Orally bioavailable styryl derivative of rohitukine-N-oxide inhibits CDK9/T1 and the growth of pancreatic cancer cells.

The chromone alkaloid is one of the classical pharmacophores for cyclin-dependent kinases (CDKs) and represents the first CDK inhibitor to reach clinical trials. Rohitukine (1), a chromone alkaloid isolated from Dysoxylum binectariferum inspired the discovery of several clinical candidates. The N-oxide derivative of rohitukine occurs naturally, with no reports on its biological activity. Herein, we report isolation, biological evaluation, and synthetic modification of rohitukine N-oxide for CDK9/T1 inhibition and antiproliferative activity in cancer cells. Rohitukine N-oxide (2) inhibits CDK9/T1 (IC50 7.6 µM) and shows antiproliferative activity in the colon and pancreatic cancer cells. The chloro-substituted styryl derivatives, 2b, and 2l, inhibit CDK9/T1 with IC50 values of 0.17 and 0.15 µM, respectively. These derivatives display cellular antiproliferative activity in HCT 116 (colon) and MIA PaCa-2 (pancreatic) cancer cells with GI50 values of 2.5-9.7 µM with excellent selectivity over HEK293 (embryonic kidney) cells. Both analogs induce cell death in MIA PaCa-2 cells via inducing intracellular ROS production, reducing mitochondrial membrane potential, and inducing apoptosis. These analogs are metabolically stable in liver microsomes and have a decent oral pharmacokinetics in BALB/c mice. The molecular modeling studies indicated their strong binding at the ATP-binding site of CDK7/H and CDK9/T1.

Discovery of Pongol, the Furanoflavonoid, as an Inhibitor of CDK7/Cyclin H/MAT1 and Its Preliminary Structure-Activity Relationship.

Natural products have been a great source of leads for cancer drug discovery. The cyclin-dependent kinases (CDKs) play a vital role in the initiation and progression of cancer. The CDK-activating kinase, CDK7/cyclin H/MAT1, has recently gained tremendous attention in targeted cancer drug discovery. Herein, we screened a small library of pure natural products in an ADP-Glo CDK7/H kinase assay that yielded a series of furano- and naphthoflavonoids among actives. Pongol (SBN-88), the hydroxy-substituted furanoflavonoid, inhibits CDK7/H as well as CDK9/T1 with IC50 values of 0.93 and 0.83 µM, respectively, and >20-fold selectivity over CDK2/E1 (IC50 > 20 µM). The molecular docking and molecular dynamics simulation revealed that the presence of phenolic -OH in pongol is vital for kinase inhibition, as its absence resulted in a significant loss in activity (e.g., lanceolatin B). The prime MM-GBSA calculations revealed the presence of strong lipophilic and H-bonding interactions of pongol with CDKs.

Multitargeted C9-substituted ester and ether derivatives of berberrubine for Alzheimer's disease: Design, synthesis, biological evaluation, metabolic stability, and pharmacokinetics.

Berberrubine is a naturally occurring isoquinoline alkaloid and a bioactive metabolite of berberine. Berberine exhibits a wide range of pharmacological activities, including cholinesterase inhibition. The cholinesterase inhibitors provide symptomatic treatment for Alzheimer's disease; however, multitarget-directed ligands have the potential as disease-modifying therapeutics. Herein, we prepared a series of C9-substituted berberrubine derivatives intending to discover dual cholinesterase and beta-site amyloid-precursor protein cleaving enzyme 1 (BACE-1) inhibitors. Most synthesized derivatives possessed balanced dual inhibition (AChE and BChE) activity in the submicromolar range and a moderate inhibition against BACE-1. Two most active ester derivatives, 12a and 11d, display inhibition of AChE, BChE, and BACE-1. The 3-methoxybenzoyl ester derivative, 12a, inhibits electric eel acetylcholinesterase (EeAChE), equine serum butyrylcholinesterase (eqBChE), and human hBACE-1 with IC50 values of 0.5, 4.3, and 11.9 µM, respectively and excellent BBB permeability (Pe = 8 × 10-6 cm/s). The ester derivative 12a is metabolically unstable; however, its ether analog 13 is stable in HLM and exhibits inhibition of AChE, BChE, and BACE-1 with IC50 values of 0.44, 3.8, and 17.9 µM, respectively. The ether analog also inhibits self-aggregation of Aß and crosses BBB (Pe = 7.3 × 10-6 cm/s). Administration of 13 at 5 mg/kg (iv) in Wistar rats showed excellent plasma exposure with AUC0-∞ of 28,834 ng min/ml. In conclusion, the multitargeted berberrubine ether derivative 13 is CNS permeable and has good ADME properties.

