Adenosine Triphosphate Binding Site Research Articles

Inducing mitochondriopathy-like damages by transformable nucleopeptide nanoparticles for targeted therapy of bladder cancer.

Mitochondriopathy inspired adenosine triphosphate (ATP) depletions have been recognized as a powerful way for controlling tumor growth. Nevertheless, selective sequestration or exhaustion of ATP under complex biological environments remains a prodigious challenge. Harnessing the advantages of in vivo self-assembled nanomaterials, we designed an Intracellular ATP Sequestration (IAS) system to specifically construct nanofibrous nanostructures on the surface of tumor nuclei with exposed ATP binding sites, leading to highly efficient suppression of bladder cancer by induction of mitochondriopathy-like damages. Briefly, the reported transformable nucleopeptide (NLS-FF-T) self-assembled into nuclear-targeted nanoparticles with ATP binding sites encapsulated inside under aqueous conditions. By interaction with KPNA2, the NLS-FF-T transformed into a nanofibrous-based ATP trapper on the surface of tumor nuclei, which prevented the production of intracellular energy. As a result, multiple bladder tumor cell lines (T24, EJ and RT-112) revealed that the half-maximal inhibitory concentration (IC50) of NLS-FF-T was reduced by approximately 4-fold when compared to NLS-T. Following intravenous administration, NLS-FF-T was found to be dose-dependently accumulated at the tumor site of T24 xenograft mice. More significantly, this IAS system exhibited an extremely antitumor efficacy according to the deterioration of T24 tumors and simultaneously prolonged the overall survival of T24 orthotopic xenograft mice. Together, our findings clearly demonstrated the therapeutic advantages of intracellular ATP sequestration-induced mitochondriopathy-like damages, which provides a potential treatment strategy for malignancies.

Rational design, synthesis and structural characterization of peptides and peptidomimetics to target Hsp90/Cdc37 interaction for treating hepatocellular carcinoma

Heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) work together as a molecular chaperone complex to regulate the activity of a multitude of client protein kinases. These kinases belong to a wide array of intracellular signaling networks that mediate multiple cellular processes including proliferation. As a result, Hsp90 and Cdc37 represent innovative therapeutic targets in various cancers (such as leukemia, multiple myeloma, and hepatocellular carcinoma (HCC)) in which their expression levels are elevated. Conventional small molecule Hsp90 inhibitors act by blocking the conserved adenosine triphosphate (ATP) binding site. However, by targeting less conserved sites in a more specific manner, peptides and peptidomimetics (modified peptides) hold potential as more efficacious and less toxic alternatives to the conventional small molecule inhibitors. Using a rational approach, we herein developed bioactive peptides targeting Hsp90/Cdc37 interaction. A six amino acid linear peptide derived from Cdc37, KTGDEK, was designed to target Hsp90. We used in silico computational docking to first define its mode of interaction, and binding orientation, and then conjugated the peptide with a cell penetrating peptide, TAT, and a fluorescent dye to confirm its ability to colocalize with Hsp90 in HCC cells. Based on the parent linear sequence, we developed a peptidomimetics library of pre-cyclic and cyclic derivatives. These peptidomimetics were evaluated for their binding affinity to Hsp90, and bioactivity in HCC cell lines. Among them, a pre-cyclic peptidomimetic demonstrates high binding affinity and bioactivity in HCC cells, causing reduced cell proliferation that is associated with induction of cell apoptosis, and down-regulation of phosphorylated MEK1/2. Overall, this generalized approach of rational design, structural optimization, and cellular validation of ‘drug-like’ peptidomimetics against Hsp90/Cdc37 offers a feasible and promising way to design novel therapeutic agents for malignancies and other diseases that are dependent on this molecular chaperone complex.

