Tsutomu Mimoto

Structure of HIV-1 protease with KNI-272, a tight-binding transition-state analog containing allophenylnorstatine.

BACKGROUND HIV-1 protease (HIV PR), an aspartic protease, cleaves Phe-Pro bonds in the Gag and Gag-Pol viral polyproteins. Substrate-based peptide mimics constitute a major class of inhibitors of HIV PR presently being developed for AIDS treatment. One such compound, KNI-272, which incorporates allophenylnorstatine (Apns)-thioproline (Thp) in place of Phe-Pro, has potent antiviral activity and is undergoing clinical trials. The structure of the enzyme-inhibitor complex should lead to an understanding of the structural basis for its tight binding properties and provide a framework for interpreting the emerging resistance to this drug. RESULTS The three-dimensional crystal structure of KNI-272 bound to HIV PR has been determined to 2.0 A resolution and used to analyze structure-activity data and drug resistance for the Arg8-->Gln and ILe84-->Val mutations in HIV PR. The conformationally constrained Apns-Thp linkage is favorably recognized in its low energy trans conformation, which results in a symmetric mode of binding to the active-site aspartic acids and also explains the unusual preference of HIV PR for the S, or syn, hydroxyl group of the Apns residue. The inhibitor recognizes the enzyme via hydrogen bonds to three bridging water molecules, including one that is coordinated directly to the catalytic Asp125 residue. CONCLUSIONS The structure of the HIV PR/KNI-272 complex illustrates the importance of limiting the conformational degrees of freedom and of using protein-bound water molecules for building potent inhibitors. The binding mode of HIV PR inhibitors can be predicted from the stereochemical relationship between adjacent hydroxyl-bearing and side chain bearing carbon atoms of the P1 substituent. Our structure also provides a framework for designing analogs targeted to drug-resistant mutant enzymes.

Synthesis of di- and tripeptide analogues containing α-ketoamide as a new core structure for inhibition of HIV-1 protease

Di- and tripeptide analogues containing alpha-ketoamide as a new core structure and incorporating allophenylnorstatine (Apns) as a transition state mimic, were designed and synthesized in the hope of obtaining a novel structural type of HIV-1 protease inhibitors. The immediate precursor, Apns-Thz-NHBu(t) was prepared by coupling of Boc-Apns with N-tert x butyl Thz-4-carboxamide hydrochloride. Removal of Boc group followed by coupling with the respective alpha-ketoacid residue (P2) gave the desired dipeptides (8-12) in almost quantitative yields. The alpha-keto tripeptides (18-21) were obtained by oxidation of the hydroxyl group of Apns (PI) in the appropriate tripeptide, iQOA-Val-Apns-(un)substituted Thz(Oxa)-NHBu(t) with DMSO/DCC. Preliminary evaluation of the activity of the synthesized derivatives was determined as percentage of enzyme inhibition at 5 microM and 50 nM levels of the di- and tripeptides respectively. The alpha-ketoamides displayed a significant enhanced potency relative to their parent isosteres as inhibitors of HIV-1 protease and are shown to be a promising new core structure for the development of enzyme inhibitors. A quantitative approach was attempted, using an LFE model, correlating the effect of structural modification and HIV-1 protease inhibition activity of the prepared dipeptides. The result indicates the contribution of the torsion angle by 84% to the activity of the inhibitors.

A Practical Use of Ligand Efficiency Indices Out of the Fragment-Based Approach: Ligand Efficiency-Guided Lead Identification of Soluble Epoxide Hydrolase Inhibitors

Ligand efficiency is frequently used to evaluate fragment compounds in fragment-based drug discovery. We applied ligand efficiency indices in a conventional virtual screening-initiated lead generation study of soluble epoxide hydrolase inhibitors. From a considerable number of screening hits, we carefully selected a compound exhibiting relatively weak inhibitory activity but high ligand efficiency. This ligand efficiency-guided selection could reveal compounds possessing preferable lead-like characteristics in terms of molecular size and lipophilicity. The following hit-to-lead medicinal chemistry campaign successfully led to a more potent, ADMET-clean, lead-like compound preserving high ligand efficiency. Retrospective analyses, including consideration of the more recently proposed indices of ligand efficiency, shed light on the validity of our hit triage and hit-to-lead studies. The present work proposes a practical methodology for lead generation using the concept of ligand efficiency.