In vitro and in vivo anticancer potential and molecular targets of the new colchicine analog IIIM-067.

OBJECTIVE: The current study evaluated various new colchicine analogs for their anticancer activity and to study the primary mechanism of apoptosis and in vivo antitumor activity of the analogs with selective anticancer properties and minimal toxicity to normal cells. METHODS: Sulforhodamine B (SRB) assay was used to screen various colchicine analogs for their in vitro cytotoxicity. The effect of N-[(7S)-1,2,3-trimethoxy-9-oxo-10-(pyrrolidine-1-yl)5,6,7,9-tetrahydrobenzo[a] heptalene-7-yl] acetamide (IIIM-067) on clonogenicity, apoptotic induction, and invasiveness of A549 cells was determined using a clonogenic assay, scratch assay, and staining with 4',6-diamidino-2-phenylindole (DAPI) and annexin V/propidium iodide. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels were observed using fluorescence microscopy. Western blot analysis was used to quantify expression of proteins involved in apoptosis, cell cycle, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling. Pharmacokinetic and in vivo efficacy studies against Ehrlich ascites carcinoma (EAC) and Ehrlich solid tumor models were conducted using Swiss albino mice. RESULTS: IIIM-067 showed potent cytotoxicity and better selectivity than all other colchicine analogs screened in this study. The selective activity of IIIM-067 toward A549 cells was higher among other cancer cell lines, with a selectivity index (SI) value of 2.28. IIIM-067 demonstrated concentration- and time-dependent cytotoxicity against A549 cells with half-maximal inhibitory concentration values of 0.207, 0.150 and 0.106 µmol/L at 24, 48 and 72 h, respectively. It also had reduced toxicity to normal cells (SI > 1) than the parent compound colchicine (SI = 1). IIIM-067 reduced the clonogenic ability of A549 cells in a dose-dependent manner. IIIM-067 enhanced ROS production from 24.6% at 0.05 µmol/L to 82.1% at 0.4 µmol/L and substantially decreased the MMP (100% in control to 5.6% at 0.4 µmol/L). The annexin V-FITC assay demonstrated 78% apoptosis at 0.4 µmol/L. IIIM-067 significantly (P < 0.5) induced the expression of various intrinsic apoptotic pathway proteins, and it differentially regulated the PI3K/AKT/mTOR signaling pathway. Furthermore, IIIM-067 exhibited remarkable in vivo anticancer activity against the murine EAC model, with tumor growth inhibition (TGI) of 67.0% at a dose of 6 mg/kg (i.p.) and a reduced mortality compared to colchicine. IIIM-067 also effectively inhibited the tumor growth in the murine solid tumor model with TGI rates of 48.10%, 55.68% and 44.00% at doses of 5 mg/kg (i.p.), 6 mg/kg (i.p.) and 7 mg/kg (p.o.), respectively. CONCLUSION: IIIM-067 exhibited significant anticancer activity with reduced toxicity both in vitro and in vivo and is a promising anticancer candidate. However, further studies are required in clinical settings to fully understand its potential.

Antimicrobial and Cytotoxic Potential of Helminthosporin from Rumex abyssiniscus Jacq. Discovered as a Novel Source of Syringic Acid and Bis(2-ethyloctyl) Phthalate.