Synthesis, Cytotoxic Evaluation, and Structure-Activity Relationship of Substituted Quinazolinones as Cyclin-Dependent Kinase 9 Inhibitors

Cyclin-dependent kinase 9 (CDK9) plays a critical role in transcriptional elongation, through which short-lived antiapoptotic proteins are overexpressed and make cancer cells resistant to apoptosis. Therefore, CDK9 inhibition depletes antiapoptotic proteins, which in turn leads to the reinstatement of apoptosis in cancer cells. Twenty-seven compounds were synthesized, and their CDK9 inhibitory and cytotoxic activities were evaluated. Compounds 7, 9, and 25 were the most potent CDK9 inhibitors, with IC50 values of 0.115, 0.131, and 0.142 μM, respectively. The binding modes of these molecules were studied via molecular docking, which shows that they occupy the adenosine triphosphate binding site of CDK9. Of these three molecules, compound 25 shows good drug-like properties, as it does not violate Lipinski's rule of five. In addition, this molecule shows promising ligand and lipophilic efficiency values and is an ideal candidate for further optimization.

Asciminib (Scemblix): A third-line treatment option for chronic myeloid leukemia in chronic phase with or without T315I mutation.

To review the pharmacology, efficacy, safety, dosing and administration, and place in therapy of asciminib, an oral tyrosine kinase inhibitor (TKI) used as a third-line treatment option for Philadelphia chromosome-positive chronic myeloid leukemia (CML) in chronic phase. CML is a rare cancer caused by a chromosomal translocation that forms a fusion of the BCR and ABL1 genes on chromosomes 22 and 9. Until recently, patients for whom first-line treatment options failed were treated with TKIs that bind to the adenosine triphosphate-binding site on BCR-ABL1. However, because of similar mechanisms of action, there continues to be an unmet need in patients for whom at least 2 TKIs have failed or those with a T315I mutation unable to tolerate ponatinib. In October 2021, the Food and Drug Administration approved asciminib (Scemblix), the first TKI specifically targeting the ABL1 myristoyl pocket (STAMP) via allosteric binding, as a third-line option for patients with chronic-phase (CP)-CML. Asciminib received accelerated approval due to meeting its primary endpoint at week 24, demonstrating a major molecular response rate of 25.5% for patients on asciminib compared to 13.2% for those receiving bosutinib. In addition, patients on asciminib achieved a higher rate of complete cytogenetic response at 40.8% compared to a rate of 24.2% for bosutinib. Clinicians prescribing asciminib should monitor for increased levels of pancreatic enzymes, hypertension, cardiovascular toxicity including ischemic and thromboembolic conditions, and decreased numbers of neutrophils and platelets, as these may require treatment interruption, dose reduction, or treatment discontinuation. Asciminib is a unique targeted TKI that provides clinicians with an additional third-line and beyond treatment option for adults with CP-CML regardless of mutation status as well as a second TKI treatment option for patients harboring a T315I mutation.

Luteolin enhances erlotinib's cell proliferation inhibitory and apoptotic effects in glioblastoma cell lines.

The epidermal growth factor (EGFR) receptor is frequently overexpressed in glioblastoma multiforme IV (GBM). Increased expression of EGFR leads to increased proliferation, decreased apoptosis, and increased resistance to chemotherapeutic agents. A small molecule called erlotinib inhibits EGFR receptors by binding to their adenosine triphosphate (ATP) binding sites. It is FDA approved to treat a variety of EGFR-mediated cancers. Several clinical trials have explored a combination of erlotinib with other agents to treat glioblastoma since it is believed that erlotinib would benefit patients with GBM with EGFR mutations or expression. Luteolin, a natural flavonoid, inhibits cell growth and induces apoptosis in cancer cells. We investigated the combined effects of erlotinib and luteolin on proliferation and apoptosis on glioblastoma cell lines overexpressing EGFR or glioma cells expressing truncated EGFR (ΔEGFR). In a concentration-dependent fashion, the combination of luteolin and erlotinib reduced cell proliferation (p < 0.05) and induced apoptosis by cleaving PARP and increasing caspase expression. In addition, the combination of luteolin and erlotinib reduced the phosphorylation of downstream EGFR cell signaling molecules such as Akt, NF kappa B, and STAT3 in a concentration-dependent manner. These findings suggest that combining luteolin with erlotinib offers a potential treatment strategy for glioblastoma multiforme IV.

