Akira Sanjoh

Structure-based design, synthesis, and evaluation of peptide-mimetic SARS 3CL protease inhibitors.

The design and evaluation of low molecular weight peptide-based severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL) protease inhibitors are described. A substrate-based peptide aldehyde was selected as a starting compound, and optimum side-chain structures were determined, based on a comparison of inhibitory activities with Michael type inhibitors. For the efficient screening of peptide aldehydes containing a specific C-terminal residue, a new approach employing thioacetal to aldehyde conversion mediated by N-bromosuccinimide was devised. Structural optimization was carried out based on X-ray crystallographic analyses of the R188I SARS 3CL protease in a complex with each inhibitor to provide a tetrapeptide aldehyde with an IC(50) value of 98 nM. The resulting compound carried no substrate sequence, except for a P(3) site directed toward the outside of the protease. X-ray crystallography provided insights into the protein-ligand interactions.

Crystal Structure of Human Importin-α1 (Rch1), Revealing a Potential Autoinhibition Mode Involving Homodimerization

In this study, we determined the crystal structure of N-terminal importin-β-binding domain (IBB)-truncated human importin-α1 (ΔIBB-h-importin-α1) at 2.63 Å resolution. The crystal structure of ΔIBB-h-importin-α1 reveals a novel closed homodimer. The homodimer exists in an autoinhibited state in which both the major and minor nuclear localization signal (NLS) binding sites are completely buried in the homodimerization interface, an arrangement that restricts NLS binding. Analytical ultracentrifugation studies revealed that ΔIBB-h-importin-α1 is in equilibrium between monomers and dimers and that NLS peptides shifted the equilibrium toward the monomer side. This finding suggests that the NLS binding sites are also involved in the dimer interface in solution. These results show that when the IBB domain dissociates from the internal NLS binding sites, e.g., by binding to importin-β, homodimerization possibly occurs as an autoinhibition state.

Synthesis and evaluation of curcumin derivatives toward an inhibitor of beta-site amyloid precursor protein cleaving enzyme 1

To research a new non-peptidyl inhibitor of beta-site amyloid precursor protein cleaving enzyme 1, we focused on the curcumin framework, two phenolic groups combined with an sp2 carbon spacer for low-molecular and high lipophilicity. The structure-activity relationship study of curcumin derivatives is described. Our results indicate that phenolic hydroxy groups and an alkenyl spacer are important structural factors for the inhibition of beta-site amyloid precursor protein cleaving enzyme 1 and, furthermore, non-competitive inhibition of enzyme activity is anticipated from an inhibitory kinetics experiment and docking simulation.

Fused-ring structure of decahydroisoquinolin as a novel scaffold for SARS 3CL protease inhibitors

Abstract The design and evaluation of a novel decahydroisoquinolin scaffold as an inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CLpro) are described. Focusing on hydrophobic interactions at the S2 site, the decahydroisoquinolin scaffold was designed by connecting the P2 site cyclohexyl group of the substrate-based inhibitor to the main-chain at the α-nitrogen atom of the P2 position via a methylene linker. Starting from a cyclohexene enantiomer obtained by salt resolution, trans-decahydroisoquinolin derivatives were synthesized. All decahydroisoquinolin inhibitors synthesized showed moderate but clear inhibitory activities for SARS 3CLpro, which confirmed the fused ring structure of the decahydroisoquinolin functions as a novel scaffold for SARS 3CLpro inhibitor. X-ray crystallographic analyses of the SARS 3CLpro in a complex with the decahydroisoquinolin inhibitor revealed the expected interactions at the S1 and S2 sites, as well as additional interactions at the N-substituent of the inhibitor.

Evaluation of superior BACE1 cleavage sequences containing unnatural amino acids

A recombinant form of BACE1 (β-site amyloid precursor protein cleaving enzyme-1) corresponding to positions 46-454 of the extracellular domain of the original membrane enzyme was prepared. The recombinant BACE1 (rBACE1) had the kinetic parameters K(m)=5.5μM and k(cat)=1719s(-1). Using several libraries of substrates containing unnatural amino acids, the specificity of rBACE1 was evaluated. LC/MS of digests derived from the libraries clarified that a dodecapeptide containing unnatural amino acids at P(2) to [Formula: see text] was a superior cleavage sequence.

