Regulation of integrin and extracellular matrix genes by HNRNPL is necessary for epidermal renewal.

Stratified epithelia such as the epidermis require coordinated regulation of stem and progenitor cell proliferation, survival, and differentiation to maintain homeostasis. Integrin-mediated anchorage of the basal layer stem cells of the epidermis to the underlying dermis through extracellular matrix (ECM) proteins is crucial for this process. It is currently unknown how the expression of these integrins and ECM genes are regulated. Here, we show that the RNA-binding protein (RBP) heterogeneous nuclear ribonucleoprotein L (HNRNPL) binds to these genes on chromatin to promote their expression. HNRNPL recruits RNA polymerase II (Pol II) to integrin/ECM genes and is required for stabilizing Pol II transcription through those genes. In the absence of HNRNPL, the basal layer of the epidermis where the stem cells reside prematurely differentiates and detaches from the underlying dermis due to diminished integrin/ECM expression. Our results demonstrate a critical role for RBPs on chromatin to maintain stem and progenitor cell fate by dictating the expression of specific classes of genes.

CDK12 Is Necessary to Promote Epidermal Differentiation Through Transcription Elongation.

Proper differentiation of the epidermis is essential to prevent water loss and to protect the body from the outside environment. Perturbations in this process can lead to a variety of skin diseases that impacts 1 in 5 people. While transcription factors that control epidermal differentiation have been well characterized, other aspects of transcription control such as elongation are poorly understood. Here we show that of the two cyclin-dependent kinases (CDK12 and CDK13), that are known to regulate transcription elongation, only CDK12 is necessary for epidermal differentiation. Depletion of CDK12 led to loss of differentiation gene expression and absence of skin barrier formation in regenerated human epidermis. CDK12 binds to genes that code for differentiation promoting transcription factors (GRHL3, KLF4, and OVOL1) and is necessary for their elongation. CDK12 is necessary for elongation by promoting Ser2 phosphorylation on the C-terminal domain of RNA polymerase II and the stabilization of binding of the elongation factor SPT6 to target genes. Our results suggest that control of transcription elongation by CDK12 plays a prominent role in adult cell fate decisions.

RAS-mediated suppression of PAR3 and its effects on SCC initiation and tissue architecture occur independently of hyperplasia.

Proper epithelial development and homeostasis depends on strict control of oriented cell division. Current evidence shows that this process is regulated by intrinsic polarity factors and external spatial cues. Owing to the lack of an appropriate model system that can recapitulate the architecture of the skin, deregulation of spindle orientation in human epithelial carcinoma has never been investigated. Here, using an inducible model of human squamous cell carcinoma (SCC), we demonstrate that RAS-dependent suppression of PAR3 (encoded by PARD3) accelerates epithelial disorganization during early tumorigenesis. Diminished PAR3 led to loss of E-cadherin-mediated cell adhesion, which in turn contributed to misoriented cell division. Pharmacological inhibition of the MAPK pathway downstream of RAS activation reversed the defects in PAR3 expression, E-cadherin-mediated cell adhesion and mitotic spindle orientation. Thus, temporal analysis of human neoplasia provides a powerful approach to study cellular and molecular transformations during early oncogenesis, which allowed identification of PAR3 as a critical regulator of tissue architecture during initial human SCC development.

Microbial biofilm ecology, in silico study of quorum sensing receptor-ligand interactions and biofilm mediated bioremediation.

Biofilms are structured microbial communities of single or multiple populations in which microbial cells adhere to a surface and get embedded in extracellular polymeric substances (EPS). This review attempts to explain biofilm architecture, development phases, and forces that drive bacteria to promote biofilm mode of growth. Bacterial chemical communication, also known as Quorum sensing (QS), which involves the production, detection, and response to small molecules called autoinducers, is highlighted. The review also provides a brief outline of interspecies and intraspecies cell-cell communication. Additionally, we have performed docking studies using Discovery Studio 4.0, which has enabled our understanding of the prominent interactions between autoinducers and their receptors in different bacterial species while also scoring their interaction energies. Receptors, such as LuxN (Phosphoreceiver domain and RecA domain), LuxP, and LuxR, interacted with their ligands (AI-1, AI-2, and AHL) with a CDocker interaction energy of - 31.6083 kcal/mole; - 34.5821 kcal/mole, - 48.2226 kcal/mole and - 41.5885 kcal/mole, respectively. Since biofilms are ideal for the remediation of contaminants due to their high microbial biomass and their potential to immobilize pollutants, this article also provides an overview of biofilm-mediated bioremediation.