Mebendazole's Conformational Space and Its Predicted Binding to Human Heat-Shock Protein 90.

Recent experimental evidence suggests that mebendazole, a popular antiparasitic drug, binds to heat shock protein 90 (Hsp90) and inhibits acute myeloid leukemia cell growth. In this study we use quantum mechanics (QM), molecular similarity, and molecular dynamics (MD) calculations to predict possible binding poses of mebendazole to the adenosine triphosphate (ATP) binding site of Hsp90. Extensive conformational searches and minimization of the five mebendazole tautomers using the MP2/aug-cc-pVTZ theory level resulted in 152 minima. Mebendazole-Hsp90 complex models were subsequently created using the QM optimized conformations and protein coordinates obtained from experimental crystal structures that were chosen through similarity calculations. Nine different poses were identified from a total of 600 ns of explicit solvent, all-atom MD simulations using two different force fields. All simulations support the hypothesis that mebendazole is able to bind to the ATP binding site of Hsp90.

Novel Small Molecule Tyrosine Kinase 2 Pseudokinase Ligands Block Cytokine-Induced TYK2-Mediated Signaling Pathways.

A member of the Janus kinase (JAK) family, Tyrosine Kinase 2 (TYK2), is crucial in mediating various cytokine-signaling pathways such as interleukin-23 (IL23), interleukin-12 (IL12) and type I Interferons (IFN) which contribute to autoimmune disorders (e.g., psoriasis, lupus, and inflammatory bowel disease). Thus, TYK2 represents an attractive target to develop small-molecule therapeutics for the treatment of cytokine-driven inflammatory diseases. Selective inhibition of TYK2 over other JAK isoforms is critical to achieve a favorable therapeutic index in the development of TYK2 inhibitors. However, designing small molecule inhibitors to target the adenosine triphosphate (ATP) binding site of TYK2 kinase has been challenging due to the substantial structural homology of the JAK family catalytic domains. Here, we employed an approach to target the JAK homology 2 (JH2) pseudokinase regulatory domain of the TYK2 protein. We developed a series of small-molecule TYK2 pseudokinase ligands, which suppress the TYK2 catalytic activity through allosteric regulation. The TYK2 pseudokinase-binding small molecules in this study simultaneously achieve high affinity-binding for the TYK2 JH2 domain while also affording significantly reduced affinity for the TYK2 JAK homology 1 (JH1) kinase domain. These TYK2 JH2 selective molecules, although possessing little effect on suppressing the catalytic activity of the isolated TYK2 JH1 catalytic domain in the kinase assays, can still significantly block the TYK2-mediated receptor-stimulated pathways by binding to the TYK2 JH2 domain and allosterically regulating the TYK2 JH1 kinase. These compounds are potent towards human T-cell lines and primary immune cells as well as in human whole-blood specimens. Moreover, TYK2 JH2-binding ligands exhibit remarkable selectivity of TYK2 over JAK isoforms not only biochemically but also in a panel of receptor-stimulated JAK1/JAK2/JAK3-driven cellular functional assays. In addition, the TYK2 JH2-targeting ligands also demonstrate high selectivity in a multi-kinase screening panel. The data in the current study underscores that the TYK2 JH2 pseudokinase is a promising therapeutic target for achieving a high degree of biological selectivity. Meanwhile, targeting the JH2 domain represents an appealing strategy for the development of clinically well-tolerated TYK2 inhibitors that would have superior efficacy and a favorable safety profile compared to the existing Janus kinase inhibitors against autoimmune diseases.

