Recent developments of topoisomerase inhibitors: Clinical trials, emerging indications, novel molecules and global sales.

The nucleic acid topoisomerases (TOP) are an evolutionary conserved mechanism to solve topological problems within DNA and RNA that have been historically well-established as a chemotherapeutic target. During investigation of trends within clinical trials, we have identified a very high number of clinical trials involving TOP inhibitors, prompting us to further evaluate the current status of this class of therapeutic agents. In total, we have identified 233 unique molecules with TOP-inhibiting activity. In this review, we provide an overview of the clinical drug development highlighting advances in current clinical uses and discussing novel drugs and indications under development. A wide range of bacterial infections, along with solid and hematologic neoplasms, represent the bulk of clinically approved indications. Negative ADR profile and drug resistance among the antibacterial TOP inhibitors and anthracycline-mediated cardiotoxicity in the antineoplastic TOP inhibitors are major points of concern, subject to continuous research efforts. Ongoing development continues to focus on bacterial infections and cancer; however, there is a degree of diversification in terms of novel drug classes and previously uncovered indications, such as glioblastoma multiforme or Clostridium difficile infections. Preclinical studies show potential in viral, protozoal, parasitic and fungal infections as well and suggest the emergence of a novel target, TOP IIIß. We predict further growth and diversification of the field thanks to the large number of experimental TOP inhibitors emerging.

Revealing the Interaction Mechanism between Mycobacterium tuberculosis GyrB and Novobiocin, SPR719 through Binding Thermodynamics and Dissociation Kinetics Analysis.

With the rapid emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), various levels of resistance against existing anti-tuberculosis (TB) drugs have developed. Consequently, the identification of new anti-TB targets and drugs is critically urgent. DNA gyrase subunit B (GyrB) has been identified as a potential anti-TB target, with novobiocin and SPR719 proposed as inhibitors targeting GyrB. Therefore, elucidating the molecular interactions between GyrB and its inhibitors is crucial for the discovery and design of efficient GyrB inhibitors for combating multidrug-resistant TB. In this study, we revealed the detailed binding mechanisms and dissociation processes of the representative inhibitors, novobiocin and SPR719, with GyrB using classical molecular dynamics (MD) simulations, tau-random acceleration molecular dynamics (τ-RAMD) simulations, and steered molecular dynamics (SMD) simulations. Our simulation results demonstrate that both electrostatic and van der Waals interactions contribute favorably to the inhibitors' binding to GyrB, with Asn52, Asp79, Arg82, Lys108, Tyr114, and Arg141 being key residues for the inhibitors' attachment to GyrB. The τ-RAMD simulations indicate that the inhibitors primarily dissociate from the ATP channel. The SMD simulation results reveal that both inhibitors follow a similar dissociation mechanism, requiring the overcoming of hydrophobic interactions and hydrogen bonding interactions formed with the ATP active site. The binding and dissociation mechanisms of GyrB with inhibitors novobiocin and SPR719 obtained in our work will provide new insights for the development of promising GyrB inhibitors.

Recent advances toward the development of Hsp90 C-terminal inhibitors.

Heat shock protein 90 (Hsp90) is a dynamic protein which serves to ensure proper folding of nascent client proteins, regulate transcriptional responses to environmental stress and guide misfolded and damaged proteins to destruction via ubiquitin proteasome pathway. Recent advances in the field of Hsp90 have been made through development of isoform selective inhibitors, Hsp90 C-terminal inhibitors and disruption of protein-protein interactions. These approaches have led to alleviation of adverse off-target effects caused by pan-inhibition of Hsp90 using N-terminal inhibitors. In this review, we provide an overview of relevant advances on targeting the Hsp90 C-terminal Domain (CTD) and the development of Hsp90 C-terminal inhibitors (CTIs) since 2015.

Synthesis of novobiocin derivatives and evaluation of their antigonococcal activity and pharmacokinetics.

