In vitro and in silico evaluation of the serrapeptase effect on biofilm and amyloids of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an emerging threat for hospitalized and cystic fibrosis patients. Biofilm, a microbial community embedded in extracellular polymeric substance, fortifies bacteria against the immune system. In biofilms, the expression of functional amyloids is linked with highly aggregative, multi-resistant strains, and chronic infections. Serrapeptase (SPT), a protease possessing similar or superior anti-microbial properties with many antibiotics, presents anti-amyloid potential. However, studies on the employment of SPT against Pseudomonas biofilms and Fap amyloid, or the possible mechanisms of action are scarce. Here, SPT inhibited biofilm formation of P. aeruginosa ATCC 27853 on both plastic and glass surfaces, with an IC50 of 11.26 µg/mL and 0.27 µg/mL, respectively. The inhibitory effect of SPT on biofilm was also verified with optical microscopy of crystal violet-stained biofilms and with confocal microscopy. Additionally, SPT caused a dose-dependent decrease of bacterial viability (IC50 of 3.07 µg/mL) as demonstrated by MTT assay. Reduction of bacterial functional amyloids was also demonstrated, employing both fluorescence microscopy with thioflavin T and photometrical determination of Congo-red-positive compounds. Both viability and functional amyloids correlated significantly with biofilm inhibition. Finally, in silico molecular docking studies provided a mechanistic insight into the interaction of SPT with FapC or FapD, proving that both peptides are possible targets of SPT. These results offer new insights into the biofilm formation of P. aeruginosa and potentiate the involvement of SPT in the prevention and eradication of Pseudomonas biofilms. KEY POINTS: • Serrapeptase inhibits biofilm formation of P. aeruginosa on plastic and glass. • Biofilm inhibition correlated with reduced viability and functional amyloid levels. • In silico studies indicated that serrapeptase may target FapC and FapD peptides.

In Silico Approach for the Evaluation of the Potential Antiviral Activity of Extra Virgin Olive Oil (EVOO) Bioactive Constituents Oleuropein and Oleocanthal on Spike Therapeutic Drug Target of SARS-CoV-2.

Since there is an urgent need for novel treatments to combat the current coronavirus disease 2019 (COVID-19) pandemic, in silico molecular docking studies were implemented as an attempt to explore the ability of selected bioactive constituents of extra virgin olive oil (EVOO) to act as potent SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) antiviral compounds, aiming to explore their ability to interact with SARS-CoV-2 Spike key therapeutic target protein. Our results suggest that EVOO constituents display substantial capacity for binding and interfering with Spike (S) protein, both wild-type and mutant, via the receptor-binding domain (RBD) of Spike, or other binding targets such as angiotensin-converting enzyme 2 (ACE2) or the RBD-ACE2 protein complex, inhibiting the interaction of the virus with host cells. This in silico study provides useful insights for the understanding of the mechanism of action of the studied compounds at a molecular level. From the present study, it could be suggested that the studied active phytochemicals could potentially inhibit the Spike protein, contributing thus to the understanding of the role that they can play in future drug designing and the development of anti-COVID-19 therapeutics.

In silico study of potential antiviral activity of copper(II) complexes with non-steroidal anti-inflammatory drugs on various SARS-CoV-2 target proteins.

In silico molecular docking studies, in vitro toxicity and in silico predictions on the biological activity profile, pharmacokinetic properties, drug-likeness, ADMET (absorption, distribution, metabolism, excretion, and toxicity) physicochemical pharmacokinetic data, and target proteins and toxicity predictions were performed on six copper(II) complexes with the non-steroidal anti-inflammatory drugs ibuprofen, loxoprofen, fenoprofen and clonixin as ligands, in order to investigate the ability of these complexes to interact with the key therapeutic target proteins of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) 3C-like cysteine main protease (3CLpro/Mpro), viral papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), and non-structural proteins (Nsps) Nsp16-Nsp10 2'-O-methyltransferase complex, and their capacity to act as antiviral agents, contributing thus to understanding the role they can play in the context of coronavirus 2019 (COVID-19) pandemic. Cytotoxic activity against five human cancer and normal cell lines were also evaluated.

Biocompatible silver(I) complexes with heterocyclic thioamide ligands for selective killing of cancer cells and high antimicrobial activity - A combined in vitro and in silico study.