Rumex abyssinicus Jacq. is a perennial medicinal herb widely used in traditional medicine to treat many diseases. Phytochemicals of the plant were isolated using column chromatography and thin layer chromatography techniques. Extract, fractions and pure compounds were screened for antimicrobial activity against sensitive and multi-drug resistant microbes and their cytotoxicity was performed on different cancer cell lines. The mechanism of action of purified helminthosporin as well as the potent fraction containing a mixture of two compounds was assessed. Fraction R7C3 was the most potent antibacterial with the lowest MIC value of 0.12 µg/mL. Helminthosporin was the most potent compound with the lowest MIC value of 1.95 µg/mL. The compound was more potent than the antibiotic chloramphenicol against multi-drug resistant (MDR) bacteria with MIC equal to 16 µg/mL. The fraction and helminthosporin were shown to destroy the cell wall of the yeast and bacteria, and DNA fragmentation effect on the genome of Candida albicans and Bacillus cereus. Helminthosporin was the most cytotoxic compound with IC50 ˂ 10 µM. Fraction R7C3 showed the most potent cytotoxic effects on all cancer cell lines, with IC50 ranging from ˂1 to 4.35 ng/mL. Our study is the first report on the mechanism of action of helminthosporin, a potent candidate in the development of new drugs against multi-resistant bacteria and cancer cells. In addition, this study uncovered Rumex abyssinicus as a new source of syringic acid and bis(2-ethyloctyl) phthalate.

Cell-intrinsic Aryl Hydrocarbon Receptor signalling is required for the resolution of injury-induced colonic stem cells.

The aryl hydrocarbon receptor (AHR) is an environmental sensor that integrates microbial and dietary cues to influence physiological processes within the intestinal microenvironment, protecting against colitis and colitis-associated colorectal cancer development. Rapid tissue regeneration upon injury is important for the reinstatement of barrier integrity and its dysregulation promotes malignant transformation. Here we show that AHR is important for the termination of the regenerative response and the reacquisition of mature epithelial cell identity post injury in vivo and in organoid cultures in vitro. Using an integrative multi-omics approach in colon organoids, we show that AHR is required for timely termination of the regenerative response through direct regulation of transcription factors involved in epithelial cell differentiation as well as restriction of chromatin accessibility to regeneration-associated Yap/Tead transcriptional targets. Safeguarding a regulated regenerative response places AHR at a pivotal position in the delicate balance between controlled regeneration and malignant transformation.

Role of basic aminoalkyl chains in the lead optimization of Indoloquinoline alkaloids.

Indoloquinoline (IQ) is an important class of naturally occurring antimalarial alkaloids, mainly represented by cryptolepine, isocryptolepine, and neocryptolepine. The IQ structural framework consists of four isomeric ring systems differing via the linkage of indole with quinoline as [3,2-b], [3,2-c], [2,3-c], and [2,3-b]. Structurally, IQs are planar and thus they bind strongly to the DNA which largely contributes to their biological properties. The structural rigidity and associated nonspecific cellular toxicity is a key shortcoming of the IQ structural framework for preclinical development. Thus, the lead optimization efforts were aimed at improving the therapeutic window and ADME properties of IQs. The structural modifications mainly involved attaching the basic aminoalkyl chains that positively modulates the vital physicochemical and topological parameters, thereby improves biological activity. Our analysis has found that the aminoalkylation consistently improved the selectivity index and provided acceptable in-vivo antimalarial/anticancer activity. Herein, we critically review the role of aminoalkylation in deciphering the antimalarial and cytotoxic activity of IQs.

Recent Developments in the Use of Kinase Inhibitors for Management of Viral Infections.

Kinases are a group of therapeutic targets involved in the progression of numerous diseases, including cancer, rheumatoid arthritis, Alzheimer's disease, and viral infections. The majority of approved antiviral agents are inhibitors of virus-specific targets that are encoded by individual viruses. These inhibitors are narrow-spectrum agents that can cause resistance development. Viruses are dependent on host cellular proteins, including kinases, for progression of their life-cycle. Thus, targeting kinases is an important therapeutic approach to discovering broad-spectrum antiviral agents. As there are a large number of FDA approved kinase inhibitors for various indications, their repurposing for viral infections is an attractive and time-sparing strategy. Many kinase inhibitors, including baricitinib, ruxolitinib, imatinib, tofacitinib, pacritinib, zanubrutinib, and ibrutinib, are under clinical investigation for COVID-19. Herein, we discuss FDA approved kinase inhibitors, along with a repertoire of clinical/preclinical stage kinase inhibitors that possess antiviral activity or are useful in the management of viral infections.

