The Hydrophobic Extract of Sorghum bicolor (L. Moench) Enriched in Apigenin-Protected Rats against Aflatoxin B1-Associated Hepatorenal Derangement.

Aflatoxin B1 (AFB1) is a recalcitrant metabolite produced by fungi species, and due to its intoxications in animals and humans, it has been classified as a Group 1 carcinogen in humans. Preserving food products with Sorghum bicolor sheath can minimise the contamination of agricultural products and avert ill health occasioned by exposure to AFB1. The current study investigated the ameliorating effect of Sorghum bicolor sheath hydrophobic extract (SBE-HP) enriched in Apigenin (API) on the hepatorenal tissues of rats exposed to AFB1. The SBE-HP was characterised using TLC and LC-MS and was found to be enriched in Apigenin and its methylated analogues. The study used adult male rats divided into four experimental cohorts co-treated with AFB1 (50 µg/kg) and SBE-HP (5 and 10 mg/kg) for 28 days. Biochemical, enzyme-linked immunosorbent assays (ELISA) and histological staining were used to examine biomarkers of hepatorenal function, oxidative status, inflammation and apoptosis, and hepatorenal tissue histo-architectural alterations. Data were analysed using GraphPad Prism 8.3.0, an independent t-test, and a one-way analysis of variance. Co-treatment with SBE-HP ameliorated an upsurge in the biomarkers of hepatorenal functionality in the sera of rats, reduced the alterations in redox balance, resolved inflammation, inhibited apoptosis, and preserved the histological features of the liver and kidney of rats exposed to AFB1. SBE-HP-containing API is an excellent antioxidant regiment. It can amply prevent the induction of oxidative stress, inflammation, and apoptosis in the hepatorenal system of rats. Therefore, supplementing animal feeds and human foods with SBE-HP enriched in Apigenin may reduce the burden of AFB1 intoxication in developing countries with a shortage of effective antifungal agents.

Synthetic methodology-enabled discovery of a tunable indole template for COX-1 inhibition and anti-cancer activity.

Establishing structure-activity relationships (SAR) for privileged pharmacophores, such as the indole scaffold, is a key step in the early stages of drug discovery. Herein, we report the synthesis and preliminary SAR studies on substituted 6-hydroxyindole-7-carboxylates as a tunable framework for COX inhibition and anti-cancer activity. To facilitate the SAR discovery, a modular synthetic methodology was employed which enabled the synthesis of the substituted indoles. From the synthesized compounds, five displayed COX-1 inhibition activity in a colorimetric assay with their intracellular activity further confirmed by a cell-based target validation assay. Following molecular docking analyses, key interactions between the active compounds and the COX enzymes were elucidated. In addition to the identified COX inhibitors, two compounds showed selective cytotoxicity against Hep-G2, MCF-7, and LnCaP. The mechanism of cell death was investigated and found to include induction of Caspase-3 activation and cleavage, down-regulation of anti-apoptotic proteins Bcl-xL and Bcl-2, and upregulation of Bax. Finally, two representative compounds were confirmed to induce cell cycle arrest at the G1/G0 stage. In summary, the 6-hydroxyindole-7-carboxylate framework shows promising versatility as a template for the discovery of anti-inflammation or anti-cancer agents, given the evidence of its COX inhibitory and anti-cancer activities herein presented.

Protocatechuic acid protects against hepatorenal toxicities in rats exposed to Furan.

Furan formed in processed food is hepatotoxic and likely carcinogenic in humans. We investigated protocatechuic acid (PCA) protective role in rats' hepatorenal function treated with furan. Rats were grouped and treated as follows: Control, PCA (50 mg/kg), furan alone (8 mg/kg), furan + PCA1 (25 + 8 mg/kg), and furan + PCA2 (50 + 8 mg/kg). Upon sacrifice, evaluation of hepatorenal function, oxidative stress status, reactive oxygen and nitrogen species (RONS), lipid peroxidation (LPO), myeloperoxidase (MPO) activity, among nitric oxide (NO) levels were performed. Cytokine levels (IL-10, IL-1ß, TNF-alpha), Caspase 3 and 9 activities, and histopathological examination were also assessed. We found that the final body and relative liver weights changed significantly (p < 0.05) in treated groups. Hepatic transaminases, urea, and creatinine increased (p < 0.05) in furan only treated group, and reduced in PCA co-treated groups. The furan-induced decrease in antioxidant status increased RONS, and LPO levels were alleviated (p < 0.05) by PCA co-treatment. Furthermore, furan-mediated increase in NO, IL-1ß, TNF-alpha levels, MPO, Cas-3, and 9 activities and suppressed IL-10 levels was reversed accordingly in rats' kidney and liver co-treated with PCA. The extent of furan-mediated hepatorenal lesions was lessened in PCA co-treated rats. Our findings suggest that PCA protects against oxido-inflammatory pathways, enhanced caspases 3 and 9 activations induced by furan in rat hepatorenal system.

