Salicylanilides and Their Anticancer Properties.

Salicylanilides are pharmacologically active compounds with a wide spectrum of biological effects. Halogenated salicylanilides, which have been used for decades in human and veterinary medicine as anthelmintics, have recently emerged as candidates for drug repurposing in oncology. The most prominent example of salicylanilide anthelmintic, that is intensively studied for its potential anticancer properties, is niclosamide. Nevertheless, recent studies have discovered extensive anticancer potential in a number of other salicylanilides. This potential of their anticancer action is mediated most likely by diverse mechanisms of action such as uncoupling of oxidative phosphorylation, inhibition of protein tyrosine kinase epidermal growth factor receptor, modulation of different signaling pathways as Wnt/ß-catenin, mTORC1, STAT3, NF-κB and Notch signaling pathways or induction of B-Raf V600E inhibition. Here we provide a comprehensive overview of the current knowledge about the proposed mechanisms of action of anticancer activity of salicylanilides based on preclinical in vitro and in vivo studies, or structural requirements for such an activity.

Design and synthesis of novel halogen rich salicylanilides as potential antileishmanial agents.

The available therapeutic treatment for leishmaniasis is inadequate and toxic due to side effects, expensive and emergence of drug resistance. Affordable and safe antileishmanial agents are urgently needed and toward this objective, we synthesized a series of 32 novel halogen rich salicylanilides including niclosamide and oxyclozanide and investigated their antileishmanial activity against amastigotes of Leishmania donovani. In vitro data showed fifteen compounds inhibited intracellular amastigotes with an IC50 of below 5 µM and selectivity index above 10. Among 15 active compounds, 14 and 24 demonstrated better activity with an IC50 of 2.89 µM and 2.09 µM respectively and selectivity index is 18. Compound 24 exhibited significant in vivo antileishmanial efficacy and reduced 65% of the splenic parasite load on day 28th post-treatment in the experimental visceral leishmaniasis golden hamster model. The data suggest that 24 can be a promising lead candidate possessing potential to be developed into a leishmanial drug candidate.

Study of Biological Activities and ADMET-Related Properties of Salicylanilide-Based Peptidomimetics.

A series of eleven benzylated intermediates and eleven target compounds derived from salicylanilide were tested against Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 as reference strains and against three clinical isolates of methicillin-resistant S. aureus (MRSA) and three isolates of vancomycin-resistant E. faecalis. In addition, the compounds were evaluated against Mycobacterium tuberculosis H37Ra and M. smegmatis ATCC 700084. The in vitro cytotoxicity of the compounds was assessed using the human monocytic leukemia cell line THP-1. The lipophilicity of the prepared compounds was experimentally determined and correlated with biological activity. The benzylated intermediates were found to be completely biologically inactive. Of the final eleven compounds, according to the number of amide groups in the molecule, eight are diamides, and three are triamides that were inactive. 5-Chloro-2-hydroxy-N-[(2S)- 4-(methylsulfanyl)-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (3e) and 5-chloro-2-hydroxy-N-[(2S)-(4-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino)pentan-2-yl)benzamide (3f) showed the broadest spectrum of activity against all tested species/isolates comparable to the used standards (ampicillin and isoniazid). Six diamides showed high antistaphylococcal activity with MICs ranging from 0.070 to 8.95 µM. Three diamides showed anti-enterococcal activity with MICs ranging from 4.66 to 35.8 µM, and the activities of 3f and 3e against M. tuberculosis and M. smegmatis were MICs of 18.7 and 35.8 µM, respectively. All the active compounds were microbicidal. It was observed that the connecting linker between the chlorsalicylic and 4-CF3-anilide cores must be substituted with a bulky and/or lipophilic chain such as isopropyl, isobutyl, or thiabutyl chain. Anticancer activity on THP-1 cells IC50 ranged from 1.4 to >10 µM and increased with increasing lipophilicity.

α-Crystallin chaperone mimetic drugs inhibit lens γ-crystallin aggregation: Potential role for cataract prevention.

Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket-binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50-150) of HγD and one spanning the "NC tail" (residues 56-61 to 168-174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins.

Investigation of Salicylanilides as Botulinum Toxin Antagonists.