Analyzing the scaffold diversity of cyclin-dependent kinase inhibitors and revisiting the clinical and preclinical pipeline.

Kinases have gained an important place in the list of vital therapeutic targets because of their overwhelming clinical success in the last two decades. Among various clinically validated kinases, the cyclin-dependent kinases (CDK) are one of the extensively studied drug targets for clinical development. Food and Drug Administration has approved three CDK inhibitors for therapeutic use, and at least 27 inhibitors are under active clinical development. In the last decade, research and development in this area took a rapid pace, and thus the analysis of scaffold diversity is essential for future drug design. Available reviews lack the systematic study and discussion on the scaffold diversity of CDK inhibitors. Herein we have reviewed and critically analyzed the chemical diversity present in the preclinical and clinical pipeline of CDK inhibitors. Our analysis has shown that although several scaffolds represent CDK inhibitors, only the amino-pyrimidine is a well-represented scaffold. The three-nitrogen framework of amino-pyrimidine is a fundamental hinge-binding unit. Further, we have discussed the selectivity aspects among CDKs, the clinical trial dose-limiting toxicities, and highlighted the most advanced clinical candidates. We also discuss the changing paradigm towards selective inhibitors and an overview of ATP-binding pockets of all druggable CDKs. We carefully analyzed the clinical pipeline to unravel the candidates that are currently under active clinical development. In addition to the plenty of dual CDK4/6 inhibitors, there are many selective CDK7, CDK9, and CDK8/19 inhibitors in the clinical pipeline.

Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors.

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

Crocetin promotes clearance of amyloid-ß by inducing autophagy via the STK11/LKB1-mediated AMPK pathway.

Alzheimer disease (AD) is usually accompanied by two prominent pathological features, cerebral accumulation of amyloid-ß (Aß) plaques and presence of MAPT/tau neurofibrillary tangles. Dysregulated clearance of Aß largely contributes to its accumulation and plaque formation in the brain. Macroautophagy/autophagy is a lysosomal degradative process, which plays an important role in the clearance of Aß. Failure of autophagic clearance of Aß is currently acknowledged as a contributing factor to increased accumulation of Aß in AD brains. In this study, we have identified crocetin, a pharmacologically active constituent from the flower stigmas of Crocus sativus, as a potential inducer of autophagy in AD. In the cellular model, crocetin induced autophagy in N9 microglial and primary neuron cells through STK11/LKB1 (serine/threonine kinase 11)-mediated AMP-activated protein kinase (AMPK) pathway activation. Autophagy induction by crocetin significantly increased Aß clearance in N9 cells. Moreover, crocetin crossed the blood-brain barrier and induced autophagy in the brains' hippocampi of wild-type male C57BL/6 mice. Further studies in transgenic male 5XFAD mice, as a model of AD, revealed that one-month treatment with crocetin significantly reduced Aß levels and neuroinflammation in the mice brains and improved memory function by inducing autophagy that was mediated by AMPK pathway activation. Our findings support further development of crocetin as a pharmacological inducer of autophagy to prevent, slow down progression, and/or treat AD.Abbreviations: Aß: amyloid-ß; ABCB1/P-gp/P-glycoprotein: ATP-binding cassette, subfamily B (MDR/TAP), member 1; AD: Alzheimer disease; AMPK/PRKAA: AMP-activated protein kinase; APP: amyloid beta (A4) precursor protein; ATG: autophagy related; BBB: blood-brain barrier; BECN1: beclin 1, autophagy related; CAMKK2/CaMKKß: calcium/calmodulin-dependent protein kinase kinase 2, beta; CSE: Crocus sativus extract; CTSB: cathepsin B; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GFAP: glial fibrillary acidic protein; GSK3B/GSK3ß: glycogen synthase kinase 3 beta; Kp: brain partition coefficient; LRP1: low density lipoprotein receptor-related protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MAPK/ERK: mitogen-activated protein kinase; MAPT/tau: microtubule-associated protein tau; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTOR: mechanistic target of rapamycin kinase; MWM: Morris water maze; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; NMDA: N-methyl-d-aspartic acid; RPTOR: regulatory associated protein of MTOR; RPS6KB1/p70S6K: ribosomal protein S6 kinase 1; SQSTM1: sequestosome 1; SRB: sulforhodamine B; STK11/LKB1: serine/threonine kinase 11; TFEB: transcription factor EB; TSC2: TSC complex subunit 2; ULK1: unc-51 like kinase 1.