Gonorrhea has become a serious problem because the number of infected people is increasing and the multi-drug resistance of the causative bacteria, Neisseria gonorrhoeae, is progressing. To develop novel drugs against resistant N. gonorrhoeae, we focused on the antibiotic novobiocin (1). This natural product has a different mechanism of action from existing drugs for gonorrhea, which may make it effective against resistant strains. Actually, it was applied to resistant N. gonorrhoeae, and moderate antibacterial activity was confirmed. Based on this result, we investigated the development of an antigonococcal drug with 1 as the lead compound. The pharmacophore is thought to be the noviose sugar moiety, especially around the 3'-position, so we derivatized this part in order to improve antibacterial activity. As a result, we found that 5 with an methylpyrrole ester structure have a very potent antibacterial activity. This derivative also showed excellent antigonococcal activity against resistant strains in vitro, however it has poor water solubility and pharmacokinetics because it is the acidic lipid-soluble compound. Therefore, we considered introduction of a basic substituent into the molecule would result in an amphoteric compound with improved water solubility, and we investigated further derivatization. As a result of synthesizing various derivatives, we found 47 containing imidazole with strong antigonococcal activity and greatly improved water solubility. This derivative has also improved metabolism and blood concentration in vivo, and is expected to be orally absorbed. Based on these results, we believe that 47 is a very promising anti-gonococcal lead compound and has great potential for further development.

Genomic and Phenotypic Analysis of Heat and Sanitizer Resistance in Escherichia coli from Beef in Relation to the Locus of Heat Resistance.

The locus of heat resistance (LHR) can confer heat resistance to Escherichia coli to various extents. This study investigated the phylogenetic relationships and the genomic and phenotypic characteristics of E. coli with or without LHR recovered from beef by direct plating or from enrichment broth at 42°C. LHR-positive E. coli isolates (n = 24) were subjected to whole-genome sequencing by short and long reads. LHR-negative isolates (n = 18) from equivalent sources as LHR-positive isolates were short-read sequenced. All isolates were assessed for decimal reduction time at 60°C (D60°C) and susceptibility to the sanitizers E-SAN and Perox-E. Selected isolates were evaluated for growth at 42°C. The LHR-positive and -negative isolates were well separated on the core genome tree, with 22/24 positive isolates clustering into three clades. Isolates within clade 1 and 2, despite their different D60°C values, were clonal, as determined by subtyping (multilocus sequence typing [MLST], core genome MLST, and serotyping). Isolates within each clade are of one serotype. The LHR-negative isolates were genetically diverse. The LHR-positive isolates had a larger (P < 0.001) median genome size by 0.3 Mbp (5.0 versus 4.7 Mbp) and overrepresentation of genes related to plasmid maintenance, stress response, and cryptic prophages but underrepresentation of genes involved in epithelial attachment and virulence. All LHR-positive isolates harbored a chromosomal copy of LHR, and all clade 2 isolates had an additional partial copy of LHR on conjugative plasmids. The growth rates at 42°C were 0.71 ± 0.02 and 0.65 ± 0.02 log(OD) h-1 for LHR-positive and -negative isolates, respectively. No meaningful difference in sanitizer susceptibility was noted between LHR-positive and -negative isolates. IMPORTANCE Resistant bacteria are serious food safety and public health concerns. Heat resistance conferred by the LHR varies largely among different strains of E. coli. The findings in this study show that genomic background and composition of LHR, in addition to the presence of LHR, play an important role in the degree of heat resistance in E. coli and that strains with certain genetic backgrounds are more likely to acquire and maintain the LHR. Also, caution should be exercised when recovering E. coli at elevated temperatures, as the presence of LHR may confer growth advantages to some strains. Interestingly, the LHR-harboring strains seem to have evolved further from their primary animal host to adapt to their secondary habitat, as reflected by fewer genes involved in virulence and epithelial attachment. The phylogenetic relationships among the isolates point toward multiple mechanisms for acquisition of LHR by E. coli, likely prior to its being deposited on meat.

Proposal of Thermoactinomyces mirandus sp. nov., a filamentous, anaerobic bacterium isolated from a biogas plant.

We isolated a filamentous, thermophilic, and first anaerobic representative of the genus Thermoactinomyces, designated strain AMNI-1T, from a biogas plant in Tyrol, Austria and report the results of a phenotypic, genetic, and phylogenetic investigation. Strain AMNI-1T was observed to form a white branching mycelium that aggregates into pellets when grown in liquid medium. Cells could primarily utilize lactose, glucose, and mannose as carbon and energy sources, with acetate accelerating and yeast extract being mandatory for growth. The optimum growth temperature and pH turned out to be 55 °C and pH 7.0, respectively, with an optimum NaCl concentration of 0-2% (w/v). 16S rRNA gene sequence comparison indicated that the genetic relatedness between strain AMNI-1T and Thermoactinomyces intermedius, Thermoactinomyces khenchelensis, and Thermoactinomyces vulgaris was less than 97%. The G + C content of the genomic DNA was 44.7 mol%. The data obtained suggest that the isolate represents a novel and first anaerobic species of the genus Thermoactinomyces, for which the name Thermoactinomyces mirandus is proposed. The type strain is AMNI-1T (= DSM 110094T = LMG 31503T). The description of the genus Thermoactinomyces is emended accordingly.

