Protein degradation by a component of the chaperonin-linked protease ClpP.

In cells, proteins are synthesized, function, and degraded (dead). Protein synthesis (spring) is important for the life of proteins. However, how proteins die is equally important for organisms. Proteases are secreted from cells and used as nutrients to break down external proteins. Proteases degrade unwanted and harmful cellular proteins. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for cellular protein degradation. Prokaryotes, such as bacteria, have similar protein degradation systems. In this review, we describe the structure and function of the ClpXP complex in the degradation system, which is an ATP-dependent protease in bacterial cells, with a particular focus on ClpP.

ACAGT-007a, an anti-cancer compound that modulates ERK MAPK signaling, induces nuclear enrichment of phosphorylated ERK in T3M4 pancreatic cancer cells.

The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT-007a (GT-7), an anti-cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT-7 stimulates ERK phosphorylation and induces apoptosis in ERK-active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT-7 in T3M4 cells. The immunoblotting showed that GT-7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase-3 was observed only after 2-h incubation with GT-7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho-ERK) in the nucleus upon 1-h incubation with GT-7. Fractionation experiments showed that GT-7 increases phospho-ERK levels in the cytoplasm within 1 h, whereas nuclear phospho-ERK accumulation is observed after 2-h incubation with GT-7. MEK inhibition by U0126 significantly diminishes nuclear phospho-ERK distribution and apoptosis induction stimulated by GT-7. Thus, GT-7 may initiate the induction of ERK phosphorylation in the cytoplasm, which leads to phospho-ERK enrichment in the nucleus. This nuclear phospho-ERK accumulation by GT-7 precedes and may underlie apoptosis induction in T3M4.

Probing for optimal photoaffinity linkers of benzophenone-based photoaffinity probes for adenylating enzymes.

The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5'-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2-8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2-8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4'-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.

Recent progress in the reprogramming of nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) biosynthesize nonribosomal peptide (NRP) natural products, which belong to the most promising resources for drug discovery and development because of their wide range of therapeutic applications. The results of genetic, biochemical, and bioinformatics analyses have enhanced our understanding of the mechanisms of the NRPS machinery. A major goal in NRP biosynthesis is to reprogram the NRPS machinery to enable the biosynthetic production of designed peptides. Reprogramming strategies for the NRPS machinery have progressed considerably in recent years, thereby increasing the yields and generating modified peptides. Here, the recent progress in NRPS reprogramming and its application in peptide synthesis are described.

Development of a chemical scaffold for inhibiting nonribosomal peptide synthetases in live bacterial cells.

The adenylation (A) domain is essential for non-ribosomal peptide synthetases (NRPSs), which synthesize various peptide-based natural products, including virulence factors, such as siderophores and genotoxins. Hence, the inhibition of A-domains could attenuate the virulence of pathogens. 5'-O-N-(Aminoacyl or arylacyl)sulfamoyladenosine (AA-AMS) is a bisubstrate small-molecule inhibitor of the A-domains of NRPSs. However, the bacterial cell permeability of AA-AMS is typically a problem owing to its high hydrophilicity. In this study, we investigated the influence of a modification of 2'-OH in the AMS scaffold with different functional groups on binding to target enzymes and bacterial cell penetration. The inhibitor 7 with a cyanomethyl group at 2'-OH showed desirable inhibitory activity against both recombinant and intracellular gramicidin S synthetase A (GrsA) in the gramicidin S-producer Aneurinibacillus migulanus ATCC 9999, providing an alternative scaffold to develop novel A-domain inhibitors.

Total Synthesis of Calanthoside, a Potential Hair Growth Stimulant: A Facile Synthetic Approach via One-Pot S- and O-Glucosidic Bond Formation.

The first total synthesis of calanthoside (1), which exhibits potent proliferative activity against human hair follicle dermal papilla cells, has been achieved in seven steps with an overall yield of 43% on a gram scale starting from anthranilic acid (11). The synthetic strategy features a one-pot process involving thioglucoside bond formation via nucleophilic substitution reaction and enol-glucosylation for building the S-,O-bisdesmoside structure of 1. Moreover, the one-pot reaction showed broad substrate adaptability to several sugar donors other than d-glucose, thus affording S,O-bisglycoside intermediates in ∼84% yield.

Down-regulation of dual-specificity phosphatase 6, a negative regulator of oncogenic ERK signaling, by ACA-28 induces apoptosis in NIH/3T3 cells overexpressing HER2/ErbB2.