Natural Products in Mitigation of SARS CoV Infections.

Severe acute respiratory syndrome (SARS) is a critical respiratory disease caused by coronaviruses (CoV). The available antiviral agents or host-specific antiinflammatory therapies are the principal treatment modalities, with drug-repurposing as the most viable approach to timely tackle the CoV pandemic. Though these approaches are successful to some extent in reducing the mortality rate, however, it is too far to see a complete escape from the current SARS CoV-2 pandemic. Plants are the primary source of diet, dietary supplements, botanical drugs, and natural products (NPs). It has been well accepted and proved via several scientific studies that plant-based therapies play a vital role in managing such infections. The faulty immune system (compromised innate immunity or aberrant immune activation) determines the severity of the respiratory distress in CoV-2 infected patients. Natural products intervene at various stages of the virus replication cycle, including inhibition of virus entry into the host cells, inhibition of serine/ cysteine proteases, RNA-dependent RNA polymerase (RdRp) or helicase. Besides, several natural products or plant-based dietary supplements have a unique ability to strengthen the immune system or alleviate the hyper-inflammatory condition. Many plant-based formulations, dietary supplements, and NPs are being investigated in clinical trials in CoV-2 infected patients, and few have already shown positive results. The review has unearthed several NP leads for medicinal chemistry programs as well as some having direct opportunity of repurposing in SARS CoV infections.

Design, synthesis and comparative analysis of triphenyl-1,2,3-triazoles as anti-proliferative agents.

Herein, a series of triaryl-1,2,3-triazoles, in order to check cytotoxicity on breast cancer cell lines have been synthesized with pendent benzyl ring to mimic the phenolic A ring of Tamoxifene. The biological results indicated that most of the compounds possessed comparative anti-proliferative activities in both ER + ve (MCF-7) and ER-ve (MDA-MB-231) breast cancer cell lines. Among synthesized derivatives, five compounds 8f, 8i, 8j, 8n and 8p showed anti-proliferative activities at <5 µM against MCF-7 cell line and three compounds 8e, 8f and 8j show IC50 value greater than 30 µM in FR-2 cells (normal cell). Moreover, to understand the mechanistic behavior of the selective compound 8f, various studies performed viz. surface morphological changes by bright field microscopic examination, nuclear morphological alteration by DAPI staining, measurement of intracellular ROS level and determination of change in mitochondrial membrane potential. It was observed that, the selective compound 8f associated with higher ROS generation along with decrease in mitochondrial membrane potential in addition to surface and nuclear morphological alterations such as reduction in number and shrinkage of cells coupled with nuclear blabbing indicating sign of apoptosis. Further, molecular docking study in comparison to tamoxifen was also carried out to investigate the interaction of 8f with ER-α which favors its possible mode of anticancer action.

