Akihiro Sugawara

Design, synthesis and biological evaluation of small molecule inhibitors of CD4-gp120 binding based on virtual screening.

The low-molecular-weight compound JRC-II-191 inhibits infection of HIV-1 by blocking the binding of the HIV-1 envelope glycoprotein gp120 to the CD4 receptor and is therefore an important lead in the development of a potent viral entry inhibitor. Reported here is the use of two orthogonal screening methods, gold docking and ROCS shape-based similarity searching, to identify amine-building blocks that, when conjugated to the core scaffold, yield novel analogs that maintain similar affinity for gp120. Use of this computational approach to expand SAR produced analogs of equal inhibitory activity but with diverse capacity to enhance viral infection. The novel analogs provide additional lead scaffolds for the development of HIV-1 entry inhibitors that employ protein-ligand interactions in the vestibule of gp120 Phe 43 cavity.

Chitinase inhibitors: extraction of the active framework from natural argifin and use of in situ click chemistry.

In situ click chemistry is a target-guided synthesis technique for discovering potent protein ligands by assembling azides and alkynes into triazoles inside the affinity site of a target protein. We report the rapid discovery of a new and potent inhibitor of bacterial chitinases by the use of in situ click chemistry. We observed a target-templated formation of a potent triazole inhibitor of the chitinase-catalyzed chitin hydrolysis, through in situ click chemistry between a biologically active azide-containing scaffold and structurally unrelated alkyne fragments. Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. Argifin, which has been isolated and characterized as a cyclopentapeptide natural product by our research group, shows strong inhibitory activity against chitinases. As a result of our efforts at developing a chitinase inhibitor from an azide-bearing argifin fragment and the application of the chitinase template and a library of alkynes, we rapidly obtained a very potent and new 1,5-disubstituted triazole inhibitor against Serratia marcescens chitinase (SmChi) B. The new inhibitor expressed 300-fold increase in the inhibitory activity against SmChiB compared with that of argifin. To the best of our knowledge, our finding of an enzyme-made 1,5-disubstituted triazole, using in situ click chemistry is the second example reported in the literature.

Novel 12-membered non-antibiotic macrolides from erythromycin A; EM900 series as novel leads for anti-inflammatory and/or immunomodulatory agents

Herein, we report the design and synthesis of the novel 12-membered non-antibiotic macrolide (8R,9S)-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM900), which was found to be a potent anti-inflammatory and/or immunomodulatory agent, capable of promoting monocyte to macrophage differentiation. This molecule shows improved acid stability, does not exhibit any anti-bacterial activity and has relatively low cytotoxicity against THP-1 cells. In addition, one of its analogues, (8R,9S)-4″,13-O-diacetyl-8,9-dihydro-6,9-epoxy-8,9-anhydropseudoerythromycin A (EM911), was found to be twice as effective as EM900.

Crystal Structures of HIV-1 gp120 Envelope Glycoprotein in Complex with NBD Analogues That Target the CD4-Binding Site.

Efforts to develop therapeutic agents that inhibit HIV-1 entry have led to the identification of several small molecule leads. One of the most promising is the NBD series, which binds within a conserved gp120 cavity and possesses para-halogen substituted aromatic rings, a central oxalamide linker, and a tetramethylpiperidine moiety. In this study, we characterized structurally the interactions of four NBD analogues containing meta-fluoro substitution on the aromatic ring and various heterocyclic ring replacements of the tetramethylpiperidine group. The addition of a meta-fluorine to the aromatic ring improved surface complementarity and did not alter the position of the analogue relative to gp120. By contrast, heterocyclic ring replacements of the tetramethylpiperidine moiety exhibited diverse positioning and interactions with the vestibule of the gp120 cavity. Overall, the biological profile of NBD-congeners was modulated by ligand interactions with the gp120-cavity vestibule. Herein, six co-crystal structures of NBD-analogues with gp120 provide a structural framework for continued small molecule-entry inhibitor optimization.

Asymmetric Total Synthesis of Neoxaline

A first asymmetric total synthesis and determination of the absolute configuration of neoxaline has been accomplished through the highly stereoselective introduction of a reverse prenyl group to create a quaternary carbon stereocenter using (-)-3a-hydroxyfuroindoline as a building block, construction of the indoline spiroaminal via cautious stepwise oxidations with cyclizations from the indoline, assembly of (Z)-dehydrohistidine, and photoisomerization of unnatural (Z)-neoxaline to the natural (E)-neoxaline as the key steps.

Macrolides with promotive activity of monocyte to macrophage differentiation.

AbstractWe have been interested in the immunomodulatory effect, to promote differentiation of the human monocytic cell line THP-1 to macrophages of EM-A. We chemically modified EM-A in order to obtain derivatives with stronger promoting activity of monocyte to macrophage differentiation and no antibacterial activity. Most of the EM701 derivatives produced, all 12-membred macrolides, were remarkably active and were free of antibacterial activity. Among them, the most potent derivative was EM703, which showed very weak gastrointestinal motor-stimulating activity. EM703 may be useful tool to study the mechanisms of action of macrophage differentiation, and may be lead candidate for the development of new therapeutic drugs for chronic airway disease.