Dual-specificity phosphatase 6 (DUSP6) is a key negative feedback regulator of the member of the RAS-ERK MAPK signaling pathway that is associated with cellular proliferation and differentiation. Deterioration of DUSP6 expression could therefore result in deregulated growth activity. We have previously discovered ACA-28, a novel anticancer compound with a unique property to stimulate ERK phosphorylation and induce apoptosis in ERK-active melanoma cells. However, the mechanism of cancer cell-specific-apoptosis by ACA-28 remains obscure. Here, we investigated the involvement of DUSP6 in the mechanisms of the ACA-28-mediated apoptosis by using the NIH/3T3 cells overexpressing HER2/ErbB2 (A4-15 cells), as A4-15 exhibited higher ERK phosphorylation and are more susceptible to ACA-28 than NIH/3T3. We showed that A4-15 exhibited high DUSP6 protein levels, which require ERK activation. Notably, the silencing of the DUDSP6 gene by siRNA inhibited proliferation and induced apoptosis in A4-15, but not in NIH/3T3, indicating that A4-15 requires high DUSP6 expression for growth. Importantly, ACA-28 preferentially down-regulated the DUSP6 protein and proliferation in A4-15 via the proteasome, while it stimulated ERK phosphorylation. Collectively, the up-regulation of DUSP6 may exert a growth-promoting role in cancer cells overexpressing HER2. DUSP6 down-regulation in ERK-active cancer cells might have the potential as a novel cancer measure.

Structure-activity relationship study of 4,5-didehydroguadiscine, an aporphine alkaloid showing potent melanogenesis-inhibitory activity in B16 melanoma cells.

Although 4,5-didehydroguadiscine (12a), an alkaloid with potent melanogenesis-inhibitory activity isolated from Hornschuchia obliqua (Annonaceae), consists of an aporphine nucleus with an aromatized B-ring, to date, it has not been utilized as a template for structure-activity relationship (SAR) studies of pharmacological activities because of its exceptional structure. Accordingly, herein, five analogs (12b-12f) of 12a and five benzylisoquinoline analogs (13b-13f) lacking the C11a-C11b bond of 12b-12f were prepared. The inhibitory effects of 12b-12f and 13b-13f on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were examined and compared with those of 12a. Melanogenesis-inhibitory activities of 12b-12f were the same as that of 12a, whereas the melanogenesis-inhibitory activities of 13b-13f were significantly inferior to those of 12a and 12b-12f. These results suggest that the C11a-C11b bond plays an essential role in the melanogenesis-inhibitory activities of 12a-12e.

Elongation of the side chain by linear alkyl groups increases the potency of salacinol, a potent α-glucosidase inhibitor from the Ayurvedic traditional medicine "Salacia," against human intestinal maltase.

Four chain-extended analogs (12a-12d) and two related de-O-sulfonated analogs (13a and 13c) by introducing alkyl groups (a: R = C3H7, b R = C6H13, c: R = C8H17, d: R = C10H21) to the side chains of salacinol (1), a natural α-glucosidase inhibitor from Ayurvedic traditional medicine "Salacia", were synthesized. The α-glucosidase inhibitory activities of all the synthesized analogs were evaluated in vitro. Against human intestinal maltase, the inhibitory activities of 12a and 13a with seven-carbon side chain were equal to that of 1. In contrast, analogs (12b-12d, and 13c) exhibited higher level of inhibitory activity against the same enzyme than 1 and had equal or higher potency than those of the clinically used anti-diabetics, voglibose, acarbose, and miglitol. Thus, elongation of the side chains of 1 was effective for specifically increasing the inhibitory activity against human intestinal maltase.

Inhibition of efflux pumps aids small-molecule probe-based fluorescence labeling and imaging in the Gram-negative bacterium Escherichia coli.

A major challenge in fluorescence imaging experiments, which are essential to determine protein activity, expression, and localization, is the penetration of small-molecule probes through the outer membrane permeability barrier of bacteria. Here, we describe a novel strategy for small-molecule probe-based fluorescence protein labeling and imaging in the Gram-negative bacterium Escherichia coli. We targeted a siderophore enterobactin biosynthetic enzyme EntE in E. coli. When coupled with an efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone, small-molecule probes were able to efficiently enter the cells, leading to the fluorescence labeling and imaging of overproduced EntE in E. coli. This study demonstrates that the combination of small-molecule probes with appropriate efflux pump inhibitors may substantially enhance their interaction with the target proteins in live bacteria.

Activity, Binding, and Modeling Studies of a Reprogrammed Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket.

