Novel autophagy inducers by accelerating lysosomal clustering against Parkinson's disease.

The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including α-synuclein (αSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble αSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.

Targeting the autophagy-NAD axis protects against cell death in Niemann-Pick type C1 disease models.

Impairment of autophagy leads to an accumulation of misfolded proteins and damaged organelles and has been implicated in plethora of human diseases. Loss of autophagy in actively respiring cells has also been shown to trigger metabolic collapse mediated by the depletion of nicotinamide adenine dinucleotide (NAD) pools, resulting in cell death. Here we found that the deficit in the autophagy-NAD axis underpins the loss of viability in cell models of a neurodegenerative lysosomal storage disorder, Niemann-Pick type C1 (NPC1) disease. Defective autophagic flux in NPC1 cells resulted in mitochondrial dysfunction due to impairment of mitophagy, leading to the depletion of both the reduced and oxidised forms of NAD as identified via metabolic profiling. Consequently, exhaustion of the NAD pools triggered mitochondrial depolarisation and apoptotic cell death. Our chemical screening identified two FDA-approved drugs, celecoxib and memantine, as autophagy activators which effectively restored autophagic flux, NAD levels, and cell viability of NPC1 cells. Of biomedical relevance, either pharmacological rescue of the autophagy deficiency or NAD precursor supplementation restored NAD levels and improved the viability of NPC1 patient fibroblasts and induced pluripotent stem cell (iPSC)-derived cortical neurons. Together, our findings identify the autophagy-NAD axis as a mechanism of cell death and a target for therapeutic interventions in NPC1 disease, with a potential relevance to other neurodegenerative disorders.

Invivo anti-cancer activity of 10-methyl-aplog-1, a simplified analog of aplysiatoxin, and its possible signaling pathway associated with G1 arrest.

Naturally occurring protein kinase C (PKC) activators such as phorbol esters, teleocidins, and aplysiatoxins, have the potential to become anti-cancer agents, since they are anti-proliferative against specific cancer cell lines in vitro. However, their potent tumor-promoting and proinflammatory activities have hampered their clinical uses. Recently, we developed 10-methyl-aplog-1 (1), a simplified analog of tumor-promoting debromoaplysiatoxin (DAT), which retained anti-proliferative activity comparable to DAT, but induced neither tumorigenesis nor inflammation on mouse skin. Our previous study suggested that PKCα and δ were involved in the cell line-selective anti-proliferative activity of 1, but the downstream signaling of PKC isozymes remained unknown. In this study, we confirmed that 1 inhibited the growth of three aplog-sensitive cancer cell lines (NCI-H460, HCC-2998, and HBC-4) without severe side effects in mice xenograft models. In addition, in vitro analysis using A549, one of the aplog-sensitive cell lines in vitro, revealed that PKCα induced PP2A-mediated attenuation of the Akt/S6 signaling axis. Since S6 inhibition in A549 was reported to result in G1 arrest, this pathway could be involved in the PKCα-dependent anti-proliferative activity of 1.

Cisplatin-induced activation of TGF-ß signaling contributes to drug resistance.

Growing evidence suggests an association between epithelial-mesenchymal transition (EMT), a hallmark of tumor malignancy, and chemoresistance to a number of anti-cancer drugs. However, the mechanism of EMT induction in the process of acquiring anti-cancer drug resistance remains unclear. To address this issue, we obtained a number of cisplatin-resistant clones from LoVo cells and found that almost all of them lost cell-cell contacts. In these clones, the epithelial marker E-cadherin was downregulated, whereas the mesenchymal marker N-cadherin was upregulated. Moreover, the expression of EMT-related transcription factors, including Slug, was elevated. On the other hand, the upregulation of other mesenchymal marker Vimentin was weak, suggesting that the mesenchymal-like phenotypic changes occurred in these cisplatin-resistant clones. These mesenchymal-like features of cisplatin-resistant clones were partially reversed to parental epithelial-like features by treatment with transforming growth factor-ß (TGF-ß) receptor kinase inhibitors, indicating that TGF-ß signaling is involved in cisplatin-induced the mesenchymal-like phenotypic changes. Moreover, cisplatin was observed to enhance the secretion of TGF-ß into the culture media without influencing TGF-ß gene transcription. These results suggest that cisplatin may induce the mesenchymal-like phenotypic changes by enhancing TGF-ß secretion, ultimately resulting in drug resistance.

