Fumika Yakushiji

Synthesis and structure-activity relationships of benzophenone-bearing diketopiperazine-type anti-microtubule agents

KPU-105 (4), a potent anti-microtubule agent that contains a benzophenone was derived from the diketopiperazine-type vascular disrupting agent (VDA) plinabulin 3, which displays colchicine-like tubulin depolymerization activity. To develop derivatives with more potent anti-microtubule and cytotoxic activities, we further modified the benzophenone moiety of 4. Accordingly, we obtained a 4-fluorobenzophenone derivative 16j that inhibited tumor cell growth in vitro with a subnanomolar IC(50) value against HT-29 cells (IC(50)=0.5 nM). Next, the effect of 16j on mitotic spindles was evaluated in HeLa cells. Treatment with 3nM of 16j partially disrupted the interphase microtubule network. By contrast, treatment with the same concentration of CA-4 barely affected the microtubule network, indicating that 16j exhibited more potent anti-mitotic effects than did CA-4.

Identification of the Minimum Peptide from Mouse Myostatin Prodomain for Human Myostatin Inhibition

Myostatin, an endogenous negative regulator of skeletal muscle mass, is a therapeutic target for muscle atrophic disorders. Here, we identified minimum peptides 2 and 7 to effectively inhibit myostatin activity, which consist of 24 and 23 amino acids, respectively, derived from mouse myostatin prodomain. These peptides, which had the propensity to form α-helix structure, interacted to myostatin with KD values of 30-36 nM. Moreover, peptide 2 significantly increased muscle mass in Duchenne muscular dystrophy model mice.

Tubulin photoaffinity labeling study with a plinabulin chemical probe possessing a biotin tag at the oxazole

A new bioactive photoaffinity probe KPU-252-B1 (4) possessing a biotin tag on the oxazole ring of a potent plinabulin derivative KPU-244 (2) was synthesized via the Cu(I)-catalyzed Huisgens cycloaddition reaction to understand the precise binding mode of the diketopiperazine-based anti-microtubule agent plinabulin on tubulin. Probe 4 showed significant binding affinity toward tubulin and cytotoxicity against an HT-29 cells. A photoaffinity labeling study suggested that probe 4 specifically recognizes tubulin at a binding site that binds plinabulin or colchicine, most likely near or at the colchicine binding site, which is located at the interfacial region formed by α-and β-tubulin association. The results also demonstrated that probe 4 may serve as a useful plinabulin chemical probe to investigate the molecular mechanism by which anti-microtubule diketopiperazine derivatives operate.

Discovery of Selective Hexapeptide Agonists to Human Neuromedin U Receptors Types 1 and 2

Neuromedin U (NMU) are bioactive peptides with a common C-terminal heptapeptide sequence (FLFRPRN-amide, 1a) among mammals, which is responsible for receptor activation, namely NMU receptor types 1 (NMUR1) and 2 (NMUR2). Among the various physiological actions of NMU, the anorexigenic effect has recently attracted attention in drug discovery efforts for treating obesity. Although several structure-activity relationship (SAR) studies have been reported, receptor-selective small peptide agonists have yet to be disclosed. Herein a SAR study of 1a-derived peptide derivatives is described. We initially screened both human NMUR1- and NMUR2-selective peptides in calcium-mobilization assays with cells transiently expressing receptors. Then we performed a precise assay with a stable expression system of receptors and consequently discovered hexapeptides 8d and 6b possessing selective agonist activity toward each respective receptor. Hexapeptide 6b, which selectively activates NMUR2 without significant NMUR1 activation, should aid in the development of anorexigenic drugs as well as advance NMU-related endocrinological research.

Development of a new benzophenone-diketopiperazine-type potent antimicrotubule agent possessing a 2-pyridine structure.

A new benzophenone-diketopiperazine-type potent antimicrotubule agent was developed by modifying the structure of the clinical candidate plinabulin (1). Although the right-hand imidazole ring with a branched alkyl chain at the 5-position in 1 was critical for the potency of the antimicrotubule activity, we successfully substituted this moiety with a simpler 2-pyridyl structure by converting the left-hand ring from a phenyl to a benzophenone structure without decreasing the potency. The resultant compound 6b (KPU-300) exhibited a potent cytotoxicity, with an IC50 value of 7.0 nM against HT-29 cells, by strongly binding to tubulin (K d = 1.3 μM) and inducing microtubule depolymerization.