The gatekeeping adenylation (A) domain of the non-ribosomal peptide synthetase (NRPS) selectively incorporates specific proteinogenic/non-proteinogenic amino acid into a growing peptide chain. The EntE of the enterobactin NRPS is a discrete aryl acid A-domain with 2,3-dihydroxybenzoic acid (DHB) substrate specificity. Reprogrammed EntE N235G variant possesses an enlarged substrate recognition site, and is capable of accepting non-native aryl acids. Biochemical characterization of this unique substrate recognition site should provide a better understanding of activi-site microenvironments. Here, we synthesized a non-hydrolysable adenylate analogue with 2-aminobenzoic acid (2-ABA), 3-aminobenzoic acid (3-ABA), and 4-aminobenzoic acid (4-ABA) and used them to calculate the apparent inhibition constants (Kiapp.). Dose-response experiments using 3-ABA-sulfamoyladenosine (AMS) provided Kiapp. values of 596 nM for wild-type EntE and 2.4 nM for the N235G variants. These results suggest that 3-amino group of benzoic acid plays an important role in substrate recognition by the N235G variant. These findings would help designing aryl acid substrates with substituents at the 2- and 3-positions.

Precise Probing of Residue Roles by NRPS Code Swapping: Mutation, Enzymatic Characterization, Modeling, and Substrate Promiscuity of Aryl Acid Adenylation Domains.

Aryl acids are most commonly found in iron-scavenging siderophores but are not limited to them. The nonribosomal peptide synthetase (NRPS) codes of aryl acids remain poorly elucidated relative to those of amino acids. Here, we defined more precisely the role of active-site residues in aryl acid adenylation domains (A-domains) by gradually grafting the NRPS codes used for salicylic acid (Sal) into an archetypal aryl acid A-domain, EntE [specific for the substrate 2,3-dihydroxybenzoic acid (DHB)]. Enzyme kinetics and modeling studies of these EntE variants demonstrated that the NRPS code residues at positions 236, 240, and 339 collectively regulate the substrate specificity toward DHB and Sal. Furthermore, the EntE variants exhibited the ability to activate the non-native aryl acids 3-hydroxybenzoic acid, 3-aminobenzoic acid, 3-fluorobenzoic acid, and 3-chlorobenzoic acid. These studies enhance our knowledge of the NRPS codes of aryl acids and could be exploited to reprogram aryl acid A-domains for non-native aryl acids.

Probing the Compatibility of an Enzyme-Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains.

An important challenge in natural product biosynthesis is the biosynthetic design and production of artificial peptides. One of the most promising strategies is reprogramming adenylation (A) domains to expand the substrate repertoire of nonribosomal peptide synthetases (NRPSs). Therefore, the precise detection of subtle structural changes in the substrate binding pockets of A domains might accelerate their reprogramming. Here we show that an enzyme-linked immunosorbent assay (ELISA) using a combination of small-molecule probes can detect the effects of substrate binding pocket residue substitutions in A-domains. When coupled with a set of aryl acid A-domain variants (total of nine variants), the ELISA can analyze the subtle differences in their active-site architectures. Furthermore, the ELISA-based screening was able to identify the variants with substrate binding pockets that accepted a non-cognate substrate from an original pool of 45. These studies demonstrate that ELISA is a reliable platform for providing insights into the active-site properties of A-domains and can be applied for the reprogramming of NRPS A-domains.

Activity-Based Protein Profiling of Non-ribosomal Peptide Synthetases.

Non-ribosomal peptide (NRP) natural products are one of the most promising resources for drug discovery and development because of their wide-ranging of therapeutic potential, and their behavior as virulence factors and signaling molecules. The NRPs are biosynthesized independently of the ribosome by enzyme assembly lines known as the non-ribosomal peptide synthetase (NRPS) machinery. Genetic, biochemical, and bioinformatics analyses have provided a detailed understanding of the mechanism of NRPS catalysis. However, proteomic techniques for natural product biosynthesis remain a developing field. New strategies are needed to investigate the proteomes of diverse producer organisms and directly analyze the endogenous NRPS machinery. Advanced platforms should verify protein expression, protein folding, and activities and also enable the profiling of the NRPS machinery in biological samples from wild-type, heterologous, and engineered bacterial systems. Here, we focus on activity-based protein profiling strategies that have been recently developed for studies aimed at visualizing and monitoring the NRPS machinery and also for rapid labeling, identification, and biochemical analysis of NRPS enzyme family members as required for proteomic chemistry in natural product sciences.

Discovery of new benzhydrol biscarbonate esters as potent and selective apoptosis inducers of human melanomas bearing the activated ERK pathway: SAR studies on an ERK MAPK signaling modulator, ACA-28.

The recent discovery that an ERK signaling modulator [ACA-28 (2a)] preferentially kills human melanoma cell lines by inducing ERK-dependent apoptosis has generated significant interest in the field of anti-cancer therapy. In the first SAR study on 2a, here, we successfully developed candidates (2b, 2c) both of which induce more potent and selective apoptosis towards ERK-active melanoma cells than 2a, thus revealing the structural basis for inducing the ERK-dependent apoptosis and proposing the therapeutic prospect of these candidates against ERK-dependent cancers represented by melanoma.