Autophagy promotes cell survival by maintaining NAD levels.

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.

Oxidative stress-induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2.

Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson's disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4-TRPML1-ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca2+ fluxes tightly regulated this system. Increased vulnerability of JIP4 KO cells to acrolein indicated that lysosomal clustering and subsequent autophagy activation served as defence mechanisms against cytotoxicity of acrolein itself. Furthermore, the JIP4-TRPML1-ALG2 pathway was also activated by H2 O2 , indicating that this system acts as a broad mechanism of the oxidative stress response. Conversely, starvation-induced lysosomal retrograde transport involved both the TMEM55B-JIP4 and TRPML1-ALG2 pathways in the absence of the JIP4 phosphorylation. Therefore, the phosphorylation status of JIP4 acts as a switch that controls the signalling pathways of lysosoma l distribution depending on the type of autophagy-inducing signal.

Dihydromaniwamycin E, a Heat-Shock Metabolite from Thermotolerant Streptomyces sp. JA74, Exhibiting Antiviral Activity against Influenza and SARS-CoV-2 Viruses.

Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50 values of 25.7 and 63.2 µM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50 values (for infection of 293TA cells) of 19.7 and 9.7 µM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50 concentrations.

Identification of the polyether ionophore lenoremycin through a new screening strategy for targeting cancer stem cells.

Targeting and eradicating cancer stem cells (CSCs), also termed tumor-initiating cells, are promising strategies for preventing cancer progression and recurrence. To identify candidate compounds targeting CSCs, we established a new screening strategy with colorectal CSC spheres and non-CSC spheres in three-dimensional (3D) culture system. Through chemical screening using our system with in-house microbial metabolite library, we identified polyether cation ionophores that selectively inhibited CSC sphere formation, whereas CSC spheres were resistant to conventional anticancer agents. One of the hit compounds, the most selective and effective microbial metabolite lenoremycin, decreased CSC populations via inducing reactive oxygen species production. This study demonstrated that our newly established screening system is useful for discovering agents that selectively eliminate CSCs.

Isolation of Caldorazole, a Thiazole-Containing Polyketide with Selective Cytotoxicity under Glucose-Restricted Conditions.

Caldorazole (1) was isolated from the marine cyanobacterium Caldora sp. collected on Ishigaki Island, Okinawa, Japan. Its structure was determined to be a new polyketide that contained two thiazole rings and an O-methylenolpyruvamide moiety. Caldorazole (1) showed strong cytotoxicity toward tumor cells that had been seeded at a high density. Cell death induced by 1 in HeLa and A431 cells was also observed only in the presence of the glycolysis blocker 2-deoxy-d-glucose (2DG). Co-treatment with 1 and 2DG remarkably decreased ATP levels in these cells. Furthermore, 1 selectively inhibited complex I in the mitochondrial respiratory chain. Thus, 1 was demonstrated to exert cytotoxicity toward human tumor cells by blocking mitochondrial respiration.

Androgen receptor antagonists produced by Streptomyces overcome resistance to enzalutamide.

Prostate cancer (PC) is a leading cause of cancer-related death in men in Western countries. Androgen receptor (AR) signaling is a major driver of PC; therefore, androgen deprivation by medical and surgical castration is the standard treatment for patients with PC. However, over time, most patients will progress to metastatic castration-resistant PC. Enzalutamide is the only AR antagonist approved by the Food and Drug Administration for the treatment of metastatic castration-resistant PC. However, resistance to enzalutamide also develops in most patients with castration-resistant PC. Thus, there is an urgent need to develop new AR antagonists with new structures. For this purpose, we conducted both in silico and natural product screenings. From the in silico screening, we obtained T5853872 and more potent compound, STK765173. From the natural product screening, the novel compound arabilin was isolated from Streptomyces sp. MK756-CF1. Unlike STK765173, arabilin could overcome resistance to enzalutamide. Furthermore, we also extracted a novel compound, antarlide A, and its geometric isomers from Streptomyces sp. BB47. Antarlides A-F have novel 22-membered-ring macrocyclic structures, while antarlides G and H have 20-membered-ring structures. Both antarlides B and G showed potent AR antagonist activity in prostate cancer cells and could overcome resistance to enzalutamide.

