Identification of Highly Potent Human Immunodeficiency Virus Type-1 Protease Inhibitors against Lopinavir and Darunavir Resistant Viruses from Allophenylnorstatine-Based Peptidomimetics with P2 Tetrahydrofuranylglycine.

The emergence of drug-resistant HIV from a widespread antiviral chemotherapy targeting HIV protease in the past decades is unavoidable and provides a challenge to develop alternative inhibitors. We synthesized a series of allophenylnorstatine-based peptidomimetics with various P3, P2, and P2́ moieties. The derivatives with P2 tetrahydrofuranylglycine (Thfg) were found to be potent against wild type HIV-1 protease and the virus, leading to a highly potent compound 21f (KNI-1657) against lopinavir/ritonavir- or darunavir-resistant strains. Co-crystal structures of 21f and the wild-type protease revealed numerous key hydrogen bonding interactions with Thfg. These results suggest that the strategy to design allophenylnorstatine-based peptidomimetics combined with Thfg residue would be promising for generating candidates to overcome multidrug resistance.

Structure-guided design and synthesis of P1' position 1-phenylcycloalkylamine-derived pentapeptidic BACE1 inhibitors.

Previously, we reported potent pentapeptidic BACE1 inhibitors with the hydroxymethylcarbonyl isostere as a substrate transition-state mimic. To improve the in vitro potency, we further reported pentapeptidic inhibitors with carboxylic acid bioisosteres at the P(4) and P1' positions. In the current study, we screened new P1' position 1-phenylcycloalkylamine analogs to find non-acidic inhibitors that possess double-digit nanomolar range IC(50) values. An extensive structure-activity relationship study was performed with various amine derivatives at the P1' position. The most potent inhibitor of this pentapeptide series, KMI-1830, possessing 1-phenylcyclopentylamine at the P1' position had an IC(50) value of 11.6 nM against BACE1 in vitro enzymatic assay.

Development of [Ile4°]HTLV-I protease inhibition assay using novel fluorogenic and chromogenic substrate.

HTLV-I is a debilitating and/or lethal retrovirus that causes HTLV-I-associated myelopathy/tropical spastic paraparesis, adult T-cell leukemia and several inflammatory diseases. HTLV-I protease is an aspartic retropepsin involved in HTLV-I replication and its inhibition could treatHTLV-I infection. A recombinant L40I mutant HTLV-I protease was designed and obtained from Escherichia coli, self-processingand purification by ion-exchange chromatography. The protease was refolded by a one-step dialysis and recovered activity. The cleavage efficiency of the [Ile4°]HTLV-I protease was at least 300 times higher for a fluorescent substratethan that of our previously reported recombinant His-tagged non-mutated HTLV-I protease. In addition, we designed and synthesized a substrate containing a highly fluorescent Mca moiety in the fragment before the scissile bond, and a chromogenic p-nitrophenylalanine moiety after the scissile bond that greatly amplified spectrometry detection and improved the HTLV-I protease inhibition potency assay. The HTLV-I protease inhibition assay with the [Ile4°]HTLV-I protease and fluorogenic substrate requires distinctively less protease, substrate, inhibitor and assay time than our previous methods. This means our new assay is more cost-effective and more time-efficient while being reproducible and less labor-intensive.

Design and synthesis of several small-size HTLV-I protease inhibitors with different hydrophilicity profiles.

The human T cell leukemia/lymphotropic virus type 1 (HTLV-I) is clinically associated with adult T cell leukemia/lymphoma, HTLV-I associated myelopathy/tropical spastic paraparesis, and a number of other chronic inflammatory diseases. To stop the replication of the virus, we developed highly potent tetrapeptidic HTLV-I protease inhibitors. In a recent X-ray crystallography study, several of our inhibitors could not form co-crystal complexes with the protease due to their high hydrophobicity. In the current study, we designed, synthesized and evaluated the HTLV-I protease inhibition potency of compounds with hydrophilic end-capping moieties with the aim of improving pharmaceutic and pharmacokinetic properties.

