Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

The small molecule genkwanine M induces single mode, mesenchymal tumor cell motility.

Individual tumor cells utilize one of two modes of motility to invade the extracellular matrix, mesenchymal or amoeboid. We have determined that the diterpenoid genkwanine M (GENK) enhances the mesenchymal mode of cell motility that is intrinsic to HT-1080 osteosarcoma cells, stimulates a mesenchymal mode of motility in stationary MDA-MB-453 breast carcinoma cells, and induces a shift to a mesenchymal mode of cell motility in LS174T colorectal adenocarcinoma cells that normally utilize the alternate amoeboid mode of motility. The ability of GENK to stimulate or induce mesenchymal motility was preceded by a rapid cell spreading, elongation and polarization that did not require new gene expression. However, these initial morphologic changes were integrin dependent and they were associated with a reorganization of focal contacts and focal adhesions as well as an activation of the focal adhesion kinase. Therefore, GENK induces a mesenchymal mode of cell motility in a wide variety of tumor cell types that may be mediated, at least in part, by an activation of integrin-associated signaling.

Structure-activity analysis of niclosamide reveals potential role for cytoplasmic pH in control of mammalian target of rapamycin complex 1 (mTORC1) signaling.

Mammalian target of rapamycin complex 1 (mTORC1) signaling is frequently dysregulated in cancer. Inhibition of mTORC1 is thus regarded as a promising strategy in the treatment of tumors with elevated mTORC1 activity. We have recently identified niclosamide (a Food and Drug Administration-approved antihelminthic drug) as an inhibitor of mTORC1 signaling. In the present study, we explored possible mechanisms by which niclosamide may inhibit mTORC1 signaling. We tested whether niclosamide interferes with signaling cascades upstream of mTORC1, the catalytic activity of mTOR, or mTORC1 assembly. We found that niclosamide does not impair PI3K/Akt signaling, nor does it inhibit mTORC1 kinase activity. We also found that niclosamide does not interfere with mTORC1 assembly. Previous studies in helminths suggest that niclosamide disrupts pH homeostasis of the parasite. This prompted us to investigate whether niclosamide affects the pH balance of cancer cells. Experiments in both breast cancer cells and cell-free systems demonstrated that niclosamide possesses protonophoric activity in cells and in vitro. In cells, niclosamide dissipated protons (down their concentration gradient) from lysosomes to the cytosol, effectively lowering cytoplasmic pH. Notably, analysis of five niclosamide analogs revealed that the structural features of niclosamide required for protonophoric activity are also essential for mTORC1 inhibition. Furthermore, lowering cytoplasmic pH by means other than niclosamide treatment (e.g. incubation with propionic acid or bicarbonate withdrawal) recapitulated the inhibitory effects of niclosamide on mTORC1 signaling, lending support to a possible role for cytoplasmic pH in the control of mTORC1. Our data illustrate a potential mechanism for chemical inhibition of mTORC1 signaling involving modulation of cytoplasmic pH.

Clionamines stimulate autophagy, inhibit Mycobacterium tuberculosis survival in macrophages, and target Pik1.

The pathogen Mycobacterium tuberculosis (Mtb) evades the innate immune system by interfering with autophagy and phagosomal maturation in macrophages, and, as a result, small molecule stimulation of autophagy represents a host-directed therapeutics (HDTs) approach for treatment of tuberculosis (TB). Here we show the marine natural product clionamines activate autophagy and inhibit Mtb survival in macrophages. A yeast chemical-genetics approach identified Pik1 as target protein of the clionamines. Biotinylated clionamine B pulled down Pik1 from yeast cell lysates and a clionamine analog inhibited phosphatidyl 4-phosphate (PI4P) production in yeast Golgi membranes. Chemical-genetic profiles of clionamines and cationic amphiphilic drugs (CADs) are closely related, linking the clionamine mode of action to co-localization with PI4P in a vesicular compartment. Small interfering RNA (siRNA) knockdown of PI4KB, a human homolog of Pik1, inhibited the survival of Mtb in macrophages, identifying PI4KB as an unexploited molecular target for efforts to develop HDT drugs for treatment of TB.