Co-administration of Luteolin mitigated toxicity in rats' lungs associated with doxorubicin treatment.

Doxorubicin (DOX), is a drug against lung malignancies with undesirable side effect including oxidative, inflammatory and apoptotic effects. Luteolin (LUT), present in fruits and vegetables is pharmacologically active against oxido-inflammatory and apoptotic responses. The present study examined the effect of LUT on DOX-induced lungs and blood dysfunction in Wistars rat (sex: male; 10 weeks old, 160 ± 5 g). Randomly grouped (n = 10) rats were treated as follows: control, LUT alone (100 mg/kg; per os), DOX (2 mg/kg; i. p), and co-treated rats with LUT (50 or 100 mg/kg) and DOX for two consecutive weeks. DOX alone adversely altered the final body and relative organ weights, red and white blood cell and platelet counts. DOX significantly (p > 0.05) reduced lungs antioxidant capacity, and anti-inflammatory cytokines; increased biomarkers of oxidative stress, caspase-3 activity, and pro-inflammatory cytokine. Morphological damages accompanied these biochemical alterations in the lung of experimental rats. Co-treatment with LUT, dose-dependently reversed DOX-mediated changes in rats' survival, toxic responses, and diminished oxidative stress in rat's lungs. Furthermore, co-treatment with LUT resulted in the reduction of pro-inflammatory cytokines and apoptotic biomarkers, increased red and white blood cell, platelet counts and abated pathological injuries in rat lungs treated with DOX alone. In essence, our findings indicate that LUT dose-dependently mitigated DOX-induced toxicities in the lungs and haematopoietic systems. Supplementation of patients on DOX-chemotherapy with phytochemicals exhibiting antioxidant activities, specifically LUT, could circumvent the onset of unintended toxic responses in the lungs and haematopoietic system exposed to DOX.

Neuroprotective role of gallic acid in aflatoxin B1 -induced behavioral abnormalities in rats.

The neurotoxic impact of dietary exposure to aflatoxin B1 (AFB1 ) is documented in experimental and epidemiological studies. Gallic acid (GA) is a triphenolic phytochemical with potent anticancer, anti-inflammatory, and antioxidant activities. There is a knowledge gap on the influence of GA on AFB1 -induced neurotoxicity. This study probed the influence of GA on neurobehavioral and biochemical abnormalities in rats orally treated with AFB1 per se (75 µg/kg body weight) or administered together with GA (20 and 40 mg/kg) for 28 uninterrupted days. Behavioral endpoints obtained with video-tracking software demonstrated significant (p < .05) abatement of AFB1 -induced anxiogenic-like behaviors (increased freezing, urination, and fecal bolus discharge), motor and locomotor inadequacies, namely increased negative geotaxis and diminished grip strength, absolute turn angle, total time mobile, body rotation, maximum speed, and total distance traveled by GA. The improvement of exploratory behavior in animals that received both AFB1 and GA was confirmed by track plots and heat maps appraisal. Abatement of AFB1 -induced decreases in acetylcholinesterase activity, antioxidant status and glutathione level by GA was accompanied by a marked reduction in oxidative stress markers in the cerebellum and cerebrum of rats. Additionally, GA treatment abrogated AFB1 -mediated decrease in interleukin-10 and elevation of inflammatory indices, namely tumor necrosis factor-α, myeloperoxidase activity, interleukin-1ß, and nitric oxide. Further, GA treatment curtailed caspase-3 activation and histological injuries in the cerebral and cerebellar tissues. In conclusion, abatement of AFB1 -induced neurobehavioral abnormalities by GA involves anti-inflammatory, antioxidant, and antiapoptotic mechanisms in rats.