Crizotinib-induced anti-cancer activity in human cervical carcinoma cells via ROS-dependent mitochondrial depolarization and induction of apoptotic pathway.

INTRODUCTION: Cervical cancer is one of the leading causes of mortality among women population worldwide. In spite of recurrent screening, vaccination, and chemotherapeutic interventions, combating cervical cancer still remains a challenge. Crizotinib is a small molecule inhibitor that targets mesenchymal epithelial transition factor (c-MET) and has been successfully studied for its anti-cancer effects in non-small cell lung cancer, pancreatic, gastric, renal, prostate, and breast carcinomas. Although c-MET is a well-known prognostic, diagnostic, and therapeutic target in cervical cancer, anti-cancer properties of its inhibitor crizotinib against cervical carcinoma, has not been explored yet. METHODS: In the present study, the anti-cancer effects of crizotinib on cervical cancer cells were evaluated using various in vitro cell-based assays, such as labelling drug-treated cells with MTT, H2 DCFDA, Annexin V5-fluorescein isothiocyanate (FITC) antibody, JC-1, PI, and analysis using fluorescence-activated cell sorting (FACS). RESULTS: The molecule was found to effectively inhibit proliferation of cervical cancer cells HeLa and SiHa with an IC50 of 0.641 ± 0.0724 and 0.871 ± 0.104 µM, respectively, and induce apoptosis in a dose-dependent manner. Further investigations showed that crizotinib-induced production of reactive oxygen species (ROS) with increasing concentrations further resulted in mitochondrial membrane depolarization. However, the drug had no effect on cell cycle progression of HeLa and SiHa cells. CONCLUSION: Thus, the study elucidates the cytotoxic effects of crizotinib in cervical cancer cells by activation of ROS-dependent apoptotic pathway via mitochondrial depolarization. These findings will further aid the evaluation of other molecular mechanisms of crizotinib and would pave the way for its implication as a chemotherapeutic option in cervical cancer.

Progress in the development of human carbonic anhydrase inhibitors and their pharmacological applications: Where are we today?

Carbonic anhydrases (CAs, EC 4.2.1.1) are widely distributed metalloenzymes in both prokaryotes and eukaryotes. They efficiently catalyze the reversible hydration of carbon dioxide to bicarbonate and H+  ions and play a crucial role in regulating many physiological processes. CAs are well-studied drug target for various disorders such as glaucoma, epilepsy, sleep apnea, and high altitude sickness. In the past decades, a large category of diverse families of CA inhibitors (CAIs) have been developed and many of them showed effective inhibition toward specific isoforms, and effectiveness in pathological conditions in preclinical and clinical settings. The discovery of isoform-selective CAIs in the last decade led to diminished side effects associated with off-target isoforms inhibition. The many new classes of such compounds will be discussed in the review, together with strategies for their development. Pharmacological advances of the newly emerged CAIs in diseases not usually associated with CA inhibition (neuropathic pain, arthritis, cerebral ischemia, and cancer) will also be discussed.

Development of novel carbazole derivatives with effective multifunctional action against Alzheimer's diseases: Design, synthesis, in silico, in vitro and in vivo investigation.

