for CXCR4 binding were defined. In this way, CXCR4 ligands with two types of recognition modes for CXCR4 have been found.

Evaluation of a synthetic C34 trimer of HIV-1 gp41 as AIDS vaccines.

An artificial antigen forming the C34 trimeric structure targeting membrane-fusion mechanism of HIV-1 has been evaluated as an HIV vaccine. The C34 trimeric molecule was previously designed and synthesized using a novel template with C3-symmetric linkers by us. The antiserum produced by immunization of the C34 trimeric form antigen showed 23-fold higher binding affinity for the C34 trimer than for the C34 monomer and showed significant neutralizing activity. The present results suggest effective strategies of the design of HIV vaccines and anti-HIV agents based on the native structure mimic of proteins targeting dynamic supramolecular mechanisms in HIV fusion.

Low‐Molecular‐Weight CXCR4 Ligands with Variable Spacers

Low‐molecular‐weight CXCR4 ligands based on known lead compounds including the 14‐mer peptide T140, the cyclic pentapeptide FC131, peptide mimetics, and dipicolylamine‐containing compounds were designed and synthesized. Three types of aromatic spacers, 1,4‐phenylenedimethanamine, naphthalene‐2,6‐diyldimethanamine, and [1,1′‐biphenyl]‐4,4′‐diyldimethanamine, were used to build four pharmacophore groups. As pharmacophore groups, 2‐pyridylmethyl and 1‐naphthylmethyl are present in all of the compounds, and several aromatic groups and a cationic group from 1‐propylguanidine and 1,1,3,3‐tetramethyl‐2‐propylguanidine were also used. Several compounds showed significant CXCR4 binding affinity, and zinc(II) complexation of bis(pyridin‐2‐ylmethyl)amine moieties resulted in a remarkable increase in CXCR4 binding affinity.

Gold-catalyzed alkylation of silyl enol ethers with ortho-alkynylbenzoic acid esters

Summary Unprecedented alkylation of silyl enol ethers has been developed by the use of ortho-alkynylbenzoic acid alkyl esters as alkylating agents in the presence of a gold catalyst. The reaction probably proceeds through the gold-induced in situ construction of leaving groups and subsequent nucleophilic attack on the silyl enol ethers. The generated leaving compound abstracts a proton to regenerate the silyl enol ether structure.

Exploration of labeling by near infrared dyes of the polyproline linker for bivalent-type CXCR4 ligands.

We have previously used poly-L-proline linkers for the development of bivalent-type ligands for the chemokine receptor, CXCR4. The bivalent ligands with optimum linkers showed specific binding to CXCR4, suggesting the existence of CXCR4 possibly as a dimer on the cell membrane, and enabled definition of the amount of CXCR4 expressed. This paper reports the synthesis by a copper-catalyzed azide-alkyne cycloaddition reaction as the key reaction, of bivalent CXCR4 ligands with near infrared (NIR) dyes at the terminus or the center of the poly-L-proline linker. Some of the NIR-labeled ligands, which would be valuable probes useful in studies of the behavior of cells expressing CXCR4, have been obtained. The information concerning the effects of the labeling positions of NIR dyes on their binding properties is useful for the design of modified bivalent-type CXCR4 ligands.

Development and validation of a cell-based assay system to assess human immunodeficiency virus type 1 integrase multimerization.

Multimerization of HIV-1 integrase (IN) subunits is required for the concerted integration of HIV-1 proviral DNA into the host genome. Thus, the disruption of IN multimerization represents a new avenue for intervening HIV-1 infection. Here, we generated a cell-based assay system to assess IN multimerization using a newly constructed bimolecular fluorescence complementation (BiFC-IN) system. BiFC-IN proteins were efficient in emitting fluorescence, and amino acid (AA) substitutions associated with IN multimerization attenuated fluorescence, suggesting that the BiFC-IN system may be useful for evaluating the profile of IN multimerization. A recently reported non-catalytic site IN inhibitor (NCINI), which allosterically induces IN over-multimerization/aggregation, significantly increased fluorescence in the BiFC-IN system. An INs substitution, A128T, associated with viral resistance to NCINIs, decreased the NCINI-induced increase of fluorescence, suggesting that A128T reduces the potential for IN over-multimerization. Moreover, E11K and F181T substitutions known to inhibit IN tetramerization also reduced the NCINI-induced fluorescence increase, suggesting that NCINI-induced IN over-multimerization was more likely to occur from tetramer subunits than from dimer subunits. The present study demonstrates that our cell-based BiFC-IN system may be useful in elucidating the profile of IN multimerization, and also help evaluate and identify novel compounds that disrupt IN multimerization in living cells.