Modulation of proteasome subunit selectivity of syringolins.

Development of selective or dual proteasome subunit inhibitors based on syringolin B as a scaffold is described. We focused our efforts on a structure-activity relationship study of inhibitors with various substituents at the 3-position of the macrolactam moiety of syringolin B analogue to evaluate whether this would be sufficient to confer subunit selectivity by using sets of analogues with hydrophobic, basic and acidic substituents, which were designed to target Met45, Glu53 and Arg45 embedded in the S1 subsite, respectively. The structure-activity relationship study using systematic analogues provided insight into the origin of the subunit-selective inhibitory activity. This strategy would be sufficient to confer subunit selectivity regarding ß5 and ß2 subunits.

Synthesis and biological evaluation of echinomycin analogues as potential colon cancer agent.

Colorectal cancer is the third most commonly diagnosed cancer and the second leading cause of cancer-related death, thus a novel chemotherapeutic agent for colon cancer therapy is needed. In this study, analogues of echinomycin, a cyclic peptide natural product with potent toxicity to several human cancer cell lines, were synthesized, and their biological activities against human colon cancer cells were investigated. Analogue 3 as well as 1 inhibit HIF-1α-mediated transcription. Notably, transcriptome analysis indicated that the cell cycle and its regulation were involved in the effects on cells treated with 3. Analogue 3 exhibited superior in vivo efficacy to echinomycin without significant toxicity in mouse xenograft model. The low dose of 3 needed to be efficacious in vivo is also noteworthy and our data suggest that 3 is an attractive and potentially novel agent for the treatment of colon cancer.

Synthetic Modifications of Histones and Their Functional Evaluation.

Post-translational modifications (PTMs) of histones play a key role in epigenetic regulation. Unraveling the roles of each epigenetic mark can provide new insights into their biological mechanisms. On the other hand, it is generally difficult to prepare homogeneously-modified histones/nucleosomes to investigate their specific functions. Therefore, synthetic approaches to acquire precisely mimicked histones/nucleosomes are in great demand, and further development of this research field is anticipated. In this review, synthetic strategies to modify histones/nucleosomes, including cysteine modifications, transformations of dehydroalanine residues and lysine acylation using a catalyst system, are cited. In addition, the functional evaluation of synthetically modified histones/nucleosomes is described.

Recent Advances in Iridoid Chemistry: Biosynthesis and Chemical Synthesis.

Iridoids are a large family of monoterpenoids found in traditional medicinal plants and show significant effects for the human species. In addition to their wide range of biological activities, such as neuroprotective and antitumor activities, the cis-fused bicyclic ring systems of iridoids are still attractive as synthetic targets to apply novel synthetic methodologies. Accordingly, recent progress regarding the biosynthesis and chemical synthesis of iridoids is covered in this minireview. Identification of new enzymes for the iridoid biosynthesis in Catharanthus roseus and ingenious synthetic strategies for the construction of the iridoid skeleton are described.

Development of cyclic peptide derivatives from the N-terminal region of LANA for targeting the nucleosome acidic patch.

Kaposi's sarcoma-associated herpesvirus (KSHV) is known to be a carcinogenic agent that causes AIDS-associated Kaposi's sarcoma (KS). When KSHV infects host's cells, one of the virus's proteins, latency-associated nuclear antigen 1 (LANA), binds to the host's nucleosomes to retain episomes and create latency circumstances. Although the infectious mechanism of KSHV is partly elucidated, the development of drug candidates for targeting KS is ongoing. In this study, we developed cyclic peptides corresponding to an N-terminal LANA sequence that disrupt the LANA-nucleosome interaction. The cyclic peptides showed a different secondary structure compared to their corresponding linear peptide derivatives, which suggests that our cyclization strategy imitates the N-terminal LANA binding conformation on nucleosomes.

Synthesis and biological evaluation of a MraY selective analogue of tunicamycins.

Tunicamycins, which are nucleoside natural products, inhibit both bacterial phospho-N-acetylmuraminic acid (MurNAc)-pentapeptide translocase (MraY) and human UDP-N-acetylglucosamine (GlcNAc): polyprenol phosphate translocase (GPT). The improved synthesis and detailed biological evaluation of an MraY-selective inhibitor, 2, where the GlcNAc moiety was modified to a MurNAc amide, has been described.

HIV-1 susceptibility of transgenic rat-derived primary macrophage/T cells and a T cell line that express human receptors, CyclinT1 and CRM1 genes.