A series of heteroleptic Ag(I) complexes bearing 4,6-dimethyl-2-pyrimidinethiol (dmp2SH), i.e., [AgCl(dmp2SH)(PPh3)2] (1), [Ag(dmp2SH)(PPh3)2]NO3 (2), [Ag(dmp2SΗ)(xantphos)]NO3 (3), [Ag(µ-dmp2S)(PPh3)]2 (4), [Ag(dmp2S)(xantphos)] (5), [Ag(µ-dmp2S)(DPEphos)]2 (6) (xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene and DPEPhos = bis[(2-diphenylphosphino)phenyl]ether) were synthesized. The complexes display systematic variation of particular structural characteristics which were proved to have a significant impact on their in vitro cytotoxicity and antimicrobial properties. A moderate-to-high potential for bacteria growth inhibition was observed for all complexes, with 2, 3 and 5 being particularly effective against Gram-(+) bacteria (IC50 = 1.6-4.5 µM). The three complexes exhibit high in vitro cytotoxicity against HeLa and MCF-7 cancer cells (IC50 = 0.32-3.00 µΜ), suggesting the importance of coordination unsaturation and cationic charge for effective bioactivity. A very low cytotoxicity against HDFa normal cells was observed, revealing a high degree of selectivity (selectivity index ~10) and, hence, biocompatibility. Fluorescence microscopy using 2 showed effective targeting on the membrane of the HeLa cancer cells, subsequently inducing cell death. Binding of the complexes to serum albumin proteins is reasonably strong for potential uptake and subsequent release to target sites. A moderate in vitro antioxidant capacity for free radicals scavenging was observed and a low potential to destroy the double-strand structure of calf-thymus DNA by intercalation, suggesting likely implication of these properties in the bioactivity mechanisms of these complexes. Further insight into possible mechanisms of bioactivity was obtained by molecular modeling calculations, by exploring their ability to act as potential inhibitors of DNA-gyrase, human estrogen receptor alpha, human cyclin-dependent kinase 6, and human papillomavirus E6 oncoprotein.

Extra Virgin Olive Oil consumption from Mild Cognitive Impairment patients attenuates oxidative and nitrative stress reflecting on the reduction of the PARP levels and DNA damage.

Oxidative/nitrative stress that results from the unbalance of the overproduction/clearance of reactive oxygen/nitrogen species (ROS/NOS), originated from a variety of endo- and/or exo-genous sources, can have detrimental effects on DNA and is involved in Alzheimer's disease (AD) pathology. An excellent marker of oxidative DNA lesions is 8-hydroxy-2'-deoxyguanosine (8-OHdG) while of nitrative stress the enzyme NOS2 (Nitric oxide synthase 2). Under massive oxidative stress, poly(ADP-ribose)polymerase 1 (PARP-1) enzyme activity, responsible for restoration of DNA damage, is augmented, DNA repair enzymes are recruited, and cell survival/or death is ensued through PARP-1 activation, which is correlated positively with neurodegenerative diseases. In this biochemical study the levels of PARP-1, 8-oxo-dG, and NOS2, Aß1-42, and p-tau in their sera determined using Enzyme-Linked Immunosorbent Assay (ELISA). Patients diagnosed with Mild Cognitive Impairment participated in MICOIL clinical trial, were daily administered with 50 ml Extra Virgin Olive Oil (EVOO) for one year. All MCI patients' biomarkers that had consumed EVOO were tantamount to those of healthy participants, contrary to MCI patients who were not administered. EVOO administration in MCI patients resulted in the restoration of DNA damage and of the well-established "hallmarks" AD biomarkers, thanks probably to its antioxidant properties exhibiting a therapeutic potentiality against AD. Molecular docking simulations of the EVOO constituents on the crystal structure of PARP-1 and NOS-2 target enzymes were also employed, to study in silico the ability of the compounds to bind to these enzymes and explain the observed in vitro activity. In silico analysis has proved the binding of EVOO constituents on PARP-1and NOS-2 enzymes and their interaction with crucial amino acids of the active sites. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT03362996. MICOIL GOV IDENTIFIER: NCT03362996.

Nickel(II)-meclofenamate complexes: Structure, in vitro and in silico DNA- and albumin-binding studies, antioxidant and anticholinergic activity.