Molecular Mechanism of HIV-1 Vpr for Binding to Importin-α.

Viral protein R (Vpr) is an accessory gene product of human immunodeficiency virus type 1 (HIV-1) that plays multiple important roles associated with viral replication. Structural studies using NMR have revealed that Vpr consists of three α-helices and contains flexible N- and C-termini. However, the molecular mechanisms associated with Vpr function have not been elucidated. To investigate Vpr multifunctionality, we performed an X-ray crystallographic study of Vpr complexes containing importin-α, a known Vpr binding partner present in host cells. Elucidation of the crystal structure revealed that the flexible C-terminus changes its conformation to a twisted β-turn via an induced-fit mechanism, enabling binding to a minor nuclear localization signal (NLS) site of importin-α. The Vpr C-terminus can also bind with major NLS sites of importin-α in an extended conformation in different ways. These results, which represent the first reported crystallographic analysis of Vpr, demonstrate the multifunctional aspects that enable Vpr interaction with a variety of cellular proteins.

Structural basis for the development of SARS 3CL protease inhibitors from a peptide mimic to an aza-decaline scaffold.

Design of inhibitors against severe acute respiratory syndrome (SARS) chymotrypsin‐like protease (3CLpro) is a potentially important approach to fight against SARS. We have developed several synthetic inhibitors by structure‐based drug design. In this report, we reveal two crystal structures of SARS 3CLpro complexed with two new inhibitors based on our previous work. These structures combined with six crystal structures complexed with a series of related ligands reported by us are collectively analyzed. To these eight complexes, the structural basis for inhibitor binding was analyzed by the COMBINE method, which is a chemometrical analysis optimized for the protein–ligand complex. The analysis revealed that the first two latent variables gave a cumulative contribution ratio of r2 = 0.971. Interestingly, scores using the second latent variables for each complex were strongly correlated with root mean square deviations (RMSDs) of side‐chain heavy atoms of Met49 from those of the intact crystal structure of SARS‐3CLpro (r = 0.77) enlarging the S2 pocket. The substantial contribution of this side chain (∼10%) for the explanation of pIC50s was dependent on stereochemistry and the chemical structure of the ligand adapted to the S2 pocket of the protease. Thus, starting from a substrate mimic inhibitor, a design for a central scaffold for a low molecular weight inhibitor was evaluated to develop a further potent inhibitor.

A closed-homodimer structure of human importin-α1

We determined the crystal structure of N-terminal importin-β-binding (IBB) domain truncated human importin-α1 (ΔIBB-importin-α1) at 2.63Å resolution. The crystal structure of ΔIBB-importin-α1 is a novel closed-homodimer. The homodimer exists in an autoinhibition state in which both of the major and minor NLS-binding sites are completely buried in the homodimerization interface to avoid NLS binding. Importin-α1 is in dimer-monomer equilibration in solution. In the dimerization sate, the P1’-binding pocket in the minor NLS binding site plays a role to stabilize the dimer formation. The external K108 binds into the P1’-binding pocket that results in the autoinhibition of the NLS binding. The present closed-homodimer of ΔIBB-importin-α1 conjured the functional aspects of multimerization of importin-α1. The further physicochemical studies using fulland ΔIBBimportin-α1 reveal that the IBB domain is involved in the monomer-dimer equilibration; thereby the NLS binding affinity is kept even in the higher concentration of importinα1. Owing to the multimerization property, importin-αs can autoinhibition the NLS binding, that may result in a variety of NLS recognition way.

Co-crystallization and X-ray studies of HIV-1 Vpr-Importin-alpha and Vpr-inhibitor complexes

4 then deliver into the arginine-rich groove. Second, our data show that the region centered around the protrusion is crucial for RNA binding and presumably is the major RNA binding site. The side chains of the arginine residues in this region are pointing towards each other, suggesting that this region may clamp the RNA into the groove. Third, we have found that an arginine rich region at the other end of the groove is also important for RNA-binding. Since 24-27 RNA nucleotides bind to an influenza NP molecule, the RNA is expected to make further contacts with NP in addition to binding along the arginine-rich groove. This work may lead to the design of inhibitors for perturbing the transcription and replication of influenza virus.