Selective pre-enrichment method to lessen time needed to recover Salmonella from commercial poultry processing samples.

Conventional Salmonella detection is time consuming, often employing a 24-h pre-enrichment step in buffered peptone water (BPW), followed by a 24-h selective enrichment in either Rappaport Vassiliadis (RV) or tetrathionate (TT) broths before streaking onto selective indicator agar. To reduce this time, we sought to optimize pre-enrichment for Salmonella recovery by evaluating the addition of selective chemicals to BPW. Duplicate samples each representative of 500 carcasses were collected by catching processing water drip under moving carcass shackle lines immediately after feather removal in each of nine commercial processing plants. Carcass drip samples were cultured under selective pre-enrichment conditions in parallel with BPW pre-enrichment followed by RV and TT selective enrichment. Addition of bile salts (1 g/L) and novobiocin (0.015 g/L) resulted in Salmonella recovery from 89% samples when plated directly after pre-enrichment compared to 67% recovery in non-selective BPW alone. Salmonella serovar identities were determined using CRISPR-SeroSeq. Overall, serovars matched between selective pre-enrichment and traditional enrichment methods. These data suggest that increasing the selectivity of Salmonella pre-enrichment step may lessen the need for a separate selective enrichment step thereby reducing time required for Salmonella isolation by 24 h.

Disparate outer membrane exclusionary properties underlie intrinsic resistance to hydrophobic substances in Pseudomonas spp. isolated from surface waters under triclosan selection.

Representative members of surface water microbiota were obtained from three unrelated municipal sites in Oklahoma by direct plating under selection by the hydrophobic biocide triclosan. Multiple methods were employed to determine if intrinsic triclosan resistance reflected resistance to hydrophobic molecules by virtue of outer membrane impermeability. While all but one organism isolated in the absence of triclosan were able to initiate growth on MacConkey agar, only one was able to initiate significant growth with triclosan present. In contrast, all bacteria selected with triclosan were identified as Pseudomonas spp. using 16S RNA gene sequencing and exhibited growth comparable to Pseudomonas aeruginosa controls in the presence of hydrophobic antibacterial agents to include triclosan. Two representative bacteria isolated in the absence of triclosan allowed for greater outer membrane association with the fluorescent hydrophobic probe 1-N-phenylnapthylamine than did two triclosan-resistant isolates. Compound 48/80 disruption of outer membrane impermeability properties for hydrophobic substances either partially or fully sensitized nine of twelve intrinsically resistant isolates to triclosan. These data suggest that outer membrane exclusion underlies intrinsic resistance to triclosan in some, but not all Pseudomonas spp. isolated by selection from municipal surface waters and implicates the involvement of concomitant triclosan resistance mechanisms.

Stimulation of heat shock protein 90 chaperone function through binding of a novobiocin analog KU-32.

Heat shock protein 90 (Hsp90) is a eukaryotic chaperone responsible for the folding and functional activation of numerous client proteins, many of which are oncoproteins. Thus, Hsp90 inhibition has been intensely pursued, resulting in the development of many potential Hsp90 inhibitors, not all of which are well-characterized. Hsp90 inhibitors not only abrogate its chaperone functions, but also could help us gain insight into the structure-function relationship of this chaperone. Here, using biochemical and cell-based assays along with isothermal titration calorimetry, we investigate KU-32, a derivative of the Hsp90 inhibitor novobiocin (NB), for its ability to modulate Hsp90 chaperone function. Although NB and KU-32 differ only slightly in structure, we found that upon binding, they induce completely opposite conformational changes in Hsp90. We observed that NB and KU-32 both bind to the C-terminal domain of Hsp90, but surprisingly, KU-32 stimulated the chaperone functions of Hsp90 via allosteric modulation of its N-terminal domain, responsible for the chaperone's ATPase activity. In vitro and in silico studies indicated that upon KU-32 binding, Hsp90 undergoes global structural changes leading to the formation of a "partially closed" intermediate that selectively binds ATP and increases ATPase activity. We also report that KU-32 promotes HeLa cell survival and enhances the refolding of an Hsp90 substrate inside the cell. This discovery explains the effectiveness of KU-32 analogs in the management of neuropathies and may facilitate the design of molecules that promote cell survival by enhancing Hsp90 chaperone function and reducing the load of misfolded proteins in cells.