Maintaining potent HTLV-I protease inhibition without the P3-cap moiety in small tetrapeptidic inhibitors.

The human T cell lymphotropic/leukemia virus type 1 (HTLV-I) causes adult T cell lymphoma/leukemia. The virus is also responsible for chronic progressive myelopathy and several inflammatory diseases. To stop the manufacturing of new viral components, in our previous reports, we derived small tetrapeptidic HTLV-I protease inhibitors with an important amide-capping moiety at the P(3) residue. In the current study, we removed the P(3)-cap moiety and, with great difficulty, optimized the P(3) residue for HTLV-I protease inhibition potency. We discovered a very potent and small tetrapeptidic HTLV-I protease inhibitor (KNI-10774a, IC(50)=13 nM).

S-acyl isopeptide method: use of allyl-type protective group for improved preparation of thioester-containing S-acyl isopeptides by Fmoc-based SPPS.

We have studied the "S-acyl isopeptide method" for the synthesis of peptides containing difficult sequences. The S-acyl isopeptide, which contains a beta-thioester instead of the native N-acyl bond at a Cys residue, can be converted into the target peptide via an S-to-N intramolecular acyl migration reaction. However, the synthesis of the S-acyl isopeptide structure by Fmoc-based SPPS is hampered by repetitive base treatments; decomposition of the thioester and the epimerization of the thioesterified residue are commonly observed. Here, we adopted allyloxycarbonyl (Aloc) protective group to avoid the problem. Catalytic amount of Pd in the presence of scavengers such as PhSiH3 and dimedone selectively removed the Aloc group with neither decomposition of the thioester structure nor epimerization at the thioesterified residue. A model pentapeptide and amylin(1-12) with difficult sequences were efficiently synthesized by the improved S-acyl isopeptides method. Finally, the isolated S-acyl isopeptides were quantitatively converted into the desired peptides via the S-to-N intramolecular acyl migration reaction. The S-acyl isopeptide method will be a usefuI method to prepare the difficult sequence-containing peptides with Cys residue.

Improvement of both plasmepsin inhibitory activity and antimalarial activity by 2-aminoethylamino substitution.

We attached 2-aminoethylamino groups to allophenylnorstatine-containing plasmepsin (Plm) inhibitors and investigated SAR of the methyl or ethyl substitutions on the amino groups. Unexpectedly, compounds 22 (KNI-10743) and 25 (KNI-10742) exhibited extremely potent Plm II inhibitory activities (K(i)<0.1 nM). Moreover, among our peptidomimetic Plm inhibitors, we identified the compounds with the highest antimalarial activity using a SYBR Green I-based fluorescence assay.

Epimerization-free synthesis of cyclic peptide by use of the O-acyl isopeptide method.

A head-to-tail cyclization of a protected linear hexapeptide with a C-terminal O-acyl isopeptide proceeded to give a cyclic O-acyl isopeptide without epimerization. The cyclic O-acyl isopeptide possessed different secondary structures compared with the native cyclic peptide. The isopeptide was then efficiently converted to the desired cyclic peptide via an O-to-N acyl migration reaction using a silica gel-anchored base.

Tetrapeptides, as small-sized peptidic inhibitors; synthesis and their inhibitory activity against BACE1.

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is known to be involved in the production of amyloid beta-peptide in Alzheimer's disease and is a major target for current drug design. We previously reported substrate-based peptidomimetics, KMI-compounds as potent BACE1 inhibitors. In this study, we designed and synthesized tetrapeptides as low molecular-sized inhibitors. These exhibited high potency against recombinant BACE1, with the highest IC(50) value of 34.6 nM from KMI-927.

Design of pentapeptidic BACE1 inhibitors with carboxylic acid bioisosteres at P1' and P4 positions.