Evaluation of rohitukine-enriched fraction of Dysoxylum binectariferum Hook.f. (leaves) as anti-arthritic phytopharmaceutical candidate: Chemical standardization, in-vivo validation, formulation development and oral pharmacokinetics.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis is a chronic inflammatory disease of joints. Dysoxylum binectariferum Hook.f (Family: Meliaceae) is a Indian medicinal plant which is traditionally being used to heal inflammation of joints. AIM OF THE STUDY: This work was aimed to carry out chemical standardization, in-vitro/in-vivo validation, oral pharmacokinetics and formulation development of anti-arthritic botanical lead, the rohitukine-enriched fraction of D. binectariferum. MATERIALS AND METHODS: The rohitukine-enriched fraction of D. binectariferum was standardized using four chemical markers and was checked for microbial load, heavy metal content, aflatoxins and pesticides. Its in-vitro inhibitory effect on the lipopolysaccharide (LPS) induced production of pro-inflammatory cytokines TNF-α and IL-6 was studied in THP-1 cells. The in-vivo anti-arthritic activity was investigated in collagen-induced arthritis model in DBA/1J mice. The sustained release capsule formulation was developed and characterized for physicochemical and pharmacokinetic properties. RESULTS: Rohitukine and schumaniofioside A were found to be major chemical constituents of the botanical lead. The rohitukine-enriched fraction of D. binectariferum significantly reduced the production of both pro-inflammatory cytokines TNF-α and IL-6 (>50% inhibition at 3.12 µg/mL) in THP-1 cells. In LPS-treated wild-type mice model, the rohitukine-enriched fraction at 200 mg/kg (PO, QD) completely reduced serum TNF-α levels. In transgenic mice model (collagen-induced arthritis in DBA/1J mice), rohitukine-enriched fraction at 100 mg/kg (PO, QD) dose has resulted in >75% reduction of TNF-α/IL-6 serum levels, 68% reduction in anti-mouse type II collagen IgG1 antibody levels, decreased joint proteoglycan loss and reduced paw edema in DBA/1J mice. The sustained release capsule formulation of rohitukine-enriched fraction showed sustained-release of rohitukine over the period of 24 h, and resulted in an improved plasma-exposure of rohitukine in SD rats. CONCLUSIONS: The data presented herein demonstrated anti-arthritic potential of rohitukine-enriched fraction of D. binectariferum and this study will serve as the benchmark for further research on this botanical lead and developed sustained release capsule formulation.

Preclinical and Clinical Studies on Bryostatins, A Class of Marine-Derived Protein Kinase C Modulators: A Mini-Review.

Bryostatins are complex macrolactones isolated from marine organisms Bryozoan Bugula neritina. They are potent modulators of protein kinase C isozymes (PKCα: ki = 1.3-188 nM), and are one of the most extensively investigated marine natural products in clinical trials. Although ~21 natural bryostatins have been isolated, however only bryostatin-1 (1) has received much interest among medicinal chemists and clinicians. The structure-activity relationship of bryostatins has been well established, with the identification of key pharmacophoric features important for PKC modulation. The low natural abundance and the long synthetic route have prompted medicinal chemists to come-up with simplified analogs. Bryostatin skeleton comprises three pyran rings connected to each other to form a macrocyclic lactone. The simplest analog 27 contains only one pyran, which is also able to modulate the PKCα activity; however, the cyclic framework appears to be essential for the desired level of potency. Another simplified analog 17 ("picolog") exhibited potent and in-vivo efficacy against lymphoma. Bryostatin-1 (1) has shown an acceptable intravenous pharmacokinetic profile in mice and displayed promising in-vivo efficacy in mice models of various cancers and Alzheimer's disease. Bryostatin-1 was investigated in numerous Phase I/II oncology clinical trials; it has shown minimal effect as a single agent, however, provided encouraging results in combination with other chemotherapy agents. FDA has granted orphan drug status to bryostatin-1 in combination with paclitaxel for esophageal cancer. Bryostatin-1 has also received orphan drug status for fragile X syndrome. Bryostatin-1 was also investigated in clinical studies for Alzheimer's disease and HIV infection. In a nutshell, the natural as well as synthetic bryostatins have generated a strong hope to emerge as treatment for cancer along with many other diseases.

Discovery of benzo[cd]indol-2-one and benzylidene-thiazolidine-2,4-dione as new classes of NLRP3 inflammasome inhibitors via ER-ß structure based virtual screening.