Carbazole based novel multifunctional agents has been rationally designed and synthesized as potential anti-Alzheimer agents. Multi-functional activity of these derivatives have been assessed by performing various in-vitro assays and these compounds appeared to be potent AChE inhibitors, Aß aggregation inhibitors, anti-oxidant and neuroprotective agents. Among the entire series, MT-1 and MT-6 were most potent multifunctional agents which displayed effective and selective AChE inhibition, Aß disaggregation, anti-oxidant and metal chelation action. Neuroprotective activity of MT-6 has been examined against H2O2 induced toxicity in SHSY-5Y cells and they have shown effective neuroprotection. Additionally, MT-6 did not display any significant toxicity in SHSY-5Y cells, indicating its non-toxic nature. Molecular docking and MD simulation studies have been also performed to explore molecular level interaction with AChE and Aß. Finally, MT-6 was evaluated against scopolamine induced dementia model of mice and this compound actively improved memory deficit and cognition impairment in scopolamine treated mice. Thus, novel carbazole derivative MT-6 has been explored as an effective and safe multifunctional agent against AD and this molecule may be used as a suitable lead for development of effective anti-Alzheimer agents in future.

Naphthalene-triazolopyrimidine hybrid compounds as potential multifunctional anti-Alzheimer's agents.

In an attempt to construct potential anti-Alzheimer's agents Naphthalene-triazolopyrimidine hybrids were synthesized and screened in vitro against the two cholinesterases (ChE)s, amyloid ß aggregation and for antioxidation activity. Single-crystal X-ray crystallography was utilized for crystal structure determination of one of the compounds. In vitro study of compounds revealed that most of the compounds are capable of inhibiting acetylcholinesterase and Butyrylcholinesterase activity. Particularly, the compounds 4e and 4d exhibited IC50 values ranging from 8.6 to 14 nM against AChE lower than the standard drug Donepezil (IC50 49 nM). Best result was found for compound 4e with IC50 of 8.6 nM (for AChE) and 150 nM (for BuChE). Selectivity upto that of Donepezil and even more was observed for 4a, 4c and 4h. Investigation by electron microscopy, transmission electron microscopy and ThT fluorescence assay unveils the fact that synthesized hybrids exhibit amyloid ß self-aggregation inhibition. The compounds 4i and 4j revealed highest inhibitory potential, 85.46% and 72.77% at 50 µM respectively; above the standard Aß disaggregating agent, Curcumin. Their antioxidation profile was also analyzed. Studies from DPPH free radical scavenging assay and ORAC assay depicts molecules to possess low antioxidation profile. Results suggest that triazolopyrimidines are potential candidate for Acetylcholinesterase (AChE), Butyrylcholinesterase (BuChE), and amyloid ß aggregation inhibition. In silico ADMET profiling indicates drug-like properties with a very low toxic influence. Such synthesized compounds provide a strong vision for further development of potential anti-Alzheimer's agents.

Design, synthesis, in silico and biological evaluation of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazine carboxamides.

A series of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazinecarboxamide derivatives has been successfully designed and synthesized to evaluate their potential as carbonic anhydrase (CA) inhibitors. The inhibitory potential of synthesized compounds against human CAI and CAII was evaluated. Compounds 3a-n exhibited [Formula: see text] values between [Formula: see text] against CAI and [Formula: see text] against CAII. Compound 3g was the most active inhibitor, with an [Formula: see text] value of [Formula: see text] against CAII. Molecular docking studies of compound 3g with CAII showed this compound fits nicely in the active site of CAII and it interacts with the zinc ion ([Formula: see text]) along with three histidine residues in the active site. Molecular dynamics simulation studies of compound 3g complexed with CAII also showed essential interactions which were maintained up to 40 ns of simulation. In vivo sub-acute toxicity study using 3g (300 mg/kg) was found non-toxic in adult Wistar rats.

Esculetin induces antiproliferative and apoptotic response in pancreatic cancer cells by directly binding to KEAP1.