We previously reported potent BACE1 inhibitors KMI-420 and KMI-570 possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. Acidic moieties at the P(1)(') and P(4) positions of KMI inhibitors are thought to be unfavorable in terms of membrane permeability across the blood-brain barrier. Herein, we replaced acidic moieties at the P(4) position with hydrogen bond accepting groups and acidic moieties at the P(1)(') position with less acidic and similar molecular-size moieties (carboxylic acid or tetrazole bioisosteres). These inhibitors exhibited improved BACE1 inhibitory activities and a thorough quantitative structure-activity relationship study was performed.

Small-sized human immunodeficiency virus type-1 protease inhibitors containing allophenylnorstatine to explore the S2' pocket.

A series of HIV protease inhibitor based on the allophenylnorstatine structure with various P(2)' moieties were synthesized. Among these analogues, we discovered that a small allyl group would maintain potent enzyme inhibitory activity compared to the o-methylbenzyl moiety in clinical candidate 1 (KNI-764, also known as JE-2147, AG-1776, or SM-319777). Introduction of an anilinic amino group to 2 (KNI-727) improved water-solubility and anti-HIV-1 activity. X-ray crystallographic analysis of 13k (KNI-1689) with a beta-methallyl group at P(2)' position revealed hydrophobic interactions with Ala28, Ile84, and Ile50' similar to that of 1. The presence of an additional methyl group on the allyl group in compound 13k significantly increased anti-HIV activity over 1 while providing a rational drug design for structural minimization and improving membrane permeability.

Synthesis of amyloid beta peptide 1-42 (E22Delta) click peptide: pH-triggered in situ production of its native form.

Amyloid beta peptide (Abeta) 1-42 is known to be involved in the onset of Alzheimer's disease (AD). We developed a click peptide of Abeta1-42 as a useful tool for AD research on the basis of an O-acyl isopeptide method. The click peptide quickly produced intact Abeta1-42 via a pH-dependent O-to-N intramolecular acyl migration (pH-click). Herein, a click peptide (26-O-acyl isoAbeta1-42 (E22Delta)) of a new mutant Abeta1-42 (E22Delta) was synthesized. The mutant click peptide was more water-soluble than Abeta1-42 (E22Delta). Moreover it quantitatively converted to the native peptide under physiological conditions (pH 7.4, 37 degrees C). CD analyses showed a conformational change from a random-coil structure of the click peptide to a beta-sheet structure of the in situ produced Abeta1-42 (E22Delta). This click peptide is a useful precursor of a mutant Abeta1-42 to establish an experiment system for investigating the properties of the mutant.

O-acyl isopeptide method: efficient synthesis of isopeptide segment and application to racemization-free segment condensation.

We report the establishment of the O-acyl isopeptide method-based racemization-free segment condensation reaction toward future chemical protein synthesis. Peptide segments containing C-terminal O-acyl Ser/Thr residues were successfully synthesized by use of a lower nucleophilic base cocktail for Fmoc removal, and then coupled to an amino group of a peptide-resin without side reactions or epimerization. We also succeeded in performing the segment condensation in a sequential manner and in solution phase conditions as well.

Inhibitory effect of a dimerization-arm-mimetic peptide on EGF receptor activation.

A cyclic decapeptide was chemically synthesized that mimics the loop structure of a beta-hairpin arm of the EGF receptor, which is highly involved in receptor dimerization upon activation by ligand binding. This peptide was revealed to reduce dimer formation of the receptor in a detergent-solubilized extract of epidermoid carcinoma A431 cells and to inhibit receptor autophosphorylation at less than 10 microM in the intact cells.

Structure of HIV-1 protease in complex with potent inhibitor KNI-272 determined by high-resolution X-ray and neutron crystallography.