The structure-guided virtual screening (VS) has proved to be successful strategy in identification of new scaffolds for biological targets. The overactivity of NLRP3 inflammasome has been implicated in variety of inflammatory diseases including Alzheimer's disease. The up-regulation of estrogen-receptor ß (ER-ß) activity has been directly linked with inhibition of NLRP3 inflammasome activity. In the present study, we report discovery of new NLRP3 inflammasome inhibitors via ER-ß crystal structure (PDB: 5TOA) guided virtual screening of 20,000 compound library. For experimental validation, top 10 ligands were selected based on structure novelty, docking score, prime MMGB/SA binding affinity and interaction pattern analysis. Amongst the tested compounds, three thiazolidin-4-ones IIIM-1268, IIIM-1269 and IIIM-1270 and benzo[cd]indol-2-one IIIM-1266 have shown 73, 69, 75 and 77% suppression of IL-1ß release in mouse macrophages (J774A.1 cells) at 10 µM. Benzylidene-thiazolidine-2,4-diones IIIM-1268 and IIIM-1270 inhibited IL-1ß release with IC50 of 2.3 and 3.5 µM and also significantly decreased the protein expression level of mature form of IL-1ß in western-blot analysis. IIIM-1266 and IIIM-1270 displayed bidentate H-bonding with Arg 346 and Glu 305 residues in the active site of ER-ß; and they also strongly occupied the ADP-binding site of NLRP3 protein. The results presented herein, indicate that ER-ß guided VS can be successfully used to identify new NLRP3 inflammasome inhibitors, which may have potential in the development of novel anti-Alzheimer agents.

Binary and ternary solid dispersions of an anticancer preclinical lead, IIIM-290: In vitro and in vivo studies.

The objective of this study was to formulate an anticancer preclinical lead, IIIM-290, loaded in solid dispersions to enhance its solubility, dissolution, and oral pharmacokinetics. IIIM-290 is an in-house preclinical anticancer lead prepared by semisynthetic modification of the natural product rohitukine. It is an orally bioavailable Cdk inhibitor showing efficacy in xenograft models of pancreatic, colon and leukemia cancer. It demonstrated in vivo efficacy at a relatively higher dose owing to its poor aqueous solubility (~8.6 µg/mL). Binary and ternary solid dispersions containing PVP K-30, xanthan gum, and PEG-PPG-PEG were selected after solubility screening of various hydrophilic polymers. Several formulations with varying ratios of polymers, alone and in combination, were prepared and investigated for their effects on the solubility enhancement of IIIM-290. The binary solid dispersion VKB-SD75, prepared with PVP K-30 at the ratio of 1:4 w/w, was identified as the optimized composition that displayed 17-fold improvement in the aqueous solubility of IIIM-290. VKB-SD75 was scaled up to a 100-g scale. IIIM-290 and VKB-SD75 were evaluated for DSC, p-XRD, FTIR, 1H NMR, SEM, in vitro dissolution, and oral pharmacokinetics, as well as for in vivo anticancer activity in the Ehrlich solid tumor model. The oral administration of VKB-SD75 in BALB/c mice resulted in a 1.9-fold improvement in plasma exposure. These findings also correlated well when the formulation was administered to mice in the Ehrlich solid tumor model. The newly developed solid dispersion is expected to reduce the dose of IIIM-290 by ~40-50% in preclinical and clinical studies.

Synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine and its bromo-derivative as dual cholinesterase inhibitors.