Botulinum neurotoxin serotype A (BoNT/A) is recognized by the Centers for Disease Control and Prevention (CDC) as the most potent toxin and as a Tier 1 biowarfare agent. The severity and longevity of botulism stemming from BoNT/A is of significant therapeutic concern, and early administration of antitoxin-antibody therapy is the only approved pharmaceutical treatment for botulism. Small molecule therapeutic strategies have targeted both the heavy chain (HC) and the light chain (LC) catalytic active site and α-/ß-exosites. The LC translocation mechanism has also been studied, but an effective, nontoxic inhibitor remains underexplored. In this work, we screened a library of salicylanilides as potential translocation inhibitors. Potential leads following a primary screen were further scrutinized to identify sal30, which has a cellular minimal concentration of a drug that is required for 50% inhibition (IC50) value of 141 nM. The inquiry of salicylanilide sal30's mechanism of action was explored through a self-quenched fluorogenic substrate conjugated to bovine serum albumin (DQ-BSA) fluorescence, confocal microscopy, and vacuolar H+-ATPase (V-ATPase) inhibition assays. The summation of these findings imply that endolysosomal proton translocation through the protonophore mechanism of sal30 causes endosome pH to increase, which in turn prevents LC translocation into cytosol, a process that requires an acidic pH. Thus, the inhibition of BoNT/A activity by salicylanilides likely occurs through disruption of pH-dependent endosomal LC translocation. We further probed BoNT inhibition by sal30 using additivity analysis studies with bafilomycin A1, a known BoNT/A LC translocation inhibitor, which indicated the absence of synergy between the two ionophores.

NSC828779 Alleviates Renal Tubulointerstitial Lesions Involving Interleukin-36 Signaling in Mice.

Renal tubulointerstitial lesions (TILs), a common pathologic hallmark of chronic kidney disease that evolves to end-stage renal disease, is characterized by progressive inflammation and pronounced fibrosis of the kidney. However, current therapeutic approaches to treat these lesions remain largely ineffectual. Previously, we demonstrated that elevated IL-36α levels in human renal tissue and urine are implicated in impaired renal function, and IL-36 signaling enhances activation of NLRP3 inflammasome in a mouse model of TILs. Recently, we synthesized NSC828779, a salicylanilide derivative (protected by U.S. patents with US 8975255 B2 and US 9162993 B2), which inhibits activation of NF-κB signaling with high immunomodulatory potency and low IC50, and we hypothesized that it would be a potential drug candidate for renal TILs. The current study validated the therapeutic effects of NSC828779 on TILs using a mouse model of unilateral ureteral obstruction (UUO) and relevant cell models, including renal tubular epithelial cells under mechanically induced constant pressure. Treatment with NSC828779 improved renal lesions, as demonstrated by dramatically reduced severity of renal inflammation and fibrosis and decreased urinary cytokine levels in UUO mice. This small molecule specifically inhibits the IL-36α/NLRP3 inflammasome pathway. Based on these results, the beneficial outcome represents synergistic suppression of both the IL-36α-activated MAPK/NLRP3 inflammasome and STAT3- and Smad2/3-dependent fibrogenic signaling. NSC828779 appears justified as a new drug candidate to treat renal progressive inflammation and fibrosis.

Structural Development of Salicylanilide-Based SPAK Inhibitors as Candidate Antihypertensive Agents.

Hypertension is an important target for drug discovery. We have focused on the with-no-lysine kinase (WNK)-oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)-NaCl cotransporter (NCC) signal cascade as a potential target, and we previously developed a screening system for inhibitors of WNK-OSR1/SPAK-NCC signaling. Herein we used this system to examine the structure-activity relationship (SAR) of salicylanilide derivatives as SPAK kinase inhibitors. Structural design and development based on our previous hit compound, aryloxybenzanilide derivative 2, and the veterinary anthelmintic closantel (3) led to the discovery of compound 10 a as a potent SPAK inhibitor with reduced toxicity. Compound 10 a decreased the phosphorylation level of NCC in mouse kidney in vivo, and appears to be a promising lead compound for a new class of antihypertensive drugs.

Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives.

The SARS-CoV-2 pandemic has triggered global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro), critical for viral replication, is a key target for therapeutic development. An organoselenium drug called ebselen has been demonstrated to have potent Mpro inhibition and antiviral activity. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of derivatives. A free selenium atom bound with cysteine of catalytic dyad has been revealed in crystallographic structures of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct and formation of a phenolic by-product, confirmed by mass spectrometry. The target engagement with selenation mechanism of inhibition suggests wider therapeutic applications of these compounds against SARS-CoV-2 and other zoonotic beta-corona viruses.