BACKGROUND: A handful of studies have exploited antitumor potential of esculetin, a dihydroxy coumarine derivative; the targets to which it binds and the possible downstream mechanism for its cytotoxicity in cancer cells remain to be elucidated. Using pancreatic cancer cell lines as a model system, herein the study was initiated to check the efficacy of esculetin in inhibiting growth of these cancer cells, to decipher mechanism of its action and to predict its direct binding target protein. METHODS: The cytotoxicity of esculetin was determined in PANC-1, MIA PaCa-2 and AsPC-1 cell lines; followed by an inspection of intracellular levels of ROS and its associated transcription factor, p65-NF-κB. The interaction between transcription factor, Nrf2 and its regulator KEAP1 was studied in the presence and absence of esculetin. The effect of Nrf2 on gene expression of antioxidant response element pathway was monitored by real time PCR. Thereafter, potential binding target of esculetin was predicted through molecular docking and then confirmed in vitro. RESULTS: Esculetin treatment in all three pancreatic cancer cell lines resulted in significant growth inhibition with G1-phase cell cycle arrest and induction of mitochondrial dependent apoptosis through activation of caspases 3, 8 and 9. A notable decrease was observed in intracellular ROS and protein levels of p65-NF-κB in PANC-1 cells on esculetin treatment. Antioxidant response regulator Nrf2 has been reportedly involved in crosstalk with NF-κB. Interaction between Nrf2 and KEAP1 was found to be lost upon esculetin treatment in PANC-1 and MIA Paca-2 cells. Nuclear accumulation of Nrf2 and an upregulation of expression of Nrf2 regulated gene NQO1, observed on esculetin treatment in PANC-1 further supported the activation of Nrf2. To account for the loss of Nrf2-KEAP1 interaction on esculetin treatment, direct binding potential between esculetin and KEAP1 was depicted in silico using molecular docking studies. Pull down assay using esculetin conjugated sepharose beads confirmed the binding between esculetin and KEAP1. CONCLUSIONS: We propose that esculetin binds to KEAP1 and inhibits its interaction with Nrf2 in pancreatic cancer cells. This thereby promotes nuclear accumulation of Nrf2 in PANC-1 cells that induces antiproliferative and apoptotic response possibly by attenuating NF-κB.

Design, synthesis and evaluation of novel indandione derivatives as multifunctional agents with cholinesterase inhibition, anti-ß-amyloid aggregation, antioxidant and neuroprotection properties against Alzheimer's disease.

A series of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)-1H-indene-1,3(2H)-diones were designed, synthesized and appraised as multifunctional anti-Alzheimer agents. In vitro studies of compounds 27-38 showed that these compounds exhibit moderate to excellent AChE, BuChE and Aß aggregation inhibitory activity. Notably, compounds 34 and 38 appeared as most active multifunctional agents in the entire series and exhibited excellent inhibition against AChE (IC50=0.048µM: 34; 0.036µM: 38), Aß aggregation (max% inhibition 82.2%, IC50=9.2µM: 34; max% inhibition 80.9%, IC50=10.11µM: 38) and displayed significant antioxidant potential in ORAC-FL assay. Both compounds also successfully diminished H2O2 induced oxidative stress in SH-SY5Y cells. Fascinatingly, compounds 34 and 38 showed admirable neuroprotective effects against H2O2 and Aß induced toxicity in SH-SY5Y cells. Additionally, both derivatives showed no considerable toxicity in neuronal cell viability assay and represented drug likeness properties in the primarily pharmacokinetics study. All these results together, propelled out that compounds 34 and 38 might serve as promising multi-functional lead candidates for treatment of AD in the future.

Development of cyanopyridine-triazine hybrids as lead multitarget anti-Alzheimer agents.

A series of new cyanopyridine-triazine hybrids were designed, synthesized and screened as multitargeted anti-Alzheimer's agents. These molecules were designed while using computational techniques and were synthesized via a feasible concurrent synthetic route. Inhibition potencies of synthetic compounds 4a-4h against cholinesterases, Aß1-42 disaggregation, oxidative stress, cytotoxicity, and neuroprotection against Aß1-42-induced toxicity of the synthesized compounds were evaluated. Compounds 4d and 4h showed promising inhibitory activity on acetylcholinesterase (AChE) with IC50 values 0.059 and 0.080µM, respectively, along with good inhibition selectivity against AChE over butyrylcholinesterase (BuChE). Molecular modelling studies revealed that these compounds interacted simultaneously with the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. The mixed type inhibition of compound 4d further confirmed their dual binding nature in kinetic studies. Furthermore, the results from neuroprotection studies of most potent compounds 4d and 4h indicate that these derivatives can reduce neuronal death induced by H2O2-mediated oxidative stress and Aß1-42 induced cytotoxicity. In addition, in silico analysis of absorption, distribution, metabolism and excretion (ADME) profile of best compounds 4d and 4h revealed that they have drug like properties. Overall, these cyanopyridine-triazine hybrids can be considered as a candidate with potential impact for further pharmacological development in Alzheimer's therapy.