Prediction of binding affinity of 1,2-diphenyline ketone analogues at adenosine triphosphate binding site of glycogen synthase kinase-3β: a molecular docking and dynamic simulation study

Glycogen synthase kinase (GSK)-3β is one of the downstream signalling molecules involved in phosphorylation of glycogen synthase, a key enzyme involved in the synthesis of glycogen from glucose. GSK-3β regulate some of the critical processes underlying structural and functional synaptic plasticity of neurons. Down regulation or inhibition of GSK-3β enhances long-term potentiation and cognitive functions in animal models of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. A number of compounds are available to inhibit GSK-3β, however none of them are in clinical practice to treat neurodegenerative diseases. The aim of our study was to predict the molecular interaction and dynamic behaviour of naturally occurring 1,2-diphenyline ketone analogues at the adenosine triphosphate binding site of glycogen synthase kinase (GSK)-3β through simulation studies. Out of all 1,2-diphenyline ketone analogues,1, 3, 5, 6-Tetrahydroxyxanthone (Rank = 1), Secalonic acid F (Rank = 2), and Trihydroxy-2-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-8-(3-methyl-2-butenyl) xanthone (Rank = 3) were found to exhibit lowest docking score of −12.07, −11.49, and −11.24 kcal/mol with dissociation constant of 1.37, 3.84, and 5.99 nM, respectively. The molecular dynamic simulation of rank 1 and rank 3 ligands indicated stable interaction throughout the simulation and interaction analyses has shown that the presence of hydroxyl groups at C1, C3, C5, and C6 around 1,2 diphenyline ketone nucleus to influence their binding affinity at the ATP-binding site of GSK-3β. We predicted that 1,3,5,6-Tetrahydroxyxanthone and 1, 3, 6-Trihydroxy-2-(2,3-dihydroxy-3-methylbutyl)-7-methoxy-8-(3-methyl-2-butenyl) xanthone may act as a potential ligand or lead compound to inhibit GSK-3β and also may play an important role in alleviating neurodegenerative diseases. Communicated by Ramaswamy H. Sarma

Dynamic Control of Microbial Movement by Photoswitchable ATP Antagonists

Adenosine triphosphate (ATP) is the energy source for various biochemical processes and biomolecular motors in living things. Development of ATP antagonists and their stimuli-controlled actions offer a novel approach to regulate biological processes. Herein, we developed azobenzene-based photoswitchable ATP antagonists for controlling the activity of motor proteins; cytoplasmic and axonemal dyneins. The new ATP antagonists showed reversible photoswitching of cytoplasmic dynein activity in an in vitro dynein-microtubule system due to the trans and cis photoisomerization of their azobenzene segment. Importantly, our ATP antagonists reversibly regulated the axonemal dynein motor activity for the force generation in a demembranated model of Chlamydomonas reinhardtii. We found that the trans and cis isomers of ATP antagonists significantly differ in their affinity to the ATP binding site.

Trial in Progress: A Multicenter, Open Label, Randomized, Phase III Study of Asciminib (80 mg Once Daily) Vs Investigator-Selected TKI in Newly Diagnosed Adult Patients with Chronic Myeloid Leukemia in Chronic Phase

Trial in Progress: A Multicenter, Open Label, Randomized, Phase III Study of Asciminib (80 mg Once Daily) Vs Investigator-Selected TKI in Newly Diagnosed Adult Patients with Chronic Myeloid Leukemia in Chronic Phase

Identification and Mechanistic Characterization of a Peptide Inhibitor of Glycogen Synthase Kinase (GSK3β) Derived from the Disrupted in Schizophrenia 1 (DISC1) Protein.

Glycogen synthase kinase 3-beta (GSK3β) is a critical regulator of several cellular pathways involved in neurodevelopment and neuroplasticity and as such is a potential focus for the discovery of new neurotherapeutics toward the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3β have been focused on developing small molecule inhibitors that compete with adenosine triphosphate (ATP) through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The disrupted in schizophrenia 1 (DISC1) protein has previously been shown to bind and inhibit GSK3β activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3β in a noncompetitive mode distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3β: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3β binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3β.

Discovery of Potent and Selective PI3Kγ Inhibitors.

The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-[(1S)-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1H-isoindol-1-one (4, IC50 = 0.064 μM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.

