Discovery and Hit to Lead Optimization of Macrocyclic Peptides as Novel Tropomyosin Receptor Kinase A Antagonists.

Tropomyosin receptor kinases (Trks) are receptor tyrosine kinases activated by neurotrophic factors, called neurotrophins. Among them, TrkA interacts with the nerve growth factor (NGF), which leads to pain induction. mRNA-display screening was carried out to discover a hit compound 2, which inhibits protein-protein interactions between TrkA and NGF. Subsequent structure optimization improving phosphorylation inhibitory activity and serum stability was pursued using a unique process that took advantage of the peptide being synthesized by translation from mRNA. This gave peptide 19, which showed an analgesic effect in a rat incisional pain model. The peptides described here can serve as a new class of analgesics, and the structure optimization methods reported provide a strategy for discovering new peptide drugs.

Chemical Modification of Phage-Displayed Helix-Loop-Helix Peptides to Construct Kinase-Focused Libraries.

Conformationally constrained peptides hold promise as molecular tools in chemical biology and as a new modality in drug discovery. The construction and screening of a target-focused library could be a promising approach for the generation of de novo ligands or inhibitors against target proteins. Here, we have prepared a protein kinase-focused library by chemically modifying helix-loop-helix (HLH) peptides displayed on phage and subsequently tethered to adenosine. The library was screened against aurora kinase A (AurA). The selected HLH peptide Bip-3 retained the α-helical structure and bound to AurA with a KD value of 13.7 µM. Bip-3 and the adenosine-tethered peptide Bip-3-Adc provided IC50 values of 103 µM and 7.7 µM, respectively, suggesting that Bip-3-Adc bivalently inhibited AurA. In addition, the selectivity of Bip-3-Adc to several protein kinases was tested, and was highest against AurA. These results demonstrate that chemical modification can enable the construction of a kinase-focused library of phage-displayed HLH peptides.

Macrocyclic Peptides as a Novel Class of NNMT Inhibitors: A SAR Study Aimed at Inhibitory Activity in the Cell.

Nicotinamide N-methyltransferase (NNMT), which catalyzes the methylation of nicotinamide, is a cytosolic enzyme that has attracted much attention as a therapeutic target for a variety of diseases. However, despite the considerable interest in this target, reports of NNMT inhibitors have still been limited to date. In this work, utilizing in vitro translated macrocyclic peptide libraries, we identified peptide 1 as a novel class of NNMT inhibitors. Further exploration based on the X-ray cocrystal structures of the peptides with NNMT provided a dramatic improvement in inhibitory activity (peptide 23: IC50 = 0.15 nM). Furthermore, by balance of the peptides' lipophilicity and biological activity, inhibitory activity against NNMT in cell-based assay was successfully achieved (peptide 26: cell-based IC50 = 770 nM). These findings illuminate the potential of cyclic peptides as a relatively new drug discovery modality even for intracellular targets.

Engineering of the cytochrome P450 monooxygenase system for benzyl maltol hydroxylation.

Maltol derivatives are utilized in a variety of fields due to their metal-chelating abilities, and modification of the 2-methyl side chain is known to effectively expand their functional diversity. In the present study, microbial enzymes were screened for hydroxylating activity towards the 2-methyl group in a maltol derivative, 3-benzyloxy-2-methyl-4-pyrone (BMAL). Novosphingobium sp. SB32149 was found to have the ability to convert BMAL into 3-benzyloxy-2-hydroxymethyl-4-pyrone (BMAL-OH). The enzymes responsible, a cytochrome P450 monooxygenase (P450nov), a ferredoxin (FDXnov), and a ferredoxin reductase (FDRnov), were identified in the SB32149 strain. In the reaction with recombinant Escherichia coli expressing P450nov, FDXnov, and FDRnov, BMAL-OH was successfully produced from BMAL. Moreover, using the directed evolution approach, four amino acid substitutions, L188P/F218L/L237M in P450nov and A10T in FDXnov, were found to enhance BMAL-OH production. Consequently, up to 5.2 g/L BMAL-OH was obtained from 8.0 g/L BMAL by bioconversion using a 250-mL jar fermenter, indicating that this strain may be useful for synthesis of maltol derivatives which could have potential applications in various fields.

Biohydrogenation of C20 polyunsaturated fatty acids by anaerobic bacteria.

The PUFAs include many bioactive lipids. The microbial metabolism of C18 PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C20 PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C20 PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C20 PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C18 and C20 PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.

Production of conjugated fatty acids by lactic acid bacteria.

Conjugated fatty acids have attracted much attention as a novel type of biologically beneficial functional lipid. Some isomers of conjugated linoleic acid (CLA) reduce carcinogenesis, atherosclerosis, and body fat. Considering the use of CLA for medicinal and nutraceutical purposes, a safe isomer-selective process is required. The introduction of biological reactions for CLA production could be an answer. We screened microbial reactions useful for CLA production, and found several unique reactions in lactic acid bacteria. Lactic acid bacteria produced CLA from linoleic acid. The produced CLA comprised a mixture of cis-9,trans-11-octadecadienoic acid (18:2) and trans-9,trans-11-18:2. Lactobacillus plantarum AKU 1009a was selected as a potential CLA producer. Using washed cells of L. plantarum AKU 1009a as a catalyst, CLA production from linoleic acid reached 40 mg/ml under the optimized conditions. The CLA-producing reaction was found to consist of two successive reactions, i.e., hydration of linoleic acid to 10-hydroxy-12-octadecenoic acid and dehydrating isomerization of the hydroxy fatty acid to CLA. On the basis of these results, the transformation of hydroxy fatty acids by lactic acid bacteria was investigated. Lactic acid bacteria transformed ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid) to CLA (a mixture of cis-9,trans-11-18:2 and trans-9,trans-11-18:2). Castor oil, which is rich in the triacylglycerol form of ricinoleic acid, was also found to act as a substrate for CLA production by lactic acid bacteria with the aid of lipase-catalyzed triacylglycerol hydrolysis. L. plantarum AKU 1009a produced conjugated trienoic fatty acids from alpha- and gamma-linolenic acid. The trienoic fatty acids produced from alpha-linolenic acid were identified as cis-9,trans-11,cis-15-octadecatrienoic acid (18:3) and trans-9,trans-11,cis-15-18:3. Those produced from gamma-linolenic were cis-6,cis-9,trans-11-18:3 and cis-6,trans-9,trans-11-18:3. The conjugated trienoic fatty acids produced from alpha- and gamma-linolenic acid were further saturated by L. plantarum AKU 1009a to trans-10,cis-15-18:2 and cis-6,trans-10-18:2, respectively.