Exploiting the HSP60/10 chaperonin system as a chemotherapeutic target for colorectal cancer.

Over the past few decades, an increasing variety of molecular chaperones have been investigated for their role in tumorigenesis and as potential chemotherapeutic targets; however, the 60 kDa Heat Shock Protein (HSP60), along with its HSP10 co-chaperone, have received little attention in this regard. In the present study, we investigated two series of our previously developed inhibitors of the bacterial homolog of HSP60/10, called GroEL/ES, for their selective cytotoxicity to cancerous over non-cancerous colorectal cells. We further developed a third "hybrid" series of analogs to identify new candidates with superior properties than the two parent scaffolds. Using a series of well-established HSP60/10 biochemical screens and cell-viability assays, we identified 24 inhibitors (14%) that exhibited > 3-fold selectivity for targeting colorectal cancer over non-cancerous cells. Notably, cell viability EC50 results correlated with the relative expression of HSP60 in the mitochondria, suggesting a potential for this HSP60-targeting chemotherapeutic strategy as emerging evidence indicates that HSP60 is up-regulated in colorectal cancer tumors. Further examination of five lead candidates indicated their ability to inhibit the clonogenicity and migration of colorectal cancer cells. These promising results are the most thorough analysis and first reported instance of HSP60/10 inhibitors being able to selectively target colorectal cancer cells and highlight the potential of the HSP60/10 chaperonin system as a viable chemotherapeutic target.

Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections.

One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies.IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.

Osalmid, a Novel Identified RRM2 Inhibitor, Enhances Radiosensitivity of Esophageal Cancer.

PURPOSE: Esophageal cancer (EC) is an aggressive malignancy and is often resistant to currently available therapies. Inhibition of ribonucleotide reductase small subunit M2 (RRM2) in tumors is speculated to mediate chemosensitization. Previous studies have reported that Osalmid could act as an RRM2 inhibitor. We explored whether RRM2 was involved in radioresistance and the antitumor effects of Osalmid in EC. METHODS AND MATERIALS: RRM2 expression was detected by immunohistochemistry in EC tissues. The effects of Osalmid on cell proliferation, apoptosis, and cell cycle were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphhenyl tetrazolium, colony formation, and flow cytometry assays. DNA damage, cell apoptosis, and senescence induced by Osalmid or ionizing radiation (IR) alone, or both, were detected with immunofluorescence, flow cytometry, Western blot, and ß-galactosidase staining. A xenograft mouse model of EC was used to investigate the potential synergistic effects of Osalmid and IR in vivo. RESULTS: The expression of RRM2 in treatment-resistant EC tissues is much higher than in treatment-sensitive EC, and strong staining of RRM2 was correlated with shorter overall survival. We observed direct cytotoxicity of Osalmid in EC cells. Osalmid also produced inhibition of the ERK1/2 signal transduction pathway and substantially enhanced IR-induced DNA damage, apoptosis, and senescence. Furthermore, treatment with Osalmid and IR significantly suppressed tumor growth in xenograft EC models without additional toxicity to the hematologic system and internal organs. CONCLUSIONS: Our study revealed that RRM2 played a vital role in radioresistance in EC, and Osalmid synergized with IR to exert its antitumor effects both in vitro and in vivo.

Identification of osalmid metabolic profile and active metabolites with anti-tumor activity in human hepatocellular carcinoma cells.