Involvement of miR-3180-3p and miR-4632-5p in palmitic acid-induced insulin resistance.

Insulin resistance is defined as a failure to trigger the activation of the PI3K-AKT pathway by normal levels of insulin; therefore, it is well linked to metabolic disorders. Although multiple mechanisms contribute to insulin resistance, one major cause is elevated concentrations of plasma free fatty acids, which are known to suppress insulin signaling. However, the underlying mechanism is still elusive. Here, we found that palmitic acid increased the expression of two miRNAs, miR-3180-3p and miR-4632-5p, in HepG2 cells. Transfection of HepG2 cells with miR-3180-3p or miR-4632-5p reduced insulin-induced activation of the PI3K-AKT pathway. Moreover, palmitic acid or two miRNAs inhibited insulin-induced phosphorylation of Tyr612 on IRS-1 without affecting insulin receptor activation. Therefore, two miRNAs are suggested to be involved in palmitic acid-induced insulin resistance through suppression of insulin-induced IRS-1 phosphorylation. Identification of miR-3180-3p and miR-4632-5p targets could provide valuable information for the development of therapeutic drugs for type 2 diabetes.

Corrigendum to "Interaction between goniothalamin and peroxisomal multifunctional enzyme type 2 triggering endoplasmic reticulum stress" [Heliyon 6 (10) (October 2020) e05200].

[This corrects the article DOI: 10.1016/j.heliyon.2020.e05200.].

Unified synthesis and assessment of tumor cell migration inhibitory activity of optically active UTKO1, originally designed moverastin analog.

UTKO1 is a synthetic analog of a natural tumor cell migration inhibitor, moverastin, isolated from microbial extracts of Aspergillus sp. 7720. UTKO1 was initially developed as a mixture of the stereoisomers. In this study, a concise and unified synthesis of the 4 optically active stereoisomers of UTKO1 was achieved from a known optically pure dihydro-α-ionone through a 5-step sequence. The key transformation in the synthesis was a Nozaki-Hiyama-Kishi (NHK) reaction between an optically active enoltriflate and a known aldehyde to install the chiral allylic hydroxy group at C2'. Simple chromatographic separation of the 2 diastereomers with regard to the allylic hydroxy group was possible by the derivatization into the corresponding acetals with Nemoto's optical resolution reagent, (S)- or (R)-5-allyl-2-oxabicyclo[3.3.0]octene (ALBO). All 4 synthetic stereoisomers of UTKO1 exhibited comparable tumor cell migration inhibitory activity.

Cyclopeptides from the Mushroom Pathogen Fungus Cladobotryum varium.

Three new cyclopeptides with serial Phe residues were identified with the aid of HPLC-DAD analysis, from the culture broth of Cladobotryum varium, a fungal pathogen causing mushroom cobweb disease. Cladoamides A (1) and B (2) have two consecutive N-methylphenylalanine units in the destruxin class cyclic depsipentapeptide framework, while cladoamide C (3) has a three consecutive Phe motif in a cyclopentapeptide structure. Of these three cyclopeptides, 1 showed potent autophagy-inducing activity at 10 µg/mL, comparable to a positive control, rapamycin. For the determination of the absolute configurations of the Ile residues in 1 and 3, new conditions for separating Ile and allo-Ile, using a pentafluorophenyl-bonded solid phase and methanolic solvent, were established within the analytical scheme of the advanced Marfey's method, thus offering a convenient alternative to the C3 Marfey's method, which requires elution with a three-solvent mixture. The sequence of two d-Phe and one l-Phe in 3 was determined through NMR chemical shift prediction by DFT-based calculations and chemical synthesis, which demonstrated the significance of noncovalent interactions in the accurate calculation of stable conformers for peptides with multiple aromatic rings.

A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers.