Borrelidin analogues with antimalarial activity: Design, synthesis and biological evaluation against Plasmodium falciparum parasites

Borrelidin, a structurally unique 18-membered macrolide, was found to express antimalarial activity against drug-resistant Plasmodium falciparum malaria parasites, with IC50 value of 0.93 ng/mL. However, it also displays strong cytotoxicity against human diploid embryonic MRC-5 cells. To investigate the issue of the cytotoxicity of borrelidin, borrelidin-based analogues were synthesized and their anti-Plasmodium properties were evaluated. In this communication, we report that a novel borrelidin analogue, bearing the CH2SPh moiety via a triazole linkage, was found to retain a potent antimalarial activity, against drug-sensitive and drug-resistant parasite strains, but possess only weak cytotoxicity against human cells.

Argifin; efficient solid phase total synthesis and evalution of analogues of acyclic peptide

An effective solid phase synthesis of Argifin, providing subsequent access to effective synthesis of analogues, was developed in 13% overall yield, as well as elucidating structure-activity relationships. The novel acyclic peptide 18b, prepared from a synthetic intermediate of Argifin, was found to be 70 times more potent as an inhibitor of Serratia marcescens chitinases B than Argifin itself.

Computational analysis of the binding affinities of the natural-product cyclopentapeptides argifin and argadin to chitinase B from Serratia marcescens.

Molecular dynamics (MD) simulations and the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method were applied to study the interaction of the natural-product cyclopentapeptide chitinase inhibitors argifin and argadin with chitinase B (ChiB) from Serratia marcescens. Argadin inhibited ChiB with an inhibition constant (K(i)) value of 20 nM, which was three orders of magnitude greater than that of argifin (K(i)=33,000 nM). The MM-PBSA free-energy analysis provided absolute binding free energies of -6.98 and -11.16 kcal/mol for the argifin and argadin complexes, respectively. These estimates were in good agreement with the free energies derived from the experimental K(i) values (-6.36 and -10.92 kcal/mol for the argifin and argadin complexes, respectively). The energetic analysis revealed that the van der Waals and nonpolar solvation energies drove the binding of both argifin and argadin. We found that the binding of argadin gained approximately 12 kcal/mol more van der Waals energy than that of argifin, which was mainly responsible for the difference in binding free energy between argifin and argadin. In particular, W220 and W403 of ChiB were found to contribute to the more favorable van der Waals interaction with argadin. We also designed argifin derivatives with better binding affinity, in which a constituent amino-acid residue of argifin was mutated to one with a bulky side chain. The derivative in which D-Ala of argifin was replaced with D-Trp appeared to possess a binding affinity that was equally potent to that of argadin.

Observation of the controlled assembly of preclick components in the in situ click chemistry generation of a chitinase inhibitor

Significance Several in situ click chemistry studies have been reported. To date, there is evidence to indicate that proteins act as mold between azide and alkyne fragments by X-ray analysis of protein–ligand complexes. However, only “postclick” structural evidence has been available. We succeeded in obtaining crystal structures of a chitinase complexed with an azide inhibitor and an O-allyl oxime fragment as a mimic of a click partner, revealing a mechanism for accelerating triazole formation in chitinase. This is an example to express the “preclick” state of in situ click chemistry and a demonstration to show that the in situ click chemistry approach will benefit from this analysis for future plans. We also performed density functional theory calculations to explore the chitinase-contributed Huisgen cycloaddition. The Huisgen cycloaddition of azides and alkynes, accelerated by target biomolecules, termed “in situ click chemistry,” has been successfully exploited to discover highly potent enzyme inhibitors. We have previously reported a specific Serratia marcescens chitinase B (SmChiB)-templated syn-triazole inhibitor generated in situ from an azide-bearing inhibitor and an alkyne fragment. Several in situ click chemistry studies have been reported. Although some mechanistic evidence has been obtained, such as X-ray analysis of [protein]–[“click ligand”] complexes, indicating that proteins act as both mold and template between unique pairs of azide and alkyne fragments, to date, observations have been based solely on “postclick” structural information. Here, we describe crystal structures of SmChiB complexed with an azide ligand and an O-allyl oxime fragment as a mimic of a click partner, revealing a mechanism for accelerating syn-triazole formation, which allows generation of its own distinct inhibitor. We have also performed density functional theory calculations based on the X-ray structure to explore the acceleration of the Huisgen cycloaddition by SmChiB. The density functional theory calculations reasonably support that SmChiB plays a role by the cage effect during the pretranslation and posttranslation states of selective syn-triazole click formation.