We developed transgenic (Tg) rats that express human CD4, CCR5, CXCR4, CyclinT1, and CRM1 genes. Tg rat macrophages were efficiently infected with HIV-1 and supported production of infectious progeny virus. By contrast, both rat primary CD4+ T cells and established T cell lines expressing human CD4, CCR5, CyclinT1, and CRM1 genes were infected inefficiently, but this was ameliorated by inhibition of cyclophilin A. The infectivity of rat T cell-derived virus was lower than that of human T cell-derived virus.

Novel Hybrid Compound of a Plinabulin Prodrug with an IgG Binding Peptide for Generating a Tumor Selective Noncovalent-Type Antibody-Drug Conjugate.

Although several approaches for making antibody-drug conjugates (ADC) have been developed, it has yet to be reported that an antibody binding peptide such as Z33 from protein A is utilized as the pivotal unit to generate the noncovalent-type ADC (NC-ADC). Herein we aim to establish a novel probe for NC-ADC by synthesizing the Z33-conjugated antitumor agent, plinabulin. Due to the different solubility of two components, including hydrophobic plinabulin and hydrophilic Z33, an innovative method with a solid-supported disulfide coupling reagent is required for the synthesis of the target compounds with prominent efficiency (29% isolated yield). We demonstrate that the synthesized hybrid exhibits a binding affinity against the anti-HER2 antibody (Herceptin) and the anti-CD71 antibody (6E1) (Kd = 46.6 ± 0.5 nM and 4.5 ± 0.56 µM, respectively) in the surface plasmon resonance (SPR) assay. In the cell-based assays, the hybrid provides a significant cytotoxicity in the presence of Herceptin against HER2 overexpressing SKBR-3 cells, but not against HER2 low-expressing MCF-7 cells. Further, it is noteworthy that the hybrid in combination with Herceptin induces cytotoxicity against Herceptin-resistant SKBR-3 (SKBR-3HR) cells. Similar results are obtained with the 6E1 antibody, suggesting that the synthesized hybrid can be widely applicable for NC-ADC using the antibody of interest. In summary, a series of evidence presented here strongly indicate that NC-ADCs have high potential for the next generation of antitumor agents.

Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 µm) more potent than parent peptide A (3.53±0.25 µm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides.

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.

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.

Development of potent dipeptide-type SARS-CoV 3CL protease inhibitors with novel P3 scaffolds: design, synthesis, biological evaluation, and docking studies.

We report the design and synthesis of a series of dipeptide-type inhibitors with novel P3 scaffolds that display potent inhibitory activity against SARS-CoV 3CLpro. A docking study involving binding between the dipeptidic lead compound 4 and 3CLpro suggested the modification of a structurally flexible P3 N-(3-methoxyphenyl)glycine with various rigid P3 moieties in 4. The modifications led to the identification of several potent derivatives, including 5c-k and 5n with the inhibitory activities (Ki or IC50) in the submicromolar to nanomolar range. Compound 5h, in particular, displayed the most potent inhibitory activity, with a Ki value of 0.006 µM. This potency was 65-fold higher than the potency of the lead compound 4 (Ki=0.39 µM). In addition, the Ki value of 5h was in very good agreement with the binding affinity (16 nM) observed in isothermal titration calorimetry (ITC). A SAR study around the P3 group in the lead 4 led to the identification of a rigid indole-2-carbonyl unit as one of the best P3 moieties (5c). Further optimization showed that a methoxy substitution at the 4-position on the indole unit was highly favorable for enhancing the inhibitory potency.

Medicinal chemistry and chemical biology of diketopiperazine-type antimicrotubule and vascular-disrupting agents.

Certain antimicrotubule agents displaying colchicine-like tubulin-depolymerizing activity can act as both cytotoxic and vascular-disrupting agents (VDAs). VDAs constitute a new class of anticancer drugs and are currently in clinical trials. We have developed a VDA clinical candidate (phase II) diketopiperazine (DKP)-type antimicrotubule agent called plinabulin (7) derived from the natural DKP phenylahistin (5), which displays colchicine-like tubulin-depolymerizing activity. To develop more potent antimicrotubule DKP derivatives, we performed an intensive structure-activity relationship study examining the phenyl group of compound 7. This study identified more potent DKP derivatives (2,5-difluoro derivatives [29] and benzophenone derivatives [36]) with vascular-disrupting activities. The benzophenone moiety of compound 36 was further modified to yield the most potent cytotoxic derivative yet discovered, the 4-fluorobenzophenone derivative 38 m, which inhibited the growth of HT-29 cells in vitro at subnanomolar levels. As both VDAs and cytotoxic agents, these potent DKP derivatives are valuable second-generation drug candidates. The chemical biology of plinabulin was examined by designing and synthesizing biotin-tagged photoaffinity probes 40-42 that could be used to indicate the binding mode of compound 7 with tubulin. A tubulin photoaffinity labeling study suggested that compound 7 binds at the interface between the α- and ß-tubulins near the colchicine-binding site and not inside the colchicine-binding cavity. A water-soluble prodrug of the poorly water-soluble 7 was next designed in an effort to improve the pharmacokinetics and chemotherapeutic indices. The lead compound 56 revealed high water solubility and a half-life profile appropriate for an injected drug.

Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: structure-activity relationship study.

This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole (8; Ki = 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CL(pro). In particular, compounds 26m and 26n exhibited good inhibitory activities with Ki values of 0.39 and 0.33 µM, respectively. These low-molecular weight compounds are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compound 26m with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1' position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the N-arylglycine unit and a backbone hydrogen bond donor at the S3-position.

Design and synthesis of new tripeptide-type SARS-CoV 3CL protease inhibitors containing an electrophilic arylketone moiety.

We describe here the design, synthesis and biological evaluation of a series of molecules toward the development of novel peptidomimetic inhibitors of SARS-CoV 3CL(pro). A docking study involving binding between the initial lead compound 1 and the SARS-CoV 3CL(pro) motivated the replacement of a thiazole with a benzothiazole unit as a warhead moiety at the P1' site. This modification led to the identification of more potent derivatives, including 2i, 2k, 2m, 2o, and 2p, with IC(50) or K(i) values in the submicromolar to nanomolar range. In particular, compounds 2i and 2p exhibited the most potent inhibitory activities, with K(i) values of 4.1 and 3.1 nM, respectively. The peptidomimetic compounds identified through this process are attractive leads for the development of potential therapeutic agents against SARS. The structural requirements of the peptidomimetics with potent inhibitory activities against SARS-CoV 3CL(pro) may be summarized as follows: (i) the presence of a benzothiazole warhead at the S1'-position; (ii) hydrogen bonding capabilities at the cyclic lactam of the S1-site; (iii) appropriate stereochemistry and hydrophobic moiety size at the S2-site and (iv) a unique folding conformation assumed by the phenoxyacetyl moiety at the S4-site.

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.

Synthesis and structure-activity relationship study of antimicrotubule agents phenylahistin derivatives with a didehydropiperazine-2,5-dione structure.

Plinabulin (11, NPI-2358) is a potent microtubule-targeting agent derived from the natural diketopiperazine "phenylahistin" (1) with a colchicine-like tubulin depolymerization activity. Compound 11 was recently developed as VDA and is now under phase II clinical trials as an anticancer drug. To develop more potent antimicrotubule and cytotoxic derivatives based on the didehydro-DKP skeleton, we performed further modification on the tert-butyl or phenyl groups of 11, and evaluated their cytotoxic and tubulin-binding activities. In the SAR study, we developed more potent derivatives 33 with 2,5-difluorophenyl and 50 with a benzophenone in place of the phenyl group. The anti-HuVEC activity of 33 and 50 exhibited a lowest effective concentration of 2 and 1 nM for microtubule depolymerization, respectively. The values of 33 and 50 were 5 and 10 times more potent than that of CA-4, respectively. These derivatives could be a valuable second-generation derivative with both vascular disrupting and cytotoxic activities.

Anti-microtubule 'plinabulin' chemical probe KPU-244-B3 labeled both alpha- and beta-tubulin.

Plinabulin (1, NPI-2358), a potent microtubule-targeting agent derived from the natural diketopiperazine 'phenylahistin' with a colchicine-like tubulin depolymerization activity, is an anticancer agent undergoing Phase II clinical trials in four countries including the United States. In order to understand the precise binding mode of plinabulin with tubulin, a new bioactive biotin-tagged photoaffinity probe 4 (KPU-244-B3) was designed and synthesized. Probe 4 showed significant binding affinity to tubulin in a binding assay, and selectively bound to tubulin in an HT-1080 cell lysate without photo-irradiation. In a tubulin photoaffinity labeling study, probe 4 labeled both alpha- and beta-tubulin subunits and these interactions were competitively inhibited by plinabulin during photo-irradiation. These results suggest that plinabulin binds in the boundary region between alpha- and beta-tubulin near the colchicine binding site, and not inside the colchicine binding cavity.