Macroautophagy/autophagy plays a critical role in the pathogenesis of various human diseases including neurodegenerative disorders such as Parkinson disease (PD) and Huntington disease (HD). Chemical autophagy inducers are expected to serve as disease-modifying agents by eliminating cytotoxic/damaged proteins. Although many autophagy inducers have been identified, their precise molecular mechanisms are not fully understood because of the complicated crosstalk among signaling pathways. To address this issue, we performed several chemical genomic analyses enabling us to comprehend the dominancy among the autophagy-associated pathways followed by an aggresome-clearance assay. In a first step, more than 400 target-established small molecules were assessed for their ability to activate autophagic flux in neuronal PC12D cells, and we identified 39 compounds as autophagy inducers. We then profiled the autophagy inducers by testing their effect on the induction of autophagy by 200 well-established signal transduction modulators. Our principal component analysis (PCA) and clustering analysis using a dataset of "autophagy profiles" revealed that two Food and Drug Administration (FDA)-approved drugs, memantine and clemastine, activate endoplasmic reticulum (ER) stress responses, which could lead to autophagy induction. We also confirmed that SMK-17, a recently identified autophagy inducer, induced autophagy via the PRKC/PKC-TFEB pathway, as had been predicted from PCA. Finally, we showed that almost all of the autophagy inducers tested in this present work significantly enhanced the clearance of the protein aggregates observed in cellular models of PD and HD. These results, with the combined approach, suggested that autophagy-activating small molecules may improve proteinopathies by eliminating nonfunctional protein aggregates.Abbreviations: ADK: adenosine kinase; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; BECN1: beclin-1; DDIT3/CHOP: DNA damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FDA: Food and Drug Administration; GSH: glutathione; HD: Huntington disease; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; JAK: Janus kinase, MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAP3K8/Tpl2: mitogen-activated protein kinase kinase kinase 8; MAPK: mitogen-activated protein kinase; MPP+: 1-methyl-4-phenylpyridinium; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; NAC: N-acetylcysteine; NGF: nerve growth factor 2; NMDA: N-methyl-D-aspartate; PCA: principal component analysis; PD: Parkinson disease; PDA: pancreatic ductal adenocarcinoma; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PMA: phorbol 12-myristate 13-acetate; PRKC/PKC: protein kinase C; ROCK: Rho-associated coiled-coil protein kinase; RR: ribonucleotide reductase; SIGMAR1: sigma non-opioid intracellular receptor 1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: Transcription factor EB; TGFB/TGF-ß: Transforming growth factor beta; ULK1: unc-51 like autophagy activating kinase 1; XBP1: X-box binding protein 1.

Interaction between goniothalamin and peroxisomal multifunctional enzyme type 2 triggering endoplasmic reticulum stress.

Endoplasmic reticulum stress is one of the pathways involved in cell cytotoxicity. In this study, goniothalamin, one of styryllactone compounds found in plant Goniothalamus spp., was observed to trigger ER stress in HeLa cell line. In addition, we demonstrated that peroxisomal multifunctional enzyme type2 (MFE2) was a specific goniothalamin-binding protein using an in vitro goniothalamin-linked bead pull-down assay. Since MFE2 has been reported to be an important mediator enzyme for peroxisomal ß-oxidation of a very long chain fatty acid metabolism, therefore computational molecular docking analysis was performed to confirm the binding of goniothalamin and MFE2. The results indicated that goniothalamin structure binds to scp-2 domain, enoyl-CoA hydratase 2 domain and (3R)-hydroxyacyl-CoA dehydrogenase domain of MFE2. To further determine the effect of MFE2 on ER stress induction, MFE2 knockdown by siRNA in HeLa cell was conducted. The results implied that MFE2 triggered CHOP, a key mediator of ER stress-induced apoptosis, expression. Therefore, these data inferred that goniothalamin may interrupt the MFE2 function resulting in lipid metabolism perturbation associated with ER stress-independent activation of unfolded protein response. This is the first report to show that goniothalamin binds directly to MFE2 triggering ER stress activation probably through the lipid metabolism perturbation.

Miclxin, a Novel MIC60 Inhibitor, Induces Apoptosis via Mitochondrial Stress in ß-Catenin Mutant Tumor Cells.