N-acetyl cysteine co-treatment abates perfluorooctanoic acid-induced reproductive toxicity in male rats.

Perfluorooctanoic acid is a synthetic perfluoroalkyl-persistent in the environment and toxic to humans. N-acetylcysteine is a pro-drug of both amino acid l-cysteine and glutathione-a non-enzymatic antioxidant. N-acetylcysteine serves as an antidote for paracetamol poisoning and alleviates cellular oxidative and inflammatory stressors. We investigated N-acetylcysteine role against reproductive toxicity in male Wistar rats (weight: 140-220 g; 10 weeks old) posed by perfluorooctanoic acid exposure. Randomised rat cohorts were dosed both with perfluorooctanoic acid (5 mg/kg; p.o) or co-dosed with N-acetylcysteine (25 and 50 mg/kg p.o) for 28 days. Sperm physiognomies, biomarkers of testicular function and reproductive hormones, oxidative stress and inflammation were evaluated. Co-treatment with N-acetylcysteine significantly (p < .05) reversed perfluorooctanoic acid-mediated decreases in reproductive enzyme activities, and adverse effect on testosterone, luteinising and follicle-stimulating hormone concentrations. N-acetylcysteine treatment alone, improved sperm motility, count and viability, and reduced total sperm abnormalities. Co-treatment with N-acetylcysteine mitigated perfluorooctanoic acid-induced alterations in sperm function parameters. N-acetylcysteine abated (p < .05) perfluorooctanoic acid-induced oxidative stress in experimental rats testes and epididymis, and generally improved antioxidant enzyme activities and cellular thiol levels. Furthermore, N-acetylcysteine suppressed inflammatory responses and remedied perfluorooctanoic acid-mediated histological injuries in rat. Cooperatively, N-acetylcysteine enhanced reproductive function in perfluorooctanoic acid dosed rats, by lessening oxidative and nitrative stressors and mitigated inflammatory responses in the examined organ.

Pyrimethamine conjugated histone deacetylase inhibitors: Design, synthesis and evidence for triple negative breast cancer selective cytotoxicity.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor which has been recognized as a promising cancer therapeutic target. Small molecule pyrimethamine (PYM) is a known direct inhibitor of activated STAT3 and it is currently under clinical trial. Also, histone deacetylase (HDAC) inhibition has been shown to indirectly attenuate STAT3 signaling through inhibition of STAT3 activation. Herein we described the design and biological profiling of two classes of PYM-conjugated HDAC inhibitors (HDACi). We observed that the class I PYM-HDACi compounds 12a-c potently inhibited HDACs 1 and 6 in cell free assays while a lead class II PYM-HDACi compound 23 showed a strong HDAC 6 selective inhibition. In a cell-based assay, 12a-c are preferentially cytotoxic to MDA-MB-231, a TNBC cell line that is highly STAT3-dependent, while 23 showed no such selective toxicity. Subsequent target validation studies revealed that a representative class I PYM-HDACi compound 12c elicited a signature of HDAC and STAT3 pathway inhibition intracellularly. Collectively, these data suggest that PYM-HDACi compounds are promising leads to develop targeted therapy for TNBC.

Lung Tissue Delivery of Virus-Like Particles Mediated by Macrolide Antibiotics.

Macrophage cells are present in high abundance in the lung to intercept invading microorganisms that gain access through airway mucosal surfaces. Several bacterial pathogens have evolved the capacity to evade the innate immune response by establishing infections within pulmonary macrophages upon phagocytosis, leading to prolonged disease. Macrolide antibiotics such as azithromycin and clarithromycin accumulate in phagocytic cells and have been shown to preferentially distribute in tissues where populations of these cells reside. We employed this class of molecules as targeting ligands to direct virus-like particles (VLPs) to lung-resident macrophages. VLP-macrolide conjugates showed enhanced uptake into RAW 264.7 macrophage cells in culture, with azithromycin displaying the greatest effect; distinct differences were also observed for different macrocycle structures and orientations on the particle surface. Activation of macrophage cells was stimulated by particle uptake toward an intermediate activation state, in contrast to previous reports using macrolide-functionalized gold nanorods that stimulated a cytotoxic macrophage response. Attached azithromycin was also able to direct VLPs to the lungs in mice, with significant accumulation within 2 h of systemic injection. These results suggest that this new class of bioconjugate could serve as an effective platform for intracellular drug delivery in the context of pulmonary infections.