Five novel nickel(II) complexes with the non-steroidal anti-inflammatory drug sodium meclofenamate (Na-mclf) have been synthesized and characterized in the absence or co-existence of the nitrogen-donors imidazole (Himi), 2,2'-bipyridylamine (bipyam), 2,2'-bipyridylketoxime (Hpko) and 2,9-dimethyl-1,10-phenanthroline (neoc); namely [Ni(mclf-O)2(Himi)2(MeOH)2], [Ni(mclf-O)2(MeOH)4], [Ni(mclf-O)(mclf-O,O')(bipyam)(MeOH)]·0.25MeOH, [Ni(mclf-O,O')2(neoc)] and [Ni(mclf-O)2(Hpko-N,N')2]·MeOH·0.5H2O. The affinity of the complexes for calf-thymus (CT) DNA was investigated by various techniques and intercalation is suggested as the most possible interaction mode. The interaction of the complexes for bovine and human serum albumins was also investigated in order to determine the binding constants, concluding that the complexes bind reversibly to albumins for the transportation towards their target cells or tissues and their release upon arrival at biotargets. The antioxidant activity of the compounds was evaluated via their ability to scavenge 1,1-diphenyl-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) free radicals and to reduce H2O2. For the determination of the anticholinergic ability of the complexes the in vitro inhibitory activity against the enzymes acetylcholinesterase and butyrylcholinesterase was evaluated and presented promising results. The in silico molecular modeling calculations employed provide useful insights for the understanding of the mechanism of action of the studied complexes at a molecular level. This applies on both the impairment of DNA by its binding with the studied complexes and transportation through serum albumins, as well as the ability of these compounds to act as anticholinergic agents.

A palladium(II) complex with the Schiff base 4-chloro-2-(N-ethyliminomethyl)-phenol: Synthesis, structural characterization, and in vitro and in silico biological activity studies.

The synthesis and characterization of the Pd(II) complex of the formula [Pd(L)2] 1 with the Schiff base 4-chloro-2-(N-ethyliminomethyl)-phenol (HL) as derived in situ via the condensation reaction of 5-chloro-salicylaldehyde and ethylamine was undertaken. The structure of 1 was verified by single-crystal X-ray crystallography. The ability of 1 to interact with calf-thymus (CT) DNA was studied by UV-vis and viscosity experiments, and its ability to displace ethidium bromide (EB) from the DNA-EB conjugate was revealed by fluorescence spectroscopy. It was found that intercalation is the most possible mode of interaction with CT DNA. Additionally, DNA electrophoretic mobility experiments showed that 1 interacts with the plasmid pBluescript SK(+) (pDNA) as proved by the formation of unusual mobility DNA bands and degradation of relaxed pDNA at concentration of 5 mM. The interaction of 1 with human (HSA) and bovine serum albumin (BSA) was monitored revealing its reversible binding to albumins. The complex showed noteworthy antimicrobial activity against one (Bacillus subtilis) of the five tested bacteria. In order to explain the described in vitro activity of the compound, we adopted molecular docking studies on the crystal structure of HSA, BSA, CT DNA and DNA-gyrase. Furthermore, in silico predictive tools have been employed to study the properties of the complex. The in silico studies are adopted on a multitude of proteins involved in cancer growth, as well as prediction of drug-induced changes of gene expression profile, protein- and mRNA-based prediction results, prediction of sites of metabolism, cytotoxicity for cancer cell lines, etc.

Manganese coordination compounds of mefenamic acid: In vitro screening and in silico prediction of biological activity.

The in vitro and in silico biological properties of two manganese complexes with the non-steroidal anti-inflammatory drug mefenamic acid (Hmef) in the presence or absence of salicylaldoxime (Η2sao), i.e. [Μn6(O)2(mef)2(sao)6(CH3OH)4] 1, and [Μn(mef)2(CH3OH)4] 2, respectively, are presented in the present contribution. More specifically, the in vitro biological activity of the complexes was investigated by studying their affinity to calf-thymus DNA (by diverse spectroscopic and physicochemical techniques) and their binding towards bovine (BSA) or human serum albumin (HSA) (by fluorescence emission spectroscopy). Molecular docking simulations on the crystal structures of HSA and DNA, exploring in silico the ability of the complexes to bind to these macromolecules, were also employed in order to explain the described in vitro activity of the compounds. Furthermore, in silico predictive tools have been employed to study the properties of the most active complex 2 to act as anticancer agent, in continuation of the previously reported cytotoxic activity. It is adopted in silico studies on a multitude of proteins involved in cancer growth, as well as prediction of drug-induced changes of gene expression profile, protein- and mRNA-based prediction results, prediction of sites of metabolism, quantitative prediction of antitarget interaction profiles etc.