Usefulness of novobiocin as a selective inhibitor of intestinal breast cancer resistance protein (Bcrp) in rats.

We investigated whether novobiocin is useful for elucidating the contribution of breast cancer resistance protein (Bcrp) to intestinal absorption without affecting the activities of P-glycoprotein (P-gp), cytochrome P450 (CYP) 3 A and hepatic organic anion transporting polypeptide (Oatp) in rats.To determine the effects of novobiocin on Bcrp, P-gp, CYP3A and Oatp activities, we used sulfasalazine, fexofenadine, bosentan and midazolam, respectively, as probe substrates. Each substrate was orally or intravenously administered to rats 15 min after oral novobiocin administration at a dose of 3 mg/kg.Pre-treatment with novobiocin significantly increased the area under the plasma concentration-time curve and the peak plasma concentration of sulfasalazine after oral administration by 3.2- and 5.9-fold, respectively, in rats, whereas its systemic clearance following intravenous dosing was not influenced. These results indicate that novobiocin selectively inhibits intestinal Bcrp-mediated efflux with limited effects on extra-intestinal Bcrp activity.In addition, novobiocin pre-treatment did not significantly alter the pharmacokinetic parameters of orally administered fexofenadine and midazolam or intravenously administered bosentan, suggesting that the effects of novobiocin on other processes were negligible.These findings demonstrate that novobiocin permits estimating the net contribution of Bcrp to intestinal absorption of drug candidates.

Novobiocin-ferrocene conjugates possessing anticancer and antiplasmodial activity independent of HSP90 inhibition.

A series of tailored novobiocin-ferrocene conjugates was prepared in moderate yields and investigated for in vitro anticancer and antiplasmodial activity against the MDA-MB-231 breast cancer line and Plasmodium falciparum 3D7 strain, respectively. While the target compounds displayed moderate anticancer activity against the breast cancer cell line with IC50 values in the mid-micromolar range, compounds 10a-c displayed promising antiplasmodial activity as low as 0.889 µM. Furthermore, the most promising compounds were tested for inhibitory effects against a postulated target, heat shock protein 90 (Hsp90). A selection of tailored novobiocin derivatives bearing the organometallic ferrocene unit were synthesized and characterized by common spectroscopic techniques. The target compounds were investigated for in vitro anticancer and antimalarial activity against the MDA-MB-231 breast cancer cell line and Plasmodium falciparum 3D7 strain, respectively.

Mitochondrial-targeted Hsp90 C-terminal inhibitors manifest anti-proliferative activity.

The development of C-terminal heat shock protein 90 kDa (Hsp90) inhibitors has emerged as a potential treatment for cancer. Similarly, small molecules that target the mitochondria have proven to be efficacious towards cancer, as the reprogramming of mitochondrial function is often associated with oncogenic transformation. Herein, we report the development of triphenylphosphonium (TPP)-conjugated Hsp90 C-terminal inhibitors, their anti-proliferative activity, and accumulation in the mitochondria. In general, TPP-conjugated Hsp90 C-terminal inhibitors were found to manifest increased activity against various cancer cell lines when compared to the parent compounds.

C-terminal modulators of heat shock protein of 90 kDa (HSP90): State of development and modes of action.

Cells constantly need to adopt to changing environmental conditions, maintaining homeostasis and proteostasis. Heat shock proteins are a diverse class of molecular chaperones that assist proteins in folding to prevent stress-induced misfolding and aggregation. The heat shock protein of 90 kDa (HSP90) is the most abundant heat shock protein. While basal expression of HSP90 is essential for cell survival, in many tumors elevated HSP90 levels are found, which is often associated with bad prognosis. Therefore, HSP90 has emerged as a major target in tumor therapy. The HSP90 machinery is very complex in that it involves large conformational changes during the chaperoning cycle and a variety of co-chaperones. At the same time, this complexity offers a plethora of possibilities to interfere with HSP90 function. The best characterized class of HSP90 modulators are competitive inhibitors targeting the N-terminal ATP-binding pocket. Nineteen compounds of this class entered clinical trials. However, due to severe adverse effects, including induction of the heat shock response, no N-terminal inhibitor has been approved by the FDA so far. As alternatives, compounds commonly referred to as "C-terminal inhibitors" have been developed, either as natural product-based analogues or by rational design, which employ multiple mechanisms to modulate HSP90 function, including modulation of the interaction with co-chaperones, induction of conformational changes that influence the chaperoning cycle, or inhibition of C-terminal dimerization. In this review, we summarize the current development state of characteristic C-terminal inhibitors, with an emphasis on their (proposed) molecular modes of action and binding sites.