Design, synthesis, and evaluation of the antiproliferative activity of hydantoin-derived antiandrogen-genistein conjugates.

Androgen receptor (AR) signaling is vital to the viability of all forms of prostate cancer (PCa). With the goal of investigating the effect of simultaneous inhibition and depletion of AR on viability of PCa cells, we designed, synthesized and characterized the bioactivities of bifunctional agents which incorporate the independent cancer killing properties of an antiandrogen and genistein, and the AR downregulation effect of genistein within a single molecular template. We observed that a representative conjugate, 9b, is much more cytotoxic to both LNCaP and DU145 cells relative to the antiandrogen and genistein building blocks as single agents or their combination. Moreover, conjugate 9b more effectively down regulates cellular AR protein levels relative to genistein and induces S phase cell cycle arrest. The promising bioactivities of these conjugates warrant further investigation.

Design, synthesis and evaluation of antiproliferative activity of melanoma-targeted histone deacetylase inhibitors.

The clinical validation of histone deacetylase inhibition as a cancer therapeutic modality has stimulated interest in the development of new generation of potent and tumor selective histone deacetylase inhibitors (HDACi). With the goal of selective delivery of the HDACi to melanoma cells, we incorporated the benzamide, a high affinity melanin-binding template, into the design of HDACi to generate a new series of compounds 10a-b and 11a-b which display high potency towards HDAC1 and HDAC6. However, these compounds have attenuated antiproliferative activities relative to the untargeted HDACi. An alternative strategy furnished compound 14, a prodrug bearing the benzamide template linked via a labile bond to a hydroxamate-based HDACi. This pro-drug compound showed promising antiproliferative activity and warrant further study.

Bifunctional conjugates with potent inhibitory activity towards cyclooxygenase and histone deacetylase.

We herein disclose a series of compounds with potent inhibitory activities towards histone deacetylases (HDAC) and cyclooxygenases (COX). These compounds potently inhibited the growth of cancer cell lines consistent with their anti-COX and anti-HDAC activities. While compound 2b showed comparable level of COX-2 selectivity as celecoxib, compound 11b outperformed indomethacin in terms of selectivity towards COX-2 relative to COX-1. An important observation with our lead compounds (2b, 8, 11b, and 17b) is their enhanced cytotoxicity towards androgen dependent prostate cancer cell line (LNCaP) relative to androgen independent prostate cancer cell line (DU-145). Interestingly, compounds 2b and 17b arrested the cell cycle progression of LNCaP in the S-phase, while compound 8 showed a G0/G1 arrest, similar to SAHA. Relative to SAHA, these compounds displayed tumor-selective cytotoxicity as they have low anti-proliferative activity towards healthy cells (VERO); an attribute that makes them attractive candidates for drug development.

Exploiting translational stalling peptides in an effort to extend azithromycin interaction within the prokaryotic ribosome nascent peptide exit tunnel.

The ribosome is the primary protein synthesis machine in the cell and is a target for treatment of a variety of diseases including bacterial infection and cancer. The ribosomal peptide exit tunnel, the route of egress for the nascent peptide, is an inviting site for drug design. Toward a rational engagement of the nascent peptide components for the design of small molecule inhibitors of ribosome function, we designed and disclosed herein a set of N-10 indole functionalized azithromycin analogs. The indole moiety of these compounds is designed to mimic the translation stalling interaction of SecM W155 side-chain with the prokaryotic (Escherichia coli) ribosome A751 residue. Many of these N-10 functionalized compounds have enhanced translation inhibition activities against E. coli ribosome relative to azithromycin while a subset inhibited the growth of representative susceptible bacteria strains to about the same extent as azithromycin. Moreover, the inclusion of bovine serum in the bacterial growth media enhanced the anti-bacterial potency of the N-10 functionalized azithromycin analogs by as high as 10-fold.

A structure-activity relationship of non-peptide macrocyclic histone deacetylase inhibitors and their anti-proliferative and anti-inflammatory activities.