Novel Hybrid-Type Antimicrobial Agents Targeting the Switch Region of Bacterial RNA Polymerase

The bacterial RNA polymerase (RNAP) is an ideal target for the development of antimicrobial agents against drug-resistant bacteria. Especially, the switch region within RNAP has been considered as an attractive binding site for drug discovery. Here, we designed and synthesized a series of novel hybrid-type inhibitors of bacterial RNAP. The antimicrobial activities were evaluated using a paper disk diffusion assay, and selected derivatives were tested to determine their MIC values. The hybrid-type antimicrobial agent 29 showed inhibitory activity against Escherichia coli RNAP. The molecular docking study suggested that the RNAP switch region would be the binding site of 29.

Discovery of potent hexapeptide agonists to human neuromedin u receptor 1 and identification of their serum metabolites.

Neuromedin U (NMU) and S (NMS) display various physiological activities, including an anorexigenic effect, and share a common C-terminal heptapeptide-amide sequence that is necessary to activate two NMU receptors (NMUR1 and NMUR2). On the basis of this knowledge, we recently developed hexapeptide agonists 2 and 3, which are highly selective to human NMUR1 and NMUR2, respectively. However, the agonists are still less potent than the endogenous ligand, hNMU. Therefore, we performed an additional structure-activity relationship study, which led to the identification of the more potent hexapeptide 5d that exhibits similar NMUR1-agonistic activity as compared to hNMU. Additionally, we studied the stability of synthesized agonists, including 5d, in rat serum, and identified two major biodegradation sites: Phe(2)-Arg(3) and Arg(5)-Asn(6). The latter was more predominantly cleaved than the former. Moreover, substitution with 4-fluorophenylalanine, as in 5d, enhanced the metabolic stability at Phe(2)-Arg(3). These results provide important information to guide the development of practical hNMU agonists.

Discovery of natural products possessing selective eukaryotic readthrough activity: 3-epi-deoxynegamycin and its leucine adduct.

Herein we report the first discovery of natural readthrough products that do not display antimicrobial activity. Two natural negamycins, 3‐epi‐deoxynegamycin and its leucine adduct, isolated 37 years ago, were found to be potent readthrough agents against nonsense mutations of eukaryotes, but not prokaryotes, without displaying antimicrobial activity. These results suggest that the compounds are valuable leads for the development of readthrough drugs against nonsense‐mediated genetic diseases without the potential for contributing to the emergence of drug‐resistant bacteria.

Negamycin analogue with readthrough-promoting activity as a potential drug candidate for duchenne muscular dystrophy.

A series of (+)-negamycin 1 analogues were synthesized, and their readthrough-promoting activity was evaluated for nonsense mutations in Duchenne muscular dystrophy (DMD). A structure-activity relationship study indicated that 11b was the most potent drug candidate. Immunohistochemical analyses suggested that treatment with 11b restored dystrophin expression in mdx mice, a DMD mouse model. Furthermore, 11b decreased serum creatine kinase (CK) levels, an indicator of muscle fiber destruction. Most importantly, 11b demonstrated lower toxicity than 1, and thus, it could be a useful candidate for long-term treatment of DMD.

Identification of a Degrading Enzyme in Human Serum that Hydrolyzes a C-terminal Core Sequence of Neuromedin U

Neuromedin U (NMU), an anorexigenic peptide, has attracted attention as a candidate for development of drugs against obesity. We recently developed several potent hexapeptidic agonists derived from NMU that share a common Pro‐Arg‐Asn‐NH2 (PRN) sequence at their C‐termini and found that the amide bond between Arg and Asn is rapidly degraded in serum. In this study, we determined that the key enzyme responsible for this biodegradation was thrombin. Both irreversible and reversible thrombin inhibitors (PPACK and argatroban, respectively) enhanced the serum stability of both hexapeptidic agonists and human NMU itself as an inherent ligand. In addition, rapid degradation did not occur in citrated human plasma because thrombin was not activated under these conditions. Furthermore, we found that an N‐terminal 2‐thienylacetyl group in hexapeptidic agonists enhanced recognition by thrombin. These findings will be valuable for future investigations of the biological functions of NMU in vivo.