Pregestational neurological disorders among women of childbearing age-Nationwide data from a 13-year period in Hungary.

OBJECTIVES: Comprehensive statistics evaluating pregnancies complicated by various medical conditions are desirable for the optimization of prenatal care and for improving maternal and fetal outcomes. The main objective of our study was to assess pregnancies during a 13-year study period with accompanying pregestational neurological disorders in medical history on a nationwide level. METHODS: In the framework of the NEUROHUN 2004-2017 project utilizing medical reports submitted for reimbursement purposes to the National Health Insurance Fund, we included women with at least one labor during 2004-2016 who had at least one pregestational diagnosis of a neurological disorder received within this time frame prior to their first pregnancy during the studied period. Three-digit codes from the 10th International Classification of Diseases (ICD) were used for the identification and classification of neurological and obstetrical conditions. RESULTS: Specific inclusion and exclusion criteria were employed during the study process. A total of 744 226 women have been identified with at least one delivery during the study period with 98 792 of them (13.3%) having at least one neurological diagnosis received during 2004-2016 before their first gestation in the time frame of the study. The vast majority of diagnosis codes were related to different types of headaches affecting 69 149 (9.3%) individuals. The most prevalent diagnoses following headaches were dizziness and giddiness (15 589 patients [2.1%]; nerve, nerve root and plexus disorders (10 375 patients [1.4%]); epileptic disorders (7028 patients [0.9%]); neurological diseases of vascular origin (6091 patients [0.8%]); other disorders of the nervous system (5358 patients [0.7%]); and demyelinating diseases of the central nervous system (2129 patients [0.3%]). The present findings of our study show high prevalence of pregestational neurological disorders, the dominance of headaches followed by the rather nonspecific diagnosis of dizziness and giddiness, the relevance of nerve, nerve root and plexus disorders and epilepsy, and the importance of cerebrovascular disorders among women of childbearing age. CONCLUSION: The present research findings can help healthcare professionals, researchers and decision makers in adopting specific health policy measures based on nationwide data and further aid the development of new diagnostic and therapeutic algorithms of various neurological manifestations concerning women of childbearing age.

Exploration of Somatostatin Binding Mechanism to Somatostatin Receptor Subtype 4.

Somatostatin (also named as growth hormone-inhibiting hormone or somatotropin release-inhibiting factor) is a regulatory peptide important for the proper functioning of the endocrine system, local inflammatory reactions, mood and motor coordination, and behavioral responses to stress. Somatostatin exerts its effects via binding to G-protein-coupled somatostatin receptors of which the fourth subtype (SSTR4) is a particularly important receptor mediating analgesic, anti-inflammatory, and anti-depressant effects without endocrine actions. Thus, SSTR4 agonists are promising drug candidates. Although the knowledge of the atomic resolution-binding modes of SST would be essential for drug development, experimental elucidation of the structures of SSTR4 and its complexes is still awaiting. In the present study, structures of the somatostatin-SSTR4 complex were produced using an unbiased, blind docking approach. Beyond the static structures, the binding mechanism of SST was also elucidated in the explicit water molecular dynamics (MD) calculations, and key binding modes (external, intermediate, and internal) were distinguished. The most important residues on both receptor and SST sides were identified. An energetic comparison of SST binding to SSTR4 and 2 offered a residue-level explanation of receptor subtype selectivity. The calculated structures show good agreement with available experimental results and indicate that somatostatin binding is realized via prerequisite binding modes and an induced fit mechanism. The identified binding modes and the corresponding key residues provide useful information for future drug design targeting SSTR4.

Binding Networks Identify Targetable Protein Pockets for Mechanism-Based Drug Design.

The human genome codes only a few thousand druggable proteins, mainly receptors and enzymes. While this pool of available drug targets is limited, there is an untapped potential for discovering new drug-binding mechanisms and modes. For example, enzymes with long binding cavities offer numerous prerequisite binding sites that may be visited by an inhibitor during migration from a bulk solution to the destination site. Drug design can use these prerequisite sites as new structural targets. However, identifying these ephemeral sites is challenging. Here, we introduce a new method called NetBinder for the systematic identification and classification of prerequisite binding sites at atomic resolution. NetBinder is based on atomistic simulations of the full inhibitor binding process and provides a networking framework on which to select the most important binding modes and uncover the entire binding mechanism, including previously undiscovered events. NetBinder was validated by a study of the binding mechanism of blebbistatin (a potent inhibitor) to myosin 2 (a promising target for cancer chemotherapy). Myosin 2 is a good test enzyme because, like other potential targets, it has a long internal binding cavity that provides blebbistatin with numerous potential prerequisite binding sites. The mechanism proposed by NetBinder of myosin 2 structural changes during blebbistatin binding shows excellent agreement with experimentally determined binding sites and structural changes. While NetBinder was tested on myosin 2, it may easily be adopted to other proteins with long internal cavities, such as G-protein-coupled receptors or ion channels, the most popular current drug targets. NetBinder provides a new paradigm for drug design by a network-based elucidation of binding mechanisms at an atomic resolution.