Review of rationale and progress toward targeting cyclin-dependent kinase 2 (CDK2) for male contraception†.

Cyclin-dependent kinase 2 (CDK2) is a member of the larger cell cycle regulating CDK family of kinases, activated by binding partner cyclins as its name suggests. Despite its canonical role in mitosis, CDK2 knockout mice are viable but sterile, suggesting compensatory mechanisms for loss of CDK2 in mitosis but not meiosis. Here, we review the literature surrounding the role of CDK2 in meiosis, particularly a cyclin-independent role in complex with another activator, Speedy 1 (SPY1). From this evidence, we suggest that CDK2 could be a viable nonhormonal male contraceptive target. Finally, we review the literature of pertinent CDK2 inhibitors from the preclinical to clinical stages, mostly developed to treat various cancers. To date, there is no potent yet selective CDK2 inhibitor that could be repurposed as a contraceptive without appreciable off-target toxicity. To achieve selectivity for CDK2 over closely related kinases, developing compounds that bind outside the conserved adenosine triphosphate-binding site may be necessary.

3′‐(4‐(Benzyloxy)phenyl)‐1′‐phenyl‐5‐(heteroaryl/aryl)‐3,4‐dihydro‐1′H,2H‐[3,4′‐bipyrazole]‐2‐carboxamides as EGFR kinase inhibitors: Synthesis, anticancer evaluation, and molecular docking studies

Pyrazoline-linked carboxamide derivatives were designed, synthesized, and evaluated for potential epidermal growth factor receptor (EGFR) kinase inhibition, anticancer activity, and apoptotic and cardiomyopathy toxicity. Compounds 6m and 6n inhibit EGFR kinase at a concentration of 6.5 ± 2.91 and 3.65 ± 0.54 µM, respectively. Some of these compounds showed effects on proliferation, which were also then evaluated against four different human cancer cell lines, that is, MCF-7 (breast cancer), A549 (non-small-cell lung tumor), HCT-116 (colon cancer), and SiHa cells (cancerous tissues of the cervix uteri). The results showedthat certainsynthetic compounds showed significant inhibitor activity; compounds 6m and 6n were more cytotoxic than doxorubicin against A549 cancer cells, with IC50 values of 10.3 ± 1.07 and 4.6 ± 0.57 µM, respectively. Additionally, compounds 6m and 6n induced apoptosis in A549 cancer cells, as evidenced by 4',6-diamidino-2-phenylindole (DAPI) staining and phase-contrast microscopy. Potency to induce apoptosis by compound 6n was further confirmed by fluorescence-activated cell sorting using Annexin V-FITC and propidium iodide labeling. Compound 6n showed normal cardiomyocytes with no marked sign of pyknotic nuclei in cardiomyopathy and also normal histological appearance of the renal cortex when compared with that of control. Results of molecular docking studies suggested that compounds 6m and 6n can bind to the hinge region of the adenosine triphosphate-binding site of EGFR kinase, like the standard drug erlotinib. Therefore, the present study suggests that compounds 6m and 6n have potent in vitro antitumor activities against the human non-small-cell lung tumor cell line A549, which can be further explored in other cancer cell lines and in animal studies.

Relative Selectivity of Covalent Inhibitors Requires Assessment of Inactivation Kinetics and Cellular Occupancy: A Case Study of Ibrutinib and Acalabrutinib.