In vitro and in silico study of the biological activity of manganese(III) inverse-[9-MC-3]-metallacrowns and manganese(II) complexes with the anti-inflammatory drugs diclofenac or indomethacin.

In the present contribution, the biological properties of four manganese complexes with the non-steroidal anti-inflammatory drugs sodium diclofenac (Nadicl) or indomethacin (Hindo) in the presence or absence of salicylaldoxime (Η2sao), i.e. [Μn6(O)2(dicl)2(sao)6(CH3OH)6] 1, [Μn6(O)2(indo)2(sao)6(H2O)4], 2, [Μn(dicl)2(CH3OH)4], 3, and [Μn(indo)2(CH3OH)4], 4 are presented. More specifically, the in vitro cytotoxic effects of the complexes were evaluated against three cancer cell lines (HeLa, MCF-7 and A549 cells) as well as their combinatory activity with the well-known chemotherapeutic drugs irinotecan, cisplatin, paclitaxel and 5-fluorouracil. The biological activity of the complexes was investigated in vitro by studying their affinity to calf-thymus DNA and their binding towards bovine or human serum albumin (HSA). Molecular docking simulations on the crystal structure of HSA and human estrogen receptor alpha (hERa) were employed in order to study in silico the ability of the studied complexes to bind to these proteins.

Amplifying and broadening the cytotoxic profile of quercetin in cancer cell lines through bioconjugation.

Quercetin is a flavonoid presenting cytotoxicity against different cancer cell lines. We hypothesized that its core could serve as a scaffold for generating more potent compounds. A quercetin-alanine bioconjugate was synthesized, its cellular internalization was monitored through confocal microscopy and its cytotoxic activity was explored against ten different cell lines. The bioconjugate consistently illustrated enhanced cytotoxic activity with respect to the parent compound. A threefold enhancement in its cytotoxicity was revealed for HeLa, A549, MCF-7 and LNCaP cells. In silico studies suggested that quercetin-alanine possesses enhanced binding affinity to human estrogen receptor alpha corroborating to its activity to MCF-7, overexpressing this receptor. Spectrofluorimetric, calorimetric and in silico studies revealed that quercetin-alanine binds primarily to Sudlow site I of serum albumin mainly through hydrogen bonding. Through this array of experiments we discovered that the specific compound bears a more refined pharmaceutical profile in contrast to quercetin in terms of cytotoxicity, while at the same time preserves its affinity to serum albumin. Natural products could thus offer a potent scaffold to develop bioconjugates with amplified therapeutic window.

Rational design and structure-activity relationship studies of quercetin-amino acid hybrids targeting the anti-apoptotic protein Bcl-xL.

Anti-apoptotic proteins, like the Bcl-2 family proteins, present an important therapeutic cancer drug target. Their activity is orchestrated through neutralization upon interaction of pro-apoptotic protein counterparts that leads to immortality of cancer cells. Therefore, generating compounds targeting these proteins is of immense therapeutic importance. Herein, Induced Fit Docking (IFD) and Molecular Dynamics (MD) simulations were performed to rationally design quercetin analogues that bind in the BH3 site of the Bcl-xL protein. IFD calculations determined their binding cavity while Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics Generalised Born Surface Area (MM-GBSA) calculations provided an insight into the binding enthalpies of the analogues. The quercetin analogues were synthesized and their binding to Bcl-xL was verified with fluorescence spectroscopy. The binding affinity and the thermodynamic parameters between Bcl-xL and quercetin-glutamic acid were estimated through Isothermal Titration Calorimetry. 2D 1H-15N HSQC NMR chemical shift perturbation mapping was used to chart the binding site of the quercetin analogues in the Bcl-xL that overlapped with the predicted poses generated by both IFD and MD calculations. Furthermore, evaluation of the four conjugates against the prostate DU-145 and PC-3 cancer cell lines, revealed quercetin-glutamic acid and quercetin-alanine as the most potent conjugates bearing the higher cytostatic activity. This pinpoints that the chemical space of natural products can be tailored to exploit new hits for difficult tractable targets such as protein-protein interactions.