BACKGROUNDS: Ribonucleotide reductase (RR) catalyzes the essential step in the formation of all four deoxynucleotides. Upregulated activity of RR plays an active role in tumor progression. As the regulatory subunit of RR, ribonucleotide reductase subunit M2 (RRM2) is regarded as one of the effective therapeutic targets for DNA replication-dependent diseases, such as cancers. Recent studies have revealed that osalmid significantly inhibits the activity of RRM2, but the metabolic profile of osalmid remains unknown. OBJECTIVE: The aim of this study was to clarify the metabolic profile including metabolites, isoenzymes and metabolic pathways of osalmid. The anti-human hepatocellular carcinoma activity and mechanism of metabolites were further investigated. MATERIALS AND METHODS: Ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was used for identifying metabolites and for characterizing phase I and phase II metabolic pathways with recombinant enzymes or in human liver microsomes of osalmid. The eHiTS docking system was used for potential RRM2 inhibitor screening among metabolites. Cytotoxicity assays were performed for evaluating cell proliferation inhibitory activity of metabolites. Cell cycle assays and cell apoptosis assays were assessed by flow cytometry. Western blotting analysis of RRM2, cyclin D1, p21, p53, phosphorylated p53, Bcl-2 and Bax was performed to explore the anti-hepatocellular carcinoma mechanism of the active metabolites. RESULTS: Ten metabolites of osalmid were identified, and none of them have been reported previously. Hydroxylation, glucuronidation, sulfonation, acetylation and degradation were recognized as the main metabolic processes of osalmid. Isozymes of CYP1A2, CYP2C9, UGT1A1, UGT1A6, UGT1A9, UGT2B7 and UGT2B15 were involved in phase I and phase II metabolism of osalmid. Metabolites M7, M8 and M10 showed higher binding affinities with the RRM2 active site than osalmid. Metabolite M7 exhibited potent inhibitory activity to hepatocellular carcinoma cell lines by both competitive inhibition and down-regulation of RRM2. Moreover, M7 significantly induced cell cycle arrest and apoptosis by activating p53-related pathways. CONCLUSIONS: The metabolic profile of osalmid was identified. M7 significantly inhibited human hepatocellular carcinoma progression by inhibiting RRM2 activity. Furthermore, M7 induced cell cycle arrest and apoptosis by activating p53-related signaling pathways.

Repositioning of Anthelmintic Drugs for the Treatment of Cancers of the Digestive System.

Tumors of the digestive system, when combined together, account for more new cases and deaths per year than tumors arising in any other system of the body and their incidence continues to increase. Despite major efforts aimed at discovering and validating novel and effective drugs against these malignancies, the process of developing such drugs remains lengthy and costly, with high attrition rates. Drug repositioning (also known as drug repurposing), that is, the process of finding new uses for approved drugs, has been gaining popularity in oncological drug development as it provides the opportunity to expedite promising anti-cancer agents into clinical trials. Among the drugs considered for repurposing in oncology, compounds belonging to some classes of anthelmintics-a group of agents acting against infections caused by parasitic worms (helminths) that colonize the mammalian intestine-have shown pronounced anti-tumor activities and attracted particular attention due to their ability to target key oncogenic signal transduction pathways. In this review, we summarize and discuss the available experimental and clinical evidence about the use of anthelmintic drugs for the treatment of cancers of the digestive system.

Metabolic profiles of Oncomelania hupensis after molluscicidal treatment: Carbohydrate metabolism targeted and energy deficiency.

Oncomelania hupensis is the intermediate host of Schistosoma japonicum, one of the Schistosoma species that can cause human schistosomiasis. Molluscicidal treatment remains the primary means to control snail. Niclosamide is the only molluscicide recommended by the World Health Organization, and it has been used throughout schistosomiasis-endemic areas in China for almost 30 years. In our previous studies on transcriptomics, morphology, and enzymology of snails after molluscicidal treatment, two effective molluscicides were used, 50% wettable powder of niclosamide ethanolamine salt (WPN) and a new molluscicide derived from niclosamide, the salt of quinoid-2', 5-dichloro-4'-nitro-salicylanilide (LDS, simplified for Liu Dai Shui Yang An). Genes involved in cell structure mintenance, inhibition of neurohumoral transmission, and energy metabolism showed significant differential expression after molluscicide treatments. Damages in the structure of liver and muscle cells were accompanied by inhibited activities of enzymes related to carbohydrate metabolism and energy supply. This study was designed to clarify the dynamic metabolic process by metabonomics, together with the previous transcriptomic and enzymological profiles, to identify potential metabolite markers and metabolism pathways that related to the toxic mechanism of the molluscicide. In total, 56 metabolites were identified for O. hupensis, and 75% of these metabolites consisted of amino acids and derivatives, organic acids, and nucleic acid components. The concentration of glucose, maltose, succinate, choline, and alanine changed significantly after molluscicide treatments. These changes in metabolites mainly occurred in the process of carbohydrate metabolism, energy metabolism, and amino acid metabolism, primarily related to glycolysis/gluconeogenesis, oxidative phosphorylation, and transamination by KEGG pathway identification. Most of the identified pathways were also related to those differentially expressed unigenes and observed enzymes from our previous studies. Inhibited aerobic respiration and oxidative phosphorylation, and energy deficiency were implied further to be the leading causes of the final death of snails after molluscicide treatments. The hypothesised mathematical model in this study identified the rational hysteresis to explain the inconsistency of responses of unigenes, enzymes, and metabolites to molluscicide treatments. This study contributes to the comprehensive understanding of the molluscicidal mechanism in the metabolic process and this could assist in improving existing molluscicide formulations or development of new molluscicides.