The Wnt signaling pathway regulates diverse cellular processes. ß-Catenin is one of the major components of this pathway, in which it plays a main role. Although it has been established that ß-catenin is mutated in a wide variety of tumors, there are currently no effective therapeutic agents that target ß-catenin. In this study, we searched for the compound that targets mutant ß-catenin and found DS37262926 (miclxin). Miclxin exhibited ß-catenin-dependent apoptosis in ß-catenin-mutated HCT116 cells and isogenic HCT116 (CTNNB1 Δ45/-) cells; however, this effect was not observed in isogenic HCT116 (CTNNB1 +/-) cells. Using miclxin-immobilized beads, MIC60, one of the major components of the mitochondrial contact site and cristae organizing system (MICOS) complex, was identified as a target protein of miclxin. We revealed that MIC60 dysfunction caused by miclxin induced a mitochondrial stress response in a mutant ß-catenin-dependent manner. Activation of the mitochondrial stress response was responsible for the downregulation of Bcl-2, leading to severe loss of mitochondrial membrane potential and subsequent apoptosis-inducing factor-dependent apoptosis. Our findings suggest that targeting MIC60 is a potential strategy with which tumor cells can be killed through induction of severe mitochondrial damage in a mutant ß-catenin-dependent manner.

BRUP-1, an intracellular bilirubin modulator, exerts neuroprotective activity in a cellular Parkinson's disease model.

Bilirubin, the end product of heme redox metabolism, has cytoprotective properties and is an essential metabolite associated with cardiovascular disease, inflammatory bowel disease, type 2 diabetes, and neurodegenerative diseases including Parkinson's disease (PD). PD is characterized by progressive degeneration of nigral dopaminergic neurons and is associated with elevated oxidative stress due to mitochondrial dysfunction. In this study, using a ratiometric bilirubin probe, we revealed that the mitochondrial inhibitor, rotenone, which is widely used to create a PD model, significantly decreased intracellular bilirubin levels in HepG2 cells. Chemical screening showed that BRUP-1 was a top hit that restored cellular bilirubin levels that were lowered by rotenone. We found that BRUP-1 up-regulated the expression level of heme oxygenase-1 (HO-1), one of the rate-limiting enzyme of bilirubin production via nuclear factor erythroid 2-related factor 2 (Nrf2) activation. In addition, we demonstrated that this Nrf2 activation was due to a direct inhibition of the interaction between Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) by BRUP-1. Both HO-1 up-regulation and bilirubin restoration by BRUP-1 treatment were significantly abrogated by Nrf2 silencing. In neuronal PC12D cells, BRUP-1 also activated the Nrf2-HO-1 axis and increased bilirubin production, resulted in the suppression of neurotoxin-induced cell death, reactive oxygen species production, and protein aggregation, which are hallmarks of PD. Furthermore, BRUP-1 showed neuroprotective activity against rotenone-treated neurons derived from induced pluripotent stem cells. These findings provide a new member of Keap1-Nrf2 direct inhibitors and suggest that chemical modulation of heme metabolism using BRUP-1 may be beneficial for PD treatment.

Discovery of "heat shock metabolites" produced by thermotolerant actinomycetes in high-temperature culture.

In actinomycetes, many secondary metabolite biosynthetic genes are not expressed under typical laboratory culture conditions and various efforts have been made to activate these dormant genes. In this study, we focused on high-temperature culture. First, we examined the thermotolerance of 3160 actinomycete strains from our laboratory culture collection and selected 57 thermotolerant actinomycetes that grew well at 45 °C. These 57 thermotolerant actinomycetes were cultured for 5 days in liquid medium at both 30 °C and 45 °C. Culture broths were extracted with 1-butanol, and each extract was subjected to LC/MS analysis. The metabolic profiles of each strain were compared between the 30 °C and 45 °C cultures. We found that almost half of these thermotolerant actinomycetes produced secondary metabolites that were detected only in the 45 °C culture. This result suggests that high-temperature culture induces the production of dormant secondary metabolites. These compounds were named "heat shock metabolites (HSMs)." To examine HSM production in more detail, 18 strains were selected at random from the initial 57 strains and cultivated in six different media at 30 °C and 45 °C; as before, metabolic profiles of each strain in each medium were compared between the 30 °C and 45 °C cultures. From this analysis, we found a total of 131 HSMs. We identified several angucycline-related compounds as HSMs from two thermotolerant Streptomyces species. Furthermore, we discovered a new compound, murecholamide, as an HSM from thermotolerant Streptomyces sp. AY2. We propose that high-temperature culture of actinomycetes is a convenient method for activating dormant secondary metabolite biosynthetic genes.