HIV-1 protease is a dimeric aspartic protease that plays an essential role in viral replication. To further understand the catalytic mechanism and inhibitor recognition of HIV-1 protease, we need to determine the locations of key hydrogen atoms in the catalytic aspartates Asp-25 and Asp-125. The structure of HIV-1 protease in complex with transition-state analog KNI-272 was determined by combined neutron crystallography at 1.9-A resolution and X-ray crystallography at 1.4-A resolution. The resulting structural data show that the catalytic residue Asp-25 is protonated and that Asp-125 (the catalytic residue from the corresponding diad-related molecule) is deprotonated. The proton on Asp-25 makes a hydrogen bond with the carbonyl group of the allophenylnorstatine (Apns) group in KNI-272. The deprotonated Asp-125 bonds to the hydroxyl proton of Apns. The results provide direct experimental evidence for proposed aspects of the catalytic mechanism of HIV-1 protease and can therefore contribute substantially to the development of specific inhibitors for therapeutic application.

"Click peptide": pH-triggered in situ production and aggregation of monomer Abeta1-42.

The intense and uncontrollable self-assembling nature of amyloid beta peptide (Abeta) 1-42 is known to cause difficulties in preparing monomeric Abeta1-42; this results in irreproducible or discrepant study outcomes. Herein, we report novel features of a pH click peptide of Abeta1-42 that was designed to overcome these problems. The click peptide is a water-soluble precursor peptide of Abeta1-42 with an O-acyl isopeptide structure between the Gly25-Ser26 sequence. The click peptide adopts and retains a monomeric, random coil state under acidic conditions. Upon change to neutral pH (pH click), the click peptide converts to Abeta1-42 promptly (t(1/2) approximately 10 s) and quantitatively through an O-to-N intramolecular acyl migration. As a result of this quick and irreversible conversion, monomer Abeta1-42 with a random coil structure is produced in situ. Moreover, the oligomerization, amyloid fibril formation and conformational changes of the produced Abeta1-42 can be observed over time. This click peptide strategy should provide a reliable experimental system to investigate the pathological role of Abeta1-42 in Alzheimer's disease.

Controlled production of amyloid beta peptide from a photo-triggered, water-soluble precursor "click peptide".

In biological experiments, poor solubility and uncontrolled assembly of amyloid beta peptide (Abeta) 1-42 pose significant obstacles to establish an experiment system that clarifies the function of Abeta1-42 in Alzheimer's disease (AD). Herein, as an experimental tool to overcome these problems, we developed a water-soluble photo-"click peptide" with a coumarin-derived photocleavable protective group that is based on an O-acyl isopeptide method. The click peptide had nearly 100-fold higher water solubility than Abeta1-42 and did not self-assemble, as the isomerized structure in its peptide backbone drastically changed the conformation that was derived from Abeta1-42. Moreover, the click peptide afforded Abeta1-42 quickly under physiological conditions (pH 7.4, 37 degrees C) by photoirradiation followed by an O-N intramolecular acyl migration. Because the in situ production of intact Abeta1-42 from the click peptide could improve the difficulties in handling Abeta1-42 caused by its poor solubility and highly aggregative nature, this click peptide strategy would provide a reliable experiment system for investigating the pathological function of Abeta1-42 in AD.

Crystallization and preliminary neutron diffraction studies of HIV-1 protease cocrystallized with inhibitor KNI-272.

This paper reports the crystallization and preliminary neutron diffraction measurements of HIV-1 protease, a potential target for anti-HIV therapy, complexed with an inhibitor (KNI-272). The aim of this neutron diffraction study is to obtain structural information about the H atoms and to determine the protonation states of the residues within the active site. The crystal was grown to a size of 1.4 mm(3) by repeated macroseeding and a slow-cooling method using a two-liquid system. Neutron diffraction data were collected at room temperature using a BIX-4 diffractometer at the JRR-3 research reactor of the Japan Atomic Energy Agency (JAEA). The data set was integrated and scaled to 2.3 A resolution in space group P2(1)2(1)2, with unit-cell parameters a = 59.5, b = 87.4, c = 46.8 A.