Kinases form an attractive class of targets for small molecule inhibitors, but similarity among their adenosine triphosphate binding sites presents difficulties for developing selective drugs. Standard methods of evaluating selectivity of most reversibly bound drugs account for binding affinity but not the two-step process, affinity and inactivation, occurring during covalent inhibition. To illustrate this concept, we assessed the selectivity of Bruton's tyrosine kinase (BTK) over TEC kinases by two novel therapeutics: ibrutinib and acalabrutinib. The two-step process and time-dependent inhibition unique to covalent inhibitors were evaluated with two biochemical assays measuring enzymatic function and inhibition kinetics. The selectivity for BTK over TEC found in these biochemical analyses was 1-1.5 for ibrutinib and 3.0-4.2 for acalabrutinib. To further assess drug selectivity in a more physiologically relevant context, we developed cell-based occupancy assays that quantify the percentage of drug-inactivated kinases. Cellular selectivity of BTK over TEC was determined after MWCL-1 cells, and samples from patients with chronic lymphocytic leukemia (CLL) were treated for durations and concentrations based on human pharmacokinetics of each drug. In MWCL-1 cells, BTK/TEC selectivities measured at 0.5, 1, and 3 hours were 2.53, 1.05, and 1.51 for ibrutinib and 0.97, 1.13, and 2.56 for acalabrutinib, respectively. The equivalent selectivity measured in samples from patients with CLL were 1.31 ± 0.27 and 1.09 ± 0.11 for ibrutinib and acalabrutinib, respectively. Collectively, our data show that when properly accounting for time-dependent factors and relevant cellular context, ibrutinib and acalabrutinib demonstrate similar selectivity for BTK over TEC. SIGNIFICANCE STATEMENT: This study shows relative selectivity of covalent inhibitors toward different kinase targets should be assessed with both affinity and inactivation kinetics to accurately account for time-dependent effects of covalent binding and assessed in a cellular matrix to reproduce the physiologic context of target inhibition. This is illustrated with a case study of ibrutinib and acalabrutinib for which selectivity assessment with appropriate assays, as opposed to measuring binding affinity with KINOMEscan alone, corroborate emerging clinical data demonstrating similar safety profiles between the therapies.

Discovery of N-pyridoyl-Δ2 -pyrazolines as Hsp90 inhibitors.

Hsp90, as a key molecular chaperone, plays an important role in modulating the activity of many cell signaling proteins and is an attractive target for anticancer therapeutics. Herein, we report the discovery of N-pyridoyl-Δ2 -pyrazoline analogs as novel Hsp90 inhibitors by integrated approaches of drug design, organic synthesis, cell biology, and qualitative proteomic analysis. Novel chemical compounds were designed and optimized in the adenosine triphosphate-binding site of Hsp90; lead optimized compounds were found to have significant interactions with Asp93 and other amino acids crucial for Hsp90 inhibition. The designed compounds were synthesized by a two-step procedure; different aromatic aldehydes were reacted with various acetophenones to form substituted 1,3-diphenyl-prop-2-enones (Ic-Io), which upon reaction with isonicotinic acid hydrazide in the presence of glacial acetic acid form N-pyridoyl-Δ2 -pyrazoline compounds (PY1-PY13). Compounds PY3, PY2, and PY1 were identified as potential leads amongst the series, with promising anticancer activity against human breast cancer and melanoma cells, and the ability to inhibit Hsp90 similar to radicicol by drug-affinity responsive target stability proteomic analysis in a whole-cell assay.

Quinazoline based 1,3,5-triazine derivatives as cancer inhibitors by impeding the phosphorylated RET tyrosine kinase pathway: Design, synthesis, docking, and QSAR study.

The present research focused on designing a quinazoline skeleton, framed via 1,3,5-triazine derivatives (QBT) through field mapping and alignment studies. The QBT derivatives were synthesized via time- and cost-effective protocol. The 3D-QSAR study, computational physicochemical properties, and ADME calculation of the derivatives were performed to establish the affinity towards the biological system. Molecular docking in the adenosine triphosphate binding site of the RET tyrosine kinase domain (PDB ID: 7IVU) was studied to elucidate vital structural residues necessary for bioactivity. The derivatives were evaluated for anticancer potency against TPC-1 cells (thyroid cancer), MCF-7 cells (breast cancer), and one normal cell line (human foreskin fibroblasts) via 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide assay followed by an in ovo CAM assay. The entire series of derivatives (8a-o) showed mild to significant anticancer potency against the selected cancer cell lines.

Identification and Structure-Activity Studies of 1,3-Dibenzyl-2-aryl imidazolidines as Novel Hsp90 Inhibitors.