Adriamycin in combination with dexamethasone and octreotide lacks activity on the treatment of a 4T1 metastatic breast cancer model.

The aim of this study was to evaluate whether the palliative treatment for metastatic disease with dexamethasone (DEX) plus octreotide (OCT) can improve the anticancer effects of the standard treatment with adriamycin (ADR) on a 4T1 metastatic breast cancer (MBC) model. 4T1 cells were first characterized for the expression of the somatostatin receptors 1-5 and were then inoculated onto the femur of BALB/C mice. Investigation protocols used 4T1 cell proliferation and invasion assays, analysis of radiographic images of the bone metastatic lesions, and overall survival of the diseased animals. The triple combination treatment regime (ADR+OCT+DEX) was ineffective for growth inhibition and showed an antagonistic effect on ADR activity in the 4T1 cell line in both proliferation and invasion assays. ADR treatment following the administration of the DEX+OCT regimen decreased the anticancer activity of ADR both on the grading of the bone metastatic lesions and on the overall survival of diseased animals. Moreover, the palliation treatment with OCT+DEX and in combination with ADR rather caused disease progression of the metastatic disease and bone lesions in a 4T1 MBC model in vivo. These results suggest that the administration of the DEX+OCT regimen, although may preserve palliative effects, neutralizes or reverses the anticancer effects of ADR on a 4T1 MBC model in vitro and in vivo. The simultaneous use of these drugs should be considered carefully in clinical practice.

Overview on the current status of virtual high-throughput screening and combinatorial chemistry approaches in multi-target anticancer drug discovery; Part I.

Conventional drug design embraces the "one gene, one drug, one disease" philosophy. Nowadays, new generation of anti- cancer drugs, able to inhibit more than one pathway, is believed to play a major role in contemporary anticancer drug research. In this way, polypharmacology, focusing on multi-target drugs, has emerged as a new paradigm in drug discovery. A number of recent successful drugs have in part or in whole emerged from a structure-based research approach. Many advances including crystallography and informatics are behind these successes. Increasing insight into the genetics and molecular biology of cancer has resulted in the identification of an increasing number of potential molecular targets, for anticancer drug discovery and development. These targets can be approached through exploitation of emerging structural biology, "rational" drug design, screening of chemical libraries, or a combination of these methods. The result is the rapid discovery of new anticancer drugs. In this article we discuss the application of molecular modeling, molecular docking and virtual high-throughput screening to multi-targeted anticancer drug discovery. Efforts have been made to employ in silico methods for facilitating the search and design of selective multi-target agents. These computer aided molecular design methods have shown promising potential in facilitating drug discovery directed at selective multiple targets and is expected to contribute to intelligent lead anticancer drugs.

Overview on the current status on virtual high-throughput screening and combinatorial chemistry approaches in multi-target anticancer drug discovery; Part II.

Conventional drug design embraces the "one gene, one drug, one disease" philosophy. Nowadays, new generation of anticancer drugs, able to inhibit more than one pathway, is believed to play a major role in contemporary anticancer drug research. In this way, polypharmacology, focusing on multi-target drugs, has emerged as a new paradigm in drug discovery. A number of recent successful drugs have in part or in whole emerged from a structure-based research approach. Many advances including crystallography and informatics are behind these successes. In this part II we will review the role and methodology of ligand-, structure- and fragment-based computer-aided drug design computer aided drug desing (CADD), virtual high throughput screening (vHTS), de novo drug design, fragment-based design and structure-based molecular docking, homology modeling, combinatorial chemistry and library design, pharmacophore model chemistry and informatics in modern drug discovery.

Safranal, a Crocus sativus L constituent suppresses the growth of K-562 cells of chronic myelogenous leukemia. In silico and in vitro study.