Alkaloids have always been a great source of cholinesterase inhibitors. Numerous studies have shown that inhibiting acetylcholinesterase as well as butyrylcholinetserase is advantageous, and have better chances of success in preclinical/ clinical settings. With the objective to discover dual cholinesterase inhibitors, herein we report synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine (1) and its bromo-derivative 2. Our study has shown that cryptolepine (1) and its 2-bromo-derivative 2 are dual inhibitors of acetylcholinesterase and butyrylcholinesterase, the enzymes which are involved in blocking the process of neurotransmission. Cryptolepine inhibits Electrophorus electricus acetylcholinesterase, recombinant human acetylcholinesterase and equine serum butyrylcholinesterase with IC50 values of 267, 485 and 699 nM, respectively. The 2-bromo-derivative of cryptolepine also showed inhibition of these enzymes, with IC50 values of 415, 868 and 770 nM, respectively. The kinetic studies revealed that cryptolepine inhibits human acetylcholinesterase in a non-competitive manner, with ki value of 0.88 µM. Additionally, these alkaloids were also tested against two other important pathological events of Alzheimer's disease viz. stopping the formation of toxic amyloid-ß oligomers (via inhibition of BACE-1), and increasing the amyloid-ß clearance (via P-gp induction). Cryptolepine displayed potent P-gp induction activity at 100 nM, in P-gp overexpressing adenocarcinoma LS-180 cells and excellent toxicity window in LS-180 as well as in human neuroblastoma SH-SY5Y cell line. The molecular modeling studies with AChE and BChE have shown that both alkaloids were tightly packed inside the active site gorge (site 1) via multiple π-π and cation-π interactions. Both inhibitors have shown interaction with the allosteric "peripheral anionic site" via hydrophobic interactions. The ADME properties including the BBB permeability were computed for these alkaloids, and were found within the acceptable range.

Alborixin clears amyloid-ß by inducing autophagy through PTEN-mediated inhibition of the AKT pathway.

Imbalance in production and clearance of amyloid beta (Aß) is the primary reason for its deposition in Alzheimer disease. Macroautophagy/autophagy is one of the important mechanisms for clearance of both intracellular and extracellular Aß. Here, through screening, we identified alborixin, an ionophore, as a potent inducer of autophagy. We found that autophagy induced by alborixin substantially cleared Aß in microglia and primary neuronal cells. Induction of autophagy was accompanied by up regulation of autophagy proteins BECN1/Beclin 1, ATG5, ATG7 and increased lysosomal activities. Autophagy induced by alborixin was associated with inhibition of the phosphoinositide 3-kinase (PI3K)-AKT pathway. A knock down of PTEN and consistent, constitutive activation of AKT inhibited alborixin-induced autophagy and consequent clearance of Aß. Furthermore, clearance of Aß by alborixin led to significant reduction of Aß-mediated cytotoxicity in primary neurons and differentiated N2a cells. Thus, our findings put forward alborixin as a potential anti-Alzheimer therapeutic lead. Abbreviations: Aß: amyloid beta; ALB: alborixin; ATG: autophagy-related; BECN1: beclin 1; DAPI: 4, 6-diamidino-2-phenylindole; DCFH-DA: 2,7-dichlorodihydrofluorescein diacetate; fAß: fibrillary form of amyloid beta; GFAP: glial fibrillary acidic protein; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MTOR: mechanistic target of rapamycin kinase; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SQSTM1: sequestosome 1; TMRE: tetramethylrhodamine, ethyl ester.

CYP enzymes, expressed within live human suspension cells, are superior to widely-used microsomal enzymes in identifying potent CYP1A1/CYP1B1 inhibitors: Identification of quinazolinones as CYP1A1/CYP1B1 inhibitors that efficiently reverse B[a]P toxicity and cisplatin resistance.

Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they may not always portray how a developmental drug would behave in human cells with intact intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293 kidney cells, grown in 'suspension' for expression of CYPs, in finding potent CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell-based assays, quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC50 values of 30/21 nM, and > 150-fold selectivity over CYP2/3 enzymes, whereas they were far less active using commercially-available CYP1A1/CYP1B1 microsomal enzymes (IC50, >10/1.3-1.7 µM). Compound 9i prevented CYP1A1-mediated benzo[a]pyrene-toxicity in normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC-3/prostate, COR-L23/lung, MIAPaCa-2/pancreatic and LS174T/colon cancer cells, underlining the human-cell-assays' potential. Our results indicate that the most potent CYP1A1/CYP1B1 inhibitors would not have been identified if one had relied merely on microsomal enzymes.