Inhibition of the enzymatic activity of histone deacetylase (HDAC) is a promising therapeutic strategy for cancer treatment and several distinct small molecule histone deacetylase inhibitors (HDACi) have been reported. We have previously identified a new class of non-peptide macrocyclic HDACi derived from 14- and 15-membered macrolide skeletons. In these HDACi, the macrocyclic ring is linked to the zinc chelating hydroxamate moiety through a para-substituted aryl-triazole cap group. To further delineate the depth of the SAR of this class of HDACi, we have synthesized series of analogous compounds and investigated the influence of various substitution patterns on their HDAC inhibitory, anti-proliferative and anti-inflammatory activities. We identified compounds 25b and 38f with robust anti-proliferative activities and compound 26f (IC50 47.2 nM) with superior anti-inflammatory (IC50 88 nM) activity relative to SAHA.

Molecular architecture of zinc chelating small molecules that inhibit spliceosome assembly at an early stage.

The removal of intervening sequences (introns) from a primary RNA transcript is catalyzed by the spliceosome, a large ribonucleoprotein complex. At the start of each splicing cycle, the spliceosome assembles anew in a sequentially ordered manner on the pre-mRNA intron to be removed. We describe here the identification of a series of naphthalen-2-yl hydroxamate compounds that inhibit pre-mRNA splicing in vitro with mid- to high-micromolar values of IC(50). These hydroxamates stall spliceosome assembly at the A complex stage. A structure-activity analysis of lead compounds revealed three pharmacophores that are essential for splicing inhibition. Specifically, a hydroxamate as a zinc-binding group and a 6-methoxynaphthalene cap group are both critical, and a linker chain comprising eight to nine methylene groups is also important, for the specific binding to the docking site of a target protein molecule and precise positioning of the zinc binding group. As we found no correlation between the inhibition patterns of known histone deacetylases on the one hand and pre-mRNA splicing on the other, we conclude that these compounds may function through the inhibition of the activities of other, at present, unknown spliceosome-associated zinc metalloprotein(s).

The antileishmanial activity of isoforms 6- and 8-selective histone deacetylase inhibitors.

Histone deacetylase inhibitors (HDACi) pleiotropy is largely due to their nonselective inhibition of various cellular HDAC isoforms. Connecting inhibition of a specific isoform to biological responses and/or phenotypes is essential toward deconvoluting HDACi pleiotropy. The contribution of classes I and II HDACs to the antileishmanial activity of HDACi was investigated using the amastigote and promastigote forms of Leishmania donovani. We observed that the antileishmanial activities of HDACi are largely due to the inhibition of HDAC6-like activity. This observation could facilitate the development of HDACi as antileishmanial agents.

Development and validation of a sensitive LC-MS/MS assay of GT-14, a novel Gαi2 inhibitor, in rat plasma, and its application in pharmacokinetic study.

A sensitive and selective LC-MS/MS method was developed and validated for the quantitation of a novel Gαi2 inhibitor, GT-14, in rat plasma using a SCIEX 6500+ triple QUAD LC-MS system equipped with an ExionLC UHPLC unit. GT-14 (m/z 265.2 → 134.1) and griseofulvin (Internal Standard, IS) (m/z 353.1 → 285.1) were detected in a positive mode by electrospray ionization (ESI) using multiple reaction monitoring (MRM). The assay was linear in the concentration range of 0.78-1000 ng/mL in rat plasma. Both accuracy and precision values were within the acceptance criteria of ±15 %, as established by FDA guidance. The matrix effect was negligible from plasma, with signal percentages of 98.5-106.9 %. The mean recovery was 104.5 %, indicating complete extraction of GT-14 from plasma. GT-14 was found to be stable under different experimental conditions. The validated method was successfully applied to evaluate plasma protein binding and in vivo pharmacokinetics of GT-14 in rats.

Gαi2 Protein Inhibition Blocks Chemotherapy- and Anti-Androgen-Induced Prostate Cancer Cell Migration.