Crocin, a main constituent of Crocus sativus L (saffron), has been found to inhibit the growth of K-562 human chronic myelogenous leukemia (CML) cells expressing Bcr-Abl protein tyrosine kinase activity. The aim of our study is to investigate the ability of the bioactive saffron's constituents, crocin (CRC) and safranal (SFR), to inhibit the Bcr-Abl protein activity employing an in silico approach, as well as the in vitro effect of these compounds on K-562 growth and gene expression of Bcr-Abl. In silico molecular docking studies revealed that mostly SFR can be attached to Bcr-Abl protein, positioned inside the protein's binding cavity at the same place with the drug used in the treatment of CML, imatinib mesylate (IM). The predicted polar interactions and hydrophobic contacts constructing a hydrophobic cavity inside the active site, explain the observed inhibitory activity. Cytotoxicity experiments showed that SFR and CRC mediate cytotoxic response to K562 cells. In vitro studies on the expression of Bcr-Abl gene revealed that SFR and in a lesser degree IM inhibited the expression of the gene, while in contrast CRC induced an increase. The ultimate goal was to evaluate the existence of a potential antitumor activity of saffron's constituents SFR and CRC.

Anticarcinogenic activity of polyphenolic extracts from grape stems against breast, colon, renal and thyroid cancer cells.

A major part of the wineries' wastes is composed of grape stems which are discarded mainly in open fields and cause environmental problems due mainly to their high polyphenolic content. The grape stem extracts' use as a source of high added value polyphenols presents great interest because this combines a profitable venture with environmental protection close to wine-producing zones. In the present study, at first, the Total Polyphenolic Content (TPC) and the polyphenolic composition of grape stem extracts from four different Greek Vitis vinifera varieties were determined by HPLC methods. Afterwards, the grape stem extracts were examined for their ability to inhibit growth of colon (HT29), breast (MCF-7 and MDA-MB-23), renal (786-0 and Caki-1) and thyroid (K1) cancer cells. The cancer cells were exposed to the extracts for 72 h and the effects on cell growth were evaluated using the SRB assay. The results indicated that all extracts inhibited cell proliferation, with IC50 values of 121-230 µg/ml (MCF-7), 121-184 µg/ml (MDA-MD-23), 175-309 µg/ml (HT29), 159-314 µg/ml (K1), 180-225 µg/ml (786-0) and 134->400 µg/ml (Caki-1). This is the first study presenting the inhibitory activity of grape stem extracts against growth of colon, breast, renal and thyroid cancer cells.

Effect of cobalt(II) complexes with dipyridylamine and salicylaldehydes on cultured tumor and non-tumor cells: synthesis, crystal structure investigations and biological activity.

The synthesis of eight mixed-ligand cobalt(II) complexes with 2,2'-dipyridylamine (dpamH) and substituted salicylaldehydes (X-saloH) was undertaken in an effort to discover new compounds with anticancer activity. The complexes with the general formula [Co(dpamH)(2)(X-salo)]Y, (Y=Br or Cl) were characterized by elemental analyses, FT-IR and UV-visible spectroscopy, magnetic and conductivity measurements. The structures of two of them [Co(dpamH)(2)(5-CH(3)-salo)]Br and [Co(dpamH)(2)(3-OCH(3)-salo)]Cl, as well as of the precursors [Co(dpamH)(3)]Br(2) and [Co(dpamH)(2)Cl(H(2)O)]Cl, were determined by X-ray crystallography revealing octahedral coordination of cobalt(II) and mononuclear complexes. The complexes were thermally stable up to 200 °C in nitrogen atmosphere, studied by simultaneous TG/DTG-DTA technique. The two precursor Co compounds, as well as four of the title compounds, were evaluated for their efficacy as anticancer agents against different cancer and normal human cell lines. The in vitro chemosensitivity of various human cell lines to these Co complexes was evaluated by measuring cell growth inhibition by employing the SRB colorimetric assay. A series of experiments showed a dose-dependent cytotoxic activity of the complexes against all cell lines used. These findings represent a prompting to search for possible interaction of these complexes with other cellular elements of fundamental importance in cell proliferation.

In silico/in vitro study of hybrid D-modified steroidal alkylator anticancer activity using uridine phosphorylase as target protein.