Design, synthesis and biological evaluation of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4-(3-substitutedphenylureido) benzenesulfonamides as human carbonic anhydrase isoenzymes I, II, VII and XII inhibitors.

A series of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4-(3-substitutedphenylureido) benzenesulfonamide derivatives has been designed, synthesized and screened for their in vitro human carbonic anhydrase (hCA; EC 4.2.1.1) inhibition potential. These newly synthesized sulfonamide compounds were assessed against isoforms hCA I, II, VII and XII, with acetazolamide (AAZ) as a reference compound. The majority of these compounds were found quite weak inhibitor against all tested isoforms. Compound 15 showed a modest inhibition potency against hCA I (Ki = 73.7 µM) and hCA VII (Ki = 85.8 µM). Compounds 19 and 25 exhibited hCA II inhibition with Ki values of 96.0 µM and 87.8 µM, respectively. The results of the present study suggest that, although the synthesized derivatives have weak inhibitory potential towards all investigated isoforms, some of them may serve as lead molecules for the further development of selective inhibitors incorporating secondary sulfonamide functionalities, a class of inhibitors for which the inhibition mechanism is poorly understood.

Design, synthesis and pharmacological evaluation of N-[4-(4-(alkyl/aryl/heteroaryl)-piperazin-1-yl)-phenyl]-carbamic acid ethyl ester derivatives as novel anticonvulsant agents.

A series of alkyl/aryl/heteroaryl piperazine derivatives (37-54) were designed and synthesized as potential anticonvulsant agents. The target compounds are endowed with satisfactory physicochemical as well as pharmacokinetic properties. The synthesized compounds were screened for their in vivo anticonvulsant activity in maximal electroshock (MES) and subcutaneous pentylenetetrazole (sc-PTZ) seizure tests. Further, neurotoxicity evaluation was carried out using rotarod method. Structure activity relationship studies showed that compounds possessing aromatic group at the piperazine ring displayed potent anticonvulsant activity. Majority of the compounds showed anti-MES activity whereas compounds 39, 41, 42, 43, 44, 50, 52, and 53 exhibited anticonvulsant activity in both seizure tests. All the compounds except 42, 46, 47, and 50 did not show neurotoxicity. The most active derivative, 45 demonstrated potent anticonvulsant activity in MES test at the dose of 30mg/kg (0.5h) and 100mg/kg (4h) and also delivered excellent protection in sc-PTZ test (100mg/kg) at both time intervals. Therefore, compound 45 was further assessed in PTZ-kindling model of epilepsy which is widely used model for studying epileptogenesis. This compound was effective in delaying onset of PTZ-evoked seizures at the dose of 5mg/kg in kindled animals and significantly reduced oxidative stress better than standard drug phenobarbital (PB). In result, compound 45 emerged as a most potent and safer anticonvulsant lead molecule.

Multifunctional novel Diallyl disulfide (DADS) derivatives with ß-amyloid-reducing, cholinergic, antioxidant and metal chelating properties for the treatment of Alzheimer's disease.