KNI‐577, a Potent Small‐Sized HIV Protease Inhibitor Based on the Dipeptide Containing the Hydroxymethylcarbonyl Isostere as an Ideal Transition‐State Mimic

The development of an effective therapeutic agent for the treatment of AIDS continues to be a challenging problem. Since the discovery that the virally encoded HIV protease is vital for propagation, inhibition of this enzyme has become a major target for AIDS chemotherapy. Consequently, numerous efforts aimed at the development of potent and selective inhibitors have been undertaken[2]. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing allophenylnorstatine [Apns; (2S,3S)‐3‐amino‐2‐hydroxy‐4‐phenylbutyric acid] with a hydroxymethylcarbonyl (HMC) isostere. Among them, the tripeptide KNI‐272 was a highly selective and superpotent HIV protease inhibitor (Ki=5.5 pM)[3]. KNI‐272 exhibited potent in vitro and in vivo antiviral activities with low cytotoxicity[4]. The NMR, X‐ray crystallography, and molecular modeling studies showed that the HMC group in KNI‐272 interacted excellently with the aspartic acid carboxyl groups of the HIV protease active site[5].

Existence of β-methylnorleucine in recombinant hirudin produced by Escherichia coli

Abstract A gene encoding for hirudin, a potent thrombin inhibitor, was expressed in Escherichia coli , which is the most widely used host. When the recombinant hirudin analog, CX-397, was overproduced by E. coli (600 mg l −1 ) in the absence of nutrient amino acids in the culture medium, the presence of two derivatives in the final product was observed with extremely increased retention times on reverse-phase high-performance liquid chromatography. Each derivative was due to methylation of an isoleucine residue at Ile29 or Ile59 in the CX-397. The structure was deducible as β-methylnorleucine (βMeNle; (2 S ,3 S )-2-amino-3-methylhexanoic acid). The modification pathway of βMeNle is not thought to be a post-translational modification of the protein because Ile has no functional group in its side-chain. Additionally, βMeNle is synthesized by mutants of Serratia marcescens that belong to the same family, Enterobacteriaceae, as E. coli (J. Antibiot. 34 (1981a) 1278). These findings suggest that the lack of nutrient amino acids in the culture medium leads to the synthesis of βMeNle in E. coli , which is then activated by E. coli isoleucyl-tRNA synthetase and incorporated into the overproduced recombinant protein.

A new class of amino protecting group removable by reductive acidolysis: the 4-methylsulphinylbenzyloxycarbonyl (Msz) group

A safety-catch type of amino protecting group, the 4-methylsulphinylbenzyloxycarbonyl (Msz) group is stable under both acidic and basic conditions, but can be smoothly removed by a one-pot reaction involving reductive acidolysis using tetrachlorosilane–trifluoroacetic acid–scavengers; this new N-Msz group was successfully applied to the synthesis of a tachykinin peptide, scyliorhinin I, by a new orthogonal protection methodology.

KNI-272, a highly selective and potent peptidic HIV protease inhibitor

Kynostatin [KNI-272; systematic name: 3-[3-benzyl-2-hydroxy-9-(isoquinolin-5-yloxy)-6-methylsulfanylmethyl-5,8-dioxo-4,7-diazanonanoyl]-N-tert-butyl-1,3-thiazolane-4-carboxamide], a highly selective and potent HIV protease inhibitor containing allophenylnorstatin [(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid], has been crystallized as the hydrate, C(33)H(41)N(5)O(6)S(2) x 0.803H(2)O, from aqueous hexylene glycol. The observed disorder of the phenyl group in the structure is related to the mode of hydration. The backbone conformation of the molecule is twisted and the overall conformation of the free inhibitor is similar to that observed in its complex with HIV protease.

Synthesis and biological evaluation of a 13N-labeled opioid peptide.

The 13N-labeled opioid tetrapeptide, Tyr-D-Met(O)-Phe-Gly-[13N]NH2 (SD-62), was synthesized by amidation of its activated p-nitrophenol ester with [13N]ammonia (total synthesis time: 25 min, radiochemical yield: 48%). When injected intravenously into mice, [13N]SD-62 was taken up by the brain and this uptake was blocked by naloxone. In addition, the time course of changes in brain radioactivity paralleled that of the analgesic activity of this compound, suggesting that SD-62 underwent binding to brain opioid receptors. Thus, [13N]SD-62 appears to hold some promise for use as a radiopharmaceutical for in vivo studies of opioid peptide behavior, using positron emission tomography.