Incidence and antimicrobial susceptibility of Staphylococcus aureus isolated from ready-to-eat foods of animal origin from tourist destinations of North-western Himalayas, Himachal Pradesh, India.

This study was aimed to determine the incidence of Staphylococcus aureus in ready-to-eat (RTE) milk (n = 120) and meat (n = 120) products from various tourist places in north western Himalayas, Himachal Pradesh, India. S. aureus isolates and its enterotoxins; A, B, D and E were characterized by conventional and molecular methods. Antimicrobial susceptibility (AMS) profiles of S. aureus isolates were determined by disk diffusion method using Clinical and Laboratory Standards Institute criteria. Overall, 6.7% (n = 16/240) food samples were positive for S. aureus. PCR amplification of nucA confirmed all biochemically characterized isolates as S. aureus. Incidence of S. aureus was higher (10.0%) in RTE milk products than meat products (3.3%). S. aureus contamination levels were highest in milk cake/khoa (26.0%, p = 0.0002) followed by ice cream/kulfi (10.0%, p = 0.4), mutton momo (10.0%, p = 0.4), burfi (3.3%, p = 0.7) and chicken momo (3.3%, p = 0.7). None of the isolates carried genes for S. aureus enterotoxins; A, B, D and E. AMS testing revealed seven different resistance patterns and 81.3% multi drug resistance. All the isolates were resistant to ampicillin. High resistance levels were observed against methicillin (93.7%), clindamycin (68.8%), erythromycin (56.3%) and vancomycin (43.8%). Vancomycin resistant (n = 7) isolates were also resistant to methicillin. All isolates were susceptible to novobiocin.

Reduction of Thermotolerance by Heat Shock Protein 90 Inhibitors in Murine Erythroleukemia Cells.

Cells induce heat shock proteins (HSPs) against various stress. However, murine erythroleukemia (MEL) cells do not express HSP72, a heat-inducible member of HSP70 family. So, it is of interest to examine how MEL cells respond to heat stress (44°C, 30 min). Heat stress-induced apoptosis was suppressed by pretreatment of heat shock (44°C, 10 min). Such suppressive effects were maximal at 6 h after heat shock and remained up to 12 h. Interestingly, such effects of heat shock were abrogated by specific inhibitors of HSP90 such as 17-allylamino-17-demethoxygeldanamycin (17-AAG) and novobiocin. From flow cytometric analysis, it was found that MEL cells arrest in G2 phase at 6 h after heat shock, but restore original cell cycle at 12 h. High expression level of HSP90 was maintained before and after heat shock. Phosphorylation of HSP90α was observed in apoptotic cells induced by heat stress, but inhibited by pretreatment of heat shock. Such inhibition was abrogated by 17-AAG. Moreover, c-Jun NH2-terminal kinase (JNK) was activated in heat stress-induced apoptotic cells. Taken together, these results suggest that HSP90α in MEL cells plays an important role in the thermotolerance, i.e., suppression of heat stress-induced apoptosis by heat shock.