Identification of a Novel Oleic Acid Analog with Protective Effects in Multiple Cellular Models of Friedreich Ataxia.

Friedreich ataxia (FRDA) is an inherited neurodegenerative disorder for which there is no cure or approved treatment. It is characterized by the loss or impaired activity of frataxin protein, which is involved in the biogenesis of iron-sulfur clusters. Our previous studies suggested that cell death in FRDA may involve ferroptosis, an iron-dependent form of cell death requiring lipid peroxidation. Based on reports that oleic acid acts as a ferroptosis inhibitor, we evaluated whether it, other fatty acids, and fatty acid derivatives could rescue viability in cellular models of FRDA. We identified a trifluoromethyl alcohol analog of oleic acid that was significantly more potent than oleic acid itself. Further evaluation indicated that the effects were stereoselective, although a specific molecular target has not yet been identified. This work provides a potential starting point for therapeutics to treat FRDA, as well as a valuable probe molecule to interrogate FRDA pathophysiology.

An Effective Bifunctional Aldehyde Linchpin for Type II Anion Relay Chemistry: Development and Application to the Synthesis of a C16-C29 Fragment of Rhizopodin.

The design, synthesis, and validation of a new bifunctional aldehyde linchpin for Type II anion relay chemistry have been achieved. For this linchpin, the initial nucleophilic addition proceeds under Felkin-Anh control to generate the syn-alkoxide, which undergoes a 1,4-Brook rearrangement to relay the negative charge, thus leading to the formation of a dithiane-stabilized carbanion. Subsequent trapping with an electrophile furnishes a tricomponent adduct with an embedded propionate subunit, a ubiquitous structural motif found in polyketides. The utility of this new linchpin is demonstrated with the construction of a potential C16-C29 fragment for the synthesis of rhizopodin, an actin-binding macrolide.

Synthesis of clionamine B, an autophagy stimulating aminosteroid isolated from the sponge Cliona celata.

Clionamine B (2), an aminosteroid isolated from the marine sponge Cliona celata, has been synthesized starting from the plant sapogenin tigogenin (5). A key step in the synsthesis is the stereoselective introduction of the C-20 α-hydroxyl substituent via oxidation of a γ-lactone enolate with molecular oxygen. Synthetic clionamine B (2) strongly stimulated autophagy in human breast cancer MCF-7 cells.

A daphnane diterpenoid isolated from Wikstroemia polyantha induces an inflammatory response and modulates miRNA activity.

MicroRNAs (miRNAs) are endogenously expressed single-stranded ~21-23 nucleotide RNAs that inhibit gene expression post-transcriptionally by binding imperfectly to elements usually within the 3'untranslated region (3'UTR) of mRNAs. Small interfering RNAs (siRNAs) mediate site-specific cleavage by binding with perfect complementarity to RNA. Here, a cell-based miRNA reporter system was developed to screen for compounds from marine and plant extracts that inhibit miRNA or siRNA activity. The daphnane diterpenoid genkwanine M (GENK) isolated from the plant Wikstroemia polyantha induces an early inflammatory response and can moderately inhibit miR-122 activity in the liver Huh-7 cell line. GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells. In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity.

Alotaketals A and B, sesterterpenoids from the marine sponge Hamigera species that activate the cAMP cell signaling pathway.

The new sesterterpenoids alotaketals A (1) and B (2) have been isolated from extracts of the marine sponge Hamigera sp. collected in Papua New Guinea. Their chemical structures were elucidated by analysis of spectroscopic data. Alotaketals A and B have the unprecedented alotane carbon skeleton, and they activate the cAMP cell signaling pathway with EC(50)'s of 18 and 240 nM, respectively.