BACKGROUND: In order to reduce toxicity and to enhance anticancer activity of nitrogen mustards, three hybrid steroidal esters were synthesized and tested in vitro against human pancreatic cancer cells expressing uridine phosphorylase (UPase). The inhibition potency against a target protein implicated in the chemotherapy of solid tumors, such as UPase, is of fundamental importance in the design and synthesis of new anticancer drugs. MATERIALS AND METHODS: MTT colorimetric assay and molecular docking were employed for the in vitro and in silico drug evaluation, respectively. RESULTS: A difference in cell sensitivity was found, which followed the known different UPase expression in the cell lines. Molecular docking studies on UPase protein, revealed the tested compounds to be bound to the binding cavity of the protein, with different affinity. Between the two D-modified compounds, the D-homo-aza (lactam)-hybrid compound (C2) was found to interact with the protein in a more efficient way. CONCLUSION: The molecular docking data were in accordance with the in vitro results, where the lactam steroid alkylator showed significantly higher cytostatic and cytotoxic activity than the non-D-modified compounds, which also correlated with the level of UPase expression in the pancreatic cancer cells.

Importance of molecular computer modeling in anticancer drug development.

Increasing insight into the genetics and molecular biology of cancer has resulted in the identification of an increasing number of potential molecular targets for anticancer drug discovery and development. These targets can be approached through exploitation of emerging structural biology, "rational" drug design, screening of chemical libraries, or a combination of these methods. The result is the rapid discovery of new anticancer drugs. The processes used by academic and industrial scientists to discover new drugs has recently experienced a true renaissance with many new and exciting techniques being developed in the past 5-10 years. In this review, we will attempt to outline these latest protocols that chemists and biomedical scientists are currently employing to rapidly bring new drugs to the clinic. Structure-based drug design is perhaps the most elegant approach for discovering compounds exhibiting high specificity and efficacy. Nowadays, a number of recent successful drugs have in part or in whole emerged from a structure-based research approach. Many advances including crystallography and informatics are behind these successes. Of great importance is also the impact these advances in structure-based drug design are likely to have on the economics of drug discovery. As the structures of more and more proteins and nucleic acids become available, molecular docking is increasingly considered for lead discovery. Recent studies consider the hit-rate enhancement of docking screens and the accuracy of docking structure predictions. As more structures are determined experimentally, docking against homology-modeled targets also becomes possible for more proteins. With more docking studies being undertaken, the "drug-likeness" and specificity of docking hits is also being examined. In this article we discuss the application of molecular modeling, molecular docking and virtual molecular high-throughput, targeted drug screening to anticancer drug discovery. Currently, scientists are focusing on designing and discovering potential inhibitors against cancer-related proteins that play critical roles in the development of a variety of tumors. Future research breakthroughs with the aid of computer-aided molecular design and chemo-bioinformatics will bring not only new hope, but also create a new class of anticancer drugs that will help millions of cancer patients.

Lactandrate: a D-homo-aza-androsterone alkylator in the treatment of breast cancer.

The sensitivity of breast neoplasms to hormonal control provides the basis of novel investigational treatments with steroidal alkylators. An androsterone D-lactam steroidal ester, the 3beta-hydroxy-13alpha-amino-13,17-seco- 5alpha-androstan-17-oic-13,17-lactam, p-bis(2-chloroethyl)amino phenyl acetate (lactandrate) was synthesized and tested for antitumor activity against six human breast cancer cell lines in vitro and against two murine and one xenograft mammary tumors in vivo. A docking study on the binding interactions of lactandrate with the ligand-binding domain (LBD) of estrogen receptor-alpha (ERalpha) was inquired. In vitro testing of lactandrate cytostatic and cytotoxic activity was performed on T47D, MCF7, MDA-MB-231, BT-549, Hs578T, MDA-MB-435 breast adenocarcinoma human cell lines. In vivo testing was performed on two murine mammary tumors, the MXT tumor and CD8F1 adenocarcinoma, as well as on human mammary carcinoma MX-1 xenograft. Molecular modeling techniques were adopted to predict a possible location and interaction mode of the molecule into LBD. Lactandrate induced significantly high antitumor effect against all tested in vitro and in vivo models. The cell lines with positive ER expression found to be significantly more sensitive to lactandrate. Moreover, lactandrate found to be positioned inside the binding cavity with its steroidal moiety, whilst the alkylating moiety protrudes out of receptor's pocket. Lactandrate produced important anticancer activity on breast cancer in vitro and in vivo. Some correlation between ER and lactandrate effect was demonstrated. Docking studies provide the basis for the structure-based design of improved steroidal alkylating esters for the treatment of estrogen-related cancers.