Hsp90 (Heat shock protein 90) is involved in various processes in cancer occurrence and development, and therefore represents a promising drug target for cancer therapy. In this work, a virtual screening strategy was employed, leading to the identification of a series of compounds bearing a scaffold of 1,3-dibenzyl-2-aryl imidazolidine as novel Hsp90 inhibitors. Compound 4a showed the highest binding affinity to Hsp90α (IC50 = 12 nM) in fluorescence polarization (FP) competition assay and the strongest anti-proliferative activity against human breast adenocarcinoma cell line (MCF-7) and human lung epithelial cell line (A549) with IC50 values of 21.58 μM and 31.22 μM, respectively. Western blotting assays revealed that these novel Hsp90 inhibitors significantly down-regulated the expression level of Her2, a client protein of Hsp90, resulting in the cytotoxicity of these novel Hsp90 inhibitors. The molecular docking study showed that these novel Hsp90 inhibitors bound to the adenosine triphosphate (ATP) binding site at the N-terminus of Hsp90. Furthermore, structure–activity relationship studies indicated that the N-benzyl group is important for the anti-cancer activity of 1,3-dibenzyl-2-aryl imidazolidines.

Measurements on ATP induced cellular fluctuations using real-time dual view transport of intensity phase microscopy.

Dual view transport of intensity phase microscopy is adopted to quantitatively study the regulation of adenosine triphosphate (ATP) on cellular mechanics. It extracts cell phases in real time from simultaneously captured under- and over-focus images. By computing the root-mean-square phase and correlation time, it is found that the cellular fluctuation amplitude and speed increased with ATP compared to those with ATP depletion. Besides, when adenylyl-imidodiphosphate (AMP-PNP) was introduced, it competed with ATP to bind to the ATP binding site, and the cellular fluctuation amplitude and speed decreased. The results prove that ATP is a factor in the regulation of cellular mechanics. To our best knowledge, it is the first time that the dual view transport of intensity phase microscopy was used for live cell phase imaging and analysis. Our work not only provides direct measurements on cellular fluctuations to study ATP regulation on cellular mechanics, but it also proves that our proposed dual view transport of intensity phase microscopy can be well used, especially in quantitative phase imaging of live cells in biological and medical applications.

Structural and binding studies of phosphopantetheine adenylyl transferase from Acinetobacter baumannii

Phosphopantetheine adenylyltransferase (PPAT, EC. 2.7.7.3) catalyzes an essential step in the reaction that transfers an adenylyl group from adenosine tri phosphate (ATP) to 4′-phosphopantetheine (pPant) yielding 3′- dephospho-coenzyme A (dPCoA) and pyrophosphate (PP) in the coenzyme A (CoA) biosynthesis pathway. The enzyme PPAT from Acinetobacter baumannii (AbPPAT) was cloned, expressed and purified. The binding studies of AbPPAT were carried out with two compounds, tri‑sodium citrate (TSC) and l-ascorbic acid (LAA, vitamin-C) using fluorescence spectroscopic (FS) and surface Plasmon resonance (SPR) methods. Both methods provided similar values of dissociation constants for TSC and LAA which were of the order of 10−8 M and 10−5 M respectively. The computer aided docking studies indicated fewer interactions of LAA with AbPPAT as compared to those of TSC. The freshly purified samples of AbPPAT were crystallized. The crystals of AbPPAT were soaked in the solutions containing TSC and LAA. However, the crystals of the complex of AbPPAT with LAA did not diffract well and hence the structure of the complex of AbPPAT with LAA could not be determined. On the other hand, the crystals of the complex of AbPPAT with TSC diffracted well and the structure was determined at 1.76 Å resolution. It showed that TSC bound to AbPPAT at the ATP binding site and formed several intermolecular contacts including 12 hydrogen bonds. The results of binding studies for both TSC and LAA and the structure of the complex of AbPPAT with TSC clearly indicated a potential role of TSC and LAA as antibacterial agents.