Clinical efficiency and safety of Hsp90 inhibitor Novobiocin in avian tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is a bone defect of broilers and other poultry birds that disturbs growth plate and it causes lameness. Previously we evaluated differential expression of multiple genes involved in growth plate angiogenesis and reported the safety and efficacious of medicinal plant root extracted for controlling TD. In this study, clinical and protective effect of an antibiotic Novobiocin (Hsp90 inhibitor) and expression of Hsp90 and proteoglycan aggrecan was examined. The chicks were divided into three groups; Control, thiram-induced TD, and Novobiocin injected TD. After the induction of TD, the Novobiocin was administered through intraperitoneal route to TD-affected birds until the end of the experiment. The expressions and localization of Hsp90 were evaluated by qRT-PCR, immunohistochemistry (IHC) and western blot, respectively. Morphological, histological examinations, and serum biomarker levels were evaluated to assess specificity and protective effects of Novobiocin. The results showed that TD causing retarded growth, enlarged growth plate, distended chondrocytes, irregular columns of cells, decreased antioxidant capacity, reduced protein levels of proteoglycan aggrecan, and upregulated in Hsp90 expression (p < 0.05) in dyschondroplastic birds as compared with control. Novobiocin treatment restored growth plate morphology, reducing width, stimulated chondrocyte differentiation, sprouting blood vessels, corrected oxidative imbalance, decreased Hsp90 expressions and increased aggrecan level. Novobiocin treatment controlled lameness and improved growth in broiler chicken induced by thiram. In conclusion, the accumulation of the cartilage and up-regulated Hsp90 are associated with TD pathogenesis and irregular chondrocyte morphology in TD is along with reduced aggrecan levels in the growth plate. Our results indicate that Novobiocin treatment has potential to reduce TD by controlling the expression of Hsp90 in addition to improve growth and hepatic toxicity in broiler chicken.

Modified biphenyl Hsp90 C-terminal inhibitors for the treatment of cancer.

Heat Shock Protein 90 (Hsp90) is a molecular chaperone under clinical investigation for the treatment of neurodegenerative diseases and cancer. Neuroprotective Hsp90 C-terminal inhibitors (novologues) contain a biaryl ring system, and include KU-596, which was modified and investigated for potential anti-cancer activity. Incorporation of a benzamide group onto the biaryl novologues in lieu of the acetamide yielded compounds that manifest anti-cancer activity. Further exploration of the central phenyl ring led to compounds with enhanced anti-proliferative activity. The design, synthesis, and evaluation of these new analogs against breast and prostate cancer cell lines is reported herein, where it was found that 8b and 10 manifest potent anti-proliferative activity and a robust degradation of Hsp90 client-dependent proteins.

Molecular docking studies of fused coumarin derivatives as inhibitors of GlcN-6.

The biological activity of heterocyclic compounds depends on their structure, the type of hetero atoms in the ring and on the type of substituents present. In this paper, some heterocyclic compounds with coumarin moieties S1-S5 and novobiocin known as coumarin antibiotic were subjected to the molecular docking studies as important tools for drug discovery. Glucosamine-6-phosphate synthase is selected as a suitable target in this study. In silico studies reveal that all synthesized compounds S1-S5 are good inhibitors of GlcN-6 and the docking results are in agreement with in vitro antibacterial evaluation of compounds S1-S5.

Novobiocin and peptide analogs of α-factor are positive allosteric modulators of the yeast G protein-coupled receptor Ste2p.

G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p-[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR.

DNA affinity capturing identifies new regulators of the heterologously expressed novobiocin gene cluster in Streptomyces coelicolor M512.

Understanding the regulation of a heterologously expressed gene cluster in a host organism is crucial for activation of silent gene clusters or overproduction of the corresponding natural product. In this study, Streptomyces coelicolor M512(nov-BG1) containing the novobiocin biosynthetic gene cluster from Streptomyces niveus NCIMB 11891 was chosen as a model. An improved DNA affinity capturing assay (DACA), combined with semi-quantitative mass spectrometry, was used to identify proteins binding to the promoter regions of the novobiocin gene cluster. Altogether, 2475 proteins were identified in DACA studies with the promoter regions of the pathway-specific regulators novE (PnovE) and novG (PnovG), of the biosynthetic genes novH-W (PnovH) and of the vegetative σ-factor hrdB (PhrdB) as a negative control. A restrictive classification for specific binding reduced this number to 17 proteins. Twelve of them were captured by PnovH, among them, NovG, two were captured by PnovE, and three by PnovG. Unexpectedly some well-known regulatory proteins, such as the global regulators NdgR, AdpA, SlbR, and WhiA were captured in similar intensities by all four tested promoter regions. Of the 17 promoter-specific proteins, three were studied in more detail by deletion mutagenesis and by overexpression. Two of them, BxlRSc and BxlR2Sc, could be identified as positive regulators of novobiocin production in S. coelicolor M512. Deletion of a third gene, sco0460, resulted in reduced novobiocin production, while overexpression had no effect. Furthermore, binding of BxlRSc to PnovH and to its own promoter region was confirmed via surface plasmon resonance spectroscopy.