We have previously shown that heterotrimeric G-protein subunit alphai2 (Gαi2) is essential for cell migration and invasion in prostate, ovarian and breast cancer cells, and novel small molecule inhibitors targeting Gαi2 block its effects on migratory and invasive behavior. In this study, we have identified potent, metabolically stable, second generation Gαi2 inhibitors which inhibit cell migration in prostate cancer cells. Recent studies have shown that chemotherapy can induce the cancer cells to migrate to distant sites to form metastases. In the present study, we determined the effects of taxanes (docetaxel), anti-androgens (enzalutamide and bicalutamide) and histone deacetylase (HDAC) inhibitors (SAHA and SBI-I-19) on cell migration in prostate cancer cells. All treatments induced cell migration, and simultaneous treatments with new Gαi2 inhibitors blocked their effects on cell migration. We concluded that a combination treatment of Gαi2 inhibitors and chemotherapy could blunt the capability of cancer cells to migrate and form metastases.

Doxorubicin-Based Ionic Nanomedicines for Combined Chemo-Phototherapy of Cancer.

Synergistic combination therapy approach offers lots of options for delivery of materials with anticancer properties, which is a very promising strategy to treat a variety of malignant lesions with enhanced therapeutic efficacy. The current study involves a detailed investigation of combination ionic nanomedicines where a chemotherapeutic drug is coupled with a photothermal agent to attain dual mechanisms (chemotherapy (chemo) and photothermal therapy (PTT)) to improve the drug's efficacy. An FDA-approved Doxorubicin hydrochloride (DOX·HCl) is electrostatically attached with a near-infrared cyanine dye (ICG, IR783, and IR820), which serves as a PTT drug using ionic liquid chemistry to develop three ionic material (IM)-based chemo-PTT drugs. Carrier-free ionic nanomedicines (INMs) are derived from ionic materials (IMs). The photophysical properties of the developed combination IMs and their INMs were studied in depth. The phototherapeutic efficiency of the combination drugs was evaluated by measuring the photothermal conversion efficiency and singlet-oxygen quantum yield. The improved photophysical properties of the combination nanomedicines in comparison to their parent compounds significantly enhanced INMs' photothermal efficiency. Cellular uptake, dark and light toxicity studies, and cell death mechanisms of the chemo-PTT nanoparticles were also studied in vitro. The combination INMs exhibited enhanced cytotoxicity compared to their respective parent compounds. Moreover, the apoptosis cell death mechanism was almost doubled for combination nanomedicine than the free DOX, which is attributed to enhanced cellular uptake. Examination of the combination index and improved in vitro cytotoxicity results revealed a great synergy between chemo and PTT drugs in the developed combination nanomedicines.

A Novel Liver Cancer-Selective Histone Deacetylase Inhibitor Is Effective Against Hepatocellular Carcinoma and Induces Durable Responses with Immunotherapy.

Hepatocellular cancer (HCC) progression is facilitated by gene-silencing chromatin histone hypoacetylation due to histone deacetylases (HDACs) activation. However, inhibiting HDACs, an effective treatment for lymphomas, has shown limited success in solid tumors. We report the discovery of a class of HDAC inhibitors (HDACi) that demonstrates exquisite selective cytotoxicity against human HCC cells. The lead compound STR-V-53 (3) showed favorable safety profile in mice and robustly suppressed tumor growth in orthotopic xenograft models of HCC. When combined with the anti-HCC drug sorafenib, STR-V-53 showed greater in vivo efficacy. Moreover, STR-V-53 combined with anti-PD1 therapy increased the CD8+ to regulatory T-cell (Treg) ratio and survival in an orthotopic HCC model in immunocompetent mice. This combination therapy resulted in durable responses in 40% of the mice. Collectively, our data demonstrate that the novel HDACi STR-V-53 is an effective anti-HCC agent that can induce profound responses when combined with standard immunotherapy.

Dual-acting histone deacetylase-topoisomerase I inhibitors.

Current chemotherapy regimens are comprised mostly of single-target drugs which are often plagued by toxic side effects and resistance development. A pharmacological strategy for circumventing these drawbacks could involve designing multivalent ligands that can modulate multiple targets while avoiding the toxicity of a single-targeted agent. Two attractive targets, histone deacetylase (HDAC) and topoisomerase I (Topo I), are cellular modulators that can broadly arrest cancer proliferation through a range of downstream effects. Both are clinically validated targets with multiple inhibitors in therapeutic use. We describe herein the design and synthesis of dual-acting histone deacetylase-topoisomerase I inhibitors. We also show that these dual-acting agents retain activity against HDAC and Topo I, and potently arrest cancer proliferation.