A series of novel Diallyl disulfide (DADS) derivatives were designed, synthesized and evaluated as chemical agents, which target and modulate multiple facets of Alzheimer's disease (AD). The results showed that the target compounds 5a-l and 7e-m exhibited significant anti-Aß aggregation activity, considerable acetylcholinesterase (AChE) inhibition, high selectivity towards AChE over butyrylcholinesterase (BuChE), potential antioxidant and metal chelating activities. Specifically, compounds 7k and 7l exhibited highest potency towards self-induced Aß aggregation (74% and 71.4%, 25 µM) and metal chelating ability. Furthermore, compounds 7k and 7l disaggregated Aß fibrils generated by Cu(2+)-induced Aß aggregation by 80.9% and 78.5%, later confirmed by transmission electron microscope (TEM) analysis. Besides, 7k and 7l had the strongest AChE inhibitory activity with IC50 values of 0.056 µM and 0.121 µM, respectively. Furthermore, molecular modelling studies showed that these compounds were capable of binding simultaneously to catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. All the target compounds displayed moderate to excellent antioxidant activity with ORAC-FL values in the range 0.546-5.86Trolox equivalents. In addition, absorption, distribution, metabolism and excretion (ADME) profile and toxicity prediction (TOPKAT) of best compounds 7k and 7l revealed that they have drug like properties and possess very low toxic effects. Collectively, the results strongly support our assertion that these compounds could provide good templates for developing new multifunctional agents for AD treatment.

Synthesis, biological evaluation and molecular docking study of novel piperidine and piperazine derivatives as multi-targeted agents to treat Alzheimer's disease.

Development of Multi-Target Directed Ligands (MTDLs) has emerged as a promising approach for targeting complex etiology of Alzheimer's disease (AD). Following this approach, a new series of N'-(4-benzylpiperidin-/piperazin-/benzhydrylpiperazin-1-yl)alkylamine derivatives were designed, synthesized and biologically evaluated as inhibitors of cholinesterases (ChEs), amyloid-beta (Aß) self aggregation and also for their radical scavenging activity. The in vitro studies showed that the majority of synthesized derivatives strongly inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC50 values in the low-nanomolar range, and were clearly more potent than the reference compound donepezil in this regard. Among them, inhibitors 5h and 5k, strongly inhibited AChE, with IC50 value of 6.83nM and 2.13nM, respectively, and particularly, compound 5k was found to be highly selective for AChE (∼38-fold). Moreover, both kinetic analysis of AChE inhibition and the docking study suggested that 5k binds simultaneously to catalytic active site and peripheral anionic site of AChE. Besides, these compounds also exhibited greater ability to inhibit self-induced Aß1-42 aggregation at 25µM with percentage inhibition from ∼54% to 89% and specially compound 5k provided highest inhibition (88.81%). Also, the derivatives containing methoxy and hydroxy groups showed potent oxygen radical absorbance capacity (ORAC) ranging from 2.2- to 4.4-fold of the Trolox value. Furthermore, results of ADMET studies suggested that all compounds exhibited appropriate drug like properties. Taken together, these results suggest that 5k might be a promising lead compound for further AD drug development.

Synthesis and characterization of Fe3O4@SiO2@PDA@Ag core-shell nanoparticles and biological application on human lung cancer cell line and antibacterial strains.

Novel magnetic and metallic nanoparticles garner much attention of researchers due to their biological, chemical and catalytic properties in many chemical reactions. In this study, we have successfully prepared a core-shell Fe3O4@SiO2@PDA nanocomposite wrapped with Ag using a simple synthesis method, characterised and tested on small cell lung cancer and antibacterial strains. Incorporating Ag in Fe3O4@SiO2@PDA provides promising advantages in biomedical applications. The magnetic Fe3O4 nanoparticles were coated with SiO2 to obtain negatively charged surface which is then coated with polydopamine (PDA). Then silver nanoparticles were assembled on Fe3O4@SiO2@PDA surface, which results in the formation core-shell nanocomposite. The synthesised nanocomposite were characterized using SEM-EDAX, dynamic light scattering, XRD, FT-IR and TEM. In this work, we report the anticancer activity of silver nanoparticles against H1299 lung cancer cell line using MTT assay. The cytotoxicity data revealed that the IC50 of Fe3O4@SiO2@PDA@Ag against H1299 lung cancer nanocomposites cells was 21.52 µg/mL. Furthermore, the biological data of nanocomposites against Gram-negative 'Pseudomonas aeruginosa' and Gram-positive 'Staphylococcus aureus' were carried out. The range of minimum inhibitory concentration was found to be 115 µg/mL where gentamicin was used as a standard drug. The synthesized AgNPs proves its supremacy as an efficient biomedical agent and AgNPs may act as potential beneficial molecule in lung cancer chemoprevention and antibacterial strains.