Chemical Strategies for Visualizing and Analyzing Endogenous Nonribosomal Peptide Synthetase (NRPS) Megasynthetases.

Nonribosomal peptide (NRP) natural products are among the most promising resources for drug discovery and development, owing to their wide range of biological activities and therapeutic applications. These peptide metabolites are biosynthesized by large multienzyme machinery known as NRP synthetases (NRPSs). The structural complexity of a number of NRPs poses an enormous challenge in their synthesis. A major issue in this field is reprogramming NRPS machineries to allow the biosynthetic production of artificial peptides. NRPS adenylation (A) domains are responsible for the incorporation of a wide variety of amino acids and can be considered as reprogramming sites; therefore, advanced methods to accelerate the functional prediction and assessment of A-domains are required. This Concept article demonstrates that activity-based protein profiling of NRPSs offers a simple, rapid, and robust analytical platform for A-domains and provides insights into enzyme-substrate candidates and active-site microenvironments. It also describes the background associated with the development and application of a method to analyze endogenous NRPS machinery in its natural environment.

Design, Synthesis and Biological Evaluation of Nitrate Derivatives of Sauropunol A and B as Potent Vasodilatory Agents.

A group of nitrate derivatives of naturally occurring sauropunol A and B were designed and synthesized. Nitric oxide (NO) releasing capacity and vasodilatory capacity studies were performed to explore the structure-activity relationship of resulted nitrates. Biological evaluation of these compounds revealed that most of the synthesized mononitrate derivatives demonstrated superior releasing capacity than isosorbide mononitrate (ISMN), and 2MNS-6 even demonstrated stronger NO releasing capacity than isosorbide dinitrate (ISDN). Two dinitrates, DNS-1 and DNS-2, showed higher NO releasing capacity than ISDN. Evaluation of inhibitory activities to the contractions in mesenteric artery rings revealed that 2MNS-8 and DNS-2 showed stronger vasorelaxation activities than ISDN. High level of NO and soluble guanylyl cyclase (sGC) may be essential for the potent vasodilatory effect of DNS-2. The vasodilatory effects of DNS-2 may result from cellular signal transduction of NO-sGC-cGMP. DNS-2 was found to be the most potent sauropunol-derived nitrate vasodilatory agent for further pharmaceutical investigation against cardiovascular diseases.

An Engineered Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket.

Adenylation (A) domains act as the gatekeepers of non-ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron-chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A-domains. We show that a single asparagine-to-glycine mutation in an aryl acid A-domain leads to an enzyme that tolerates a wide range of non-native aryl acids. The engineered catalyst is capable of activating non-native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild-type enzyme was observed toward these substrates. Co-crystal structures with non-hydrolysable aryl-AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis.

Practical Route to Neokotalanol and Its Natural Analogues: Sulfonium Sugars with Antidiabetic Activities.

An efficient and divergent approach toward the synthesis of all four de-O-sulfonated sulfonium type α-glucosidase inhibitors, originally isolated from plants of genus Salacia, is reported for the first time. The key strategy features a coupling reaction between thiol derivatives and a diiodide counterpart. The newly designed thiol coupling partner presents high chemical stability, while the diiodide partner could be easily obtained with increased overall yields compared with conventional routes. The intermolecular nucleophilic substitution reaction followed by a diastereoselective intramolecular cyclization provided the target five-member sulfonium salt structure, which was connected in an α-orientation to a polyhydroxylated side-chain moiety.

Structural Basis of Protein-Protein Interactions between a trans-Acting Acyltransferase and Acyl Carrier Protein in Polyketide Disorazole Biosynthesis.

Acyltransferases (ATs) are responsible for the selection and incorporation of acyl building blocks in the biosynthesis of various polyketide natural products. The trans-AT modular polyketide synthases have a discrete trans-acting AT for the loading of an acyl unit onto the acyl carrier protein (ACP) located within each module. Despite the importance of protein-protein interactions between ATs and ACPs in trans-AT assembly lines, the dynamic actions of ACPs and trans-acting ATs remain largely uncharacterized because of the inherently transient nature of ACP-enzyme interactions. Herein, we report the crystal structure of the AT-ACP complex of disorazole trans-AT polyketide synthase. We used a bromoacetamide pantetheine cross-linking probe in combination with a Cys mutation to trap the transient AT-ACP complex, allowing the determination of the crystal structure of the disorazole AT-ACP complex at 2.03 Å resolution. On the basis of the cross-linked AT-ACP complex structure, ACP residues recognized by trans-acting AT were identified and validated by mutational studies, which demonstrated that the disorazole AT recognizes the loop 1 and helix III' residues of disorazole ACP. The disorazole AT-ACP complex structure presents a foundation for defining the dynamic processes associated with trans-acting ATs and provides detailed mechanistic insights into their ability to recognize ACPs.