Salicylanilide Analog Minimizes Relapse of Clostridioides difficile Infection in Mice.

Clostridioides difficile infection (CDI) causes serious and sometimes fatal symptoms like diarrhea and pseudomembranous colitis. Although antibiotics for CDI exist, they are either expensive or cause recurrence of the infection due to their altering the colonic microbiota, which is necessary to suppress the infection. Here, we leverage a class of known membrane-targeting compounds that we previously showed to have broad inhibitory activity across multiple Clostridioides difficile strains while preserving the microbiome to develop an efficacious agent. A new series of salicylanilides was synthesized, and the most potent analog was selected through an in vitro inhibitory assay to evaluate its pharmacokinetic parameters and potency in a CDI mouse model. The results revealed reduced recurrence of CDI and diminished disturbance of the microbiota in mice compared to standard-of-care vancomycin, thus paving the way for novel therapy that can potentially target the cell membrane of C. difficile to minimize relapse in the recovering patient.

Ring-Substituted 1-Hydroxynaphthalene-2-Carboxanilides Inhibit Proliferation and Trigger Mitochondria-Mediated Apoptosis.

Ring-substituted 1-hydroxynaphthalene-2-carboxanilides were previously investigated for their antimycobacterial properties. In our study, we have shown their antiproliferative and cell death-inducing effects in cancer cell lines. Cell proliferation and viability were assessed by WST-1 assay and a dye exclusion test, respectively. Cell cycle distribution, phosphatidylserine externalization, levels of reactive oxygen or nitrogen species (RONS), mitochondrial membrane depolarization, and release of cytochrome c were estimated by flow cytometry. Levels of regulatory proteins were determined by Western blotting. Our data suggest that the ability to inhibit the proliferation of THP-1 or MCF-7 cells might be referred to meta- or para-substituted derivatives with electron-withdrawing groups -F, -Br, or -CF3 at anilide moiety. This effect was accompanied by accumulation of cells in G1 phase. Compound 10 also induced apoptosis in THP-1 cells in association with a loss of mitochondrial membrane potential and production of mitochondrial superoxide. Our study provides a new insight into the action of salicylanilide derivatives, hydroxynaphthalene carboxamides, in cancer cells. Thus, their structure merits further investigation as a model moiety of new small-molecule compounds with potential anticancer properties.

Discovery and Structure Relationships of Salicylanilide Derivatives as Potent, Non-acidic P2X1 Receptor Antagonists.

Antagonists for the ATP-gated ion channel receptor P2X1 have potential as antithrombotics and for treating hyperactive bladder and inflammation. In this study, salicylanilide derivatives were synthesized based on a screening hit. P2X1 antagonistic potency was assessed in 1321N1 astrocytoma cells stably transfected with the human P2X1 receptor by measuring inhibition of the ATP-induced calcium influx. Structure-activity relationships were analyzed, and selectivity versus other P2X receptor subtypes was assessed. The most potent compounds, N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (1, IC50 0.0192 µM) and N-[3,5-bis(trifluoromethyl)phenyl]-4-chloro-2-hydroxybenzamide (14, IC50 0.0231 µM), displayed >500-fold selectivity versus P2X2 and P2X3, and 10-fold selectivity versus P2X4 and P2X7 receptors, and inhibited collagen-induced platelet aggregation. They behaved as negative allosteric modulators, and molecular modeling studies suggested an extracellular binding site. Besides selective P2X1 antagonists, compounds with ancillary P2X4 and/or P2X7 receptor inhibition were discovered. These compounds represent the first potent, non-acidic, allosteric P2X1 receptor antagonists reported to date.

Novel cyclophosphamide of natural products osalmide and pterostilbene induces cytotoxicity and cell cycle arrest in diffuse large B-cell lymphoma cells.