In the present study, we have successfully prepared a core-shell Fe₃O₄@SiO₂@PDA@Ag nanocomposite.We have investigated the dose-dependent cellular toxicity of silver nanocomposite in the nonsmall cell lung cancer cell line H1299 using MTT assay.Also, we have evaluated the mode of cell death using apoptosis.We have also evaluated the bioactivity of AgNPs on both Gram-positive and Gram-negative bacterial cells with highly efficient antibacterial potency.

Multitarget action of Benzothiazole-piperazine small hybrid molecule against Alzheimer's disease: In silico, In vitro, and In vivo investigation.

A novel small molecule based on benzothiazole-piperazine has been identified as an effective multi-target-directed ligand (MTDL) against Alzheimer's disease (AD). Employing a medicinal chemistry approach, combined with molecular docking, MD simulation, and binding free energy estimation, compound 1 emerged as a potent MTDL against AD. Notably, compound 1 demonstrated efficient binding to both AChE and Aß1-42, involving crucial molecular interactions within their active sites. It displayed a binding free energy (ΔGbind) -18.64± 0.16 and -16.10 ± 0.18 kcal/mol against AChE and Aß1-42, respectively. In-silico findings were substantiated through rigorous in vitro and in vivo studies. In vitro analysis confirmed compound 1 (IC50=0.42 µM) as an effective, mixed-type, and selective AChE inhibitor, binding at both the enzyme's catalytic and peripheral anionic sites. Furthermore, compound 1 demonstrated a remarkable ability to reduce the aggregation propensity of Aß, as evidenced by Confocal laser scanning microscopy and TEM studies. Remarkably, in vivo studies exhibited the promising therapeutic potential of compound 1. In a scopolamine-induced memory deficit mouse model of AD, compound 1 showed significantly improved spatial memory and cognition. These findings collectively underscore the potential of compound 1 as a promising therapeutic candidate for the treatment of AD.

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell.

Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K(d), for the heterodimer in living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K(d) values of mCherry2- and EGFP-fused p50 and p65 were determined to be 0.46 µM in the cytoplasm and 1.06 µM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.

Structure-based pharmacophore mapping and virtual screening of natural products to identify polypharmacological inhibitor against c-MET/EGFR/VEGFR-2.

Three receptor tyrosine kinases (RTKs), c-MET, EGFR, and VEGFR-2 have been identified as potential oncogenic targets involved in tumor development, metastasis, and invasion. Designing inhibitors that can simultaneously interact with multiple targets is a promising approach, therefore, inhibiting these three RTKs with a single chemical component might give an effective chemotherapeutic strategy for addressing the disease while limiting adverse effects. The in-silico methods have been developed to identify the polypharmacological inhibitors particularly for drug repurposing and multitarget drug design. Here, to find a viable inhibitor from natural source against these three RTKs, structure-based pharmacophore mapping and virtual screening of SN-II database were carried out. The filtered compound SN00020821, identified as Cedeodarin, from different computational approaches, demonstrated good interactions with all the three targets, c-MET/EGFR/VEGFR-2, with interaction energies of -42.35 kcal/mol, -49.32 kcal/mol and -44.83 kcal/mol, respectively. SN00020821displayed stable key interactions with critical amino acids of all the three receptors' kinase catalytic domains including "DFG motif" explored through the MD simulations. Furthermore, it also met the ADMET requirements and was determined to be drug-like as predicted from the Lipinski's rule of five and Veber's rule. Finally, SN00020821 provides a novel molecular scaffold that could be investigated further as a polypharmacological anticancer therapeutic candidate that targets the three RTKs.Communicated by Ramaswamy H. Sarma.