Diffuse large B-cell lymphoma (DLBCL) is the most common category and disease entity of non-Hodgkin lymphoma. Osalmide and pterostilbene are natural products with anticancer activities via different mechanism. In this study, using a new synthetic strategy for the two natural products, we obtained the compound DCZ0801, which was previously found to have anti-multiple myeloma activity. We performed both in vitro and in vivo assays to investigate its bioactivity and explore its underlying mechanism against DLBCL cells. The results showed that DCZ0801 treatment gave rise to a dose- and time-dependent inhibition of cell viability as determined by CCK-8 assay and flow cytometry assay. Western blot analysis results showed that the expression of caspase-3, caspase-8, caspase-9 and Bax was increased, while BCL-2 and BCL-XL levels were decreased, which suggested that DCZ0801 inhibited cell proliferation and promoted intrinsic apoptosis. In addition, DCZ0801 induced G0/G1 phase arrest by downregulating the protein expression levels of CDK4, CDK6 and cyclin D1. Furthermore, DCZ0801 exerted an anti-tumor effect by down-regulating the expressions of p-PI3K and p-AKT. There also existed a trend that the expression of p-JNK and p-P38 was restrained. Intraperitoneal injection of DCZ0801 suppressed tumor development in xenograft mouse models. The preliminary metabolic study showed that DCZ0801 displayed a rapid metabolism within 30 min. These results demonstrated that DCZ0801 may be a new potential anti-DLBCL agent in DLBCL therapy.

Screening for potent and selective anticlostridial leads among FDA-approved drugs.

Clostridium difficile is a leading cause of morbidity and mortality particularly in hospital settings. In addition, treatment is very challenging due to the scarcity of effective therapeutic options. Thus, there remains an unmet need to identify new therapeutic agents capable of treating C. difficile infections. In the current study, we screened two FDA-approved drug libraries against C. difficile. Out of almost 3200 drugs screened, 50 drugs were capable of inhibiting the growth of C. difficile. Remarkably, some of the potent inhibitors have never been reported before and showed activity in a clinically achievable range. Structure-activity relationship analysis of the active hits clustered the potent inhibitors into four chemical groups; nitroimidazoles (MIC50 = 0.06-2.7 µM), salicylanilides (MIC50 = 0.2-0.6 µM), imidazole antifungals (MIC50 = 4.8-11.6 µM), and miscellaneous group (MIC50 = 0.4-22.2 µM). The most potent drugs from the initial screening were further evaluated against additional clinically relevant strains of C. difficile. Moreover, we tested the activity of potent inhibitors against representative strains of human normal gut microbiota to investigate the selectivity of the inhibitors towards C. difficile. Overall, this study provides a platform that could be used for further development of potent and selective anticlostridial antibiotics.

A Novel Salicylanilide Derivative Induces Autophagy Cell Death in Castration-Resistant Prostate Cancer via ER Stress-Activated PERK Signaling Pathway.

Metastatic castration-resistant prostate cancer (CRPC) is currently incurable. Cancer growth and progression is intimately affected by its interaction with host microenvironment. Cotargeting of the stroma and prostate cancer is therefore an emerging therapeutic strategy for metastatic CRPC. Cancer-induced osteoclastogenesis is known to contribute to CRPC bone metastasis. This study is to extend pharmacologic value of our synthesized LCC03, a derivative of 5-(2',4'-difluorophenyl)-salicylanilide that has previously testified for its osteoclastogenesis activity, by exploring its additional cytotoxic properties and underlying mechanism in CRPC cells. LCC03 was chemically synthesized and examined for cell growth inhibition in a serial of CRPC cell lines. We demonstrated that LCC03 dose-dependently suppressed proliferation and retarded cell-cycle progression in CRPC cells. The classical autophagy features, including autophagosome formation and LC3-II conversion, were dramatically shown in LCC03-treated CRPC cells, and it was associated with the suppressed AKT/mTOR signaling pathways, a major negative regulator of autophagy. Moreover, an expanded morphology of the endoplasmic reticulum (ER), increased expression of the ER stress markers GRP78 and PERK, and eIF2α phosphorylation were observed. Blockage of autophagy and PERK pathways using small molecule inhibitors or shRNA knockdown reversed LCC03-induced autophagy and cell death, thus indicating that the PERK-eIF2α pathway contributed to the LCC03-induced autophagy. Furthermore, treatment of tumor-bearing mice with intraperitoneal administered LCC03 suppressed the growth of CRPC xenografts in mouse bone without systemic toxicity. The dual action of 5-(2',4'-difluorophenyl)-salicylanilide on targeting both the osteoclasts and the tumor cells strongly indicates that LCC03 is a promising anticancer candidate for preventing and treating metastatic CRPC.