Age and gender characteristics of patients affected with multiple sclerosis in Hungary between 2004 and 2016 / A hazai sclerosis multiplex betegpopuláció életkori és nemi megoszlása 2004 és 2016 között.

Összefoglaló. Bevezetés: Mivel hazánkban a sclerosis multiplex gyakoriságáról, valamint életkori és nemi jellegzetességeirol az elmúlt évtizedekben - egészen 2020-ig - csak regionális jellegu felmérések készültek egy-egy centrum betegforgalma alapján, az újonnan diagnosztizált és már ismert betegek országos koreloszlásáról és annak idobeli változásairól nincsenek ismereteink. Célkituzés: Jelen munkánkban több mint 14 000 beteg adatainak elemzésével a prevalens és incidens betegek koreloszlásának változását vizsgáljuk 2004-2016 során, és eredményeinket összevetjük az elmúlt évtizedekben közölt hazai adatokkal. Módszer: Munkacsoportunk az egészségbiztosítási pénztár anonimizált NEUROHUN adatbázisát elemezte, amely tartalmazza a 2004 és 2016 között az összes hazai, államilag finanszírozott, a fekvo- és járóbeteg-szakellátásból neurológiai diagnózissal jelentett esetet. A sclerosis multiplex BNO-kódjának elofordulása alapján korábban létrehoztuk a betegség adminisztratív definícióját, és megbecsültük a sclerosis multiplex országos prevalenciáját és incidenciáját. Eredmények: A prevalens betegek átlagéletkora 2015-ben 47,9 év, ugyanebben az évben az incidens betegek átlagéletkora 37,4 év volt. Vizsgálatunk szerint a prevalens betegek átlagéletkora szignifikánsan - évente egyötöd-egyharmad évvel (p<0,001) - emelkedik, mégpedig a nok esetében nagyobb mértékben. A nok átlagosan fél évvel idosebbek, mint a férfi páciensek (szignifikáns különbség: p = 0,002). A prevalens betegekben a legnépesebb korosztály az ötvenévesek felol a fiatalabb, 35-40 éves korosztály felé mozdul. Az incidens betegek átlagéletkora lassan, de szignifikánsan - évente átlagosan egyharmad évvel (p<0,001) - csökken. Következtetés: Eredményeink szerint az újonnan diagnosztizált sclerosis multiplexes páciensek átlagosan egyre fiatalabbak, és a prevalens betegek között is egyre fiatalabb korosztályok a legnépesebbek, de a javuló túlélés és a hosszabb élettartam miatt a prevalens betegek átlagéletkora összességében valószínuleg fokozatosan emelkedik. Orv Hetil. 2021; 162(19): 746-753. INTRODUCTION: The nationwide age and gender distribution of newly diagnosed and prevalent multiple sclerosis patients has been unknown in Hungary, as until 2020 only regional studies had been reported about the frequency and age characteristics of subjects with multiple sclerosis, based on single-center patient registries. OBJECTIVE: In the present study with the analysis of over 14 000 patients, we describe the changes in age distribution of prevalent and incident subjects between 2004 and 2016 and compare our results with the data published on the subject during the last decades in Hungary. METHOD: We have analyzed the pseudonymized NEUROHUN database provided by the single-payer National Health Insurance Fund, that contains each claim submitted by public hospitals and outpatient services for neurologic diseases between 2004 and 2016. Using the ICD10-code of multiple sclerosis, we have previously established the administrative definition of the illness and estimated its prevalence and incidence in the country. RESULTS: The mean age of prevalent patients was 47.9 years in 2015, whereas in the same year the mean age of incident cases was 37.4 years. The average age of prevalent patients shows a significant rise - with an annual increase of one fifth-one third year (p<0.001) - with a more pronounced increase among women. The age of women is higher by half a year (p = 0.002). The most populous age groups among prevalent subjects shift from the fifties towards the younger generations between 35-40 years of age. The average age of incident subjects slowly, but significantly decreases, with a mean annual decrease of about one third year (p<0.001). CONCLUSION: Our results suggest that though new patients are younger year-by-year and the most populous age groups are also younger, altogether the average age of prevalent subjects continuously increases, probably due to the longer survival and lifespan of patients with multiple sclerosis. Orv Hetil. 2021; 162(19): 746-753.

Small molecule somatostatin receptor subtype 4 (sst4) agonists are novel anti-inflammatory and analgesic drug candidates.

We provided strong proof of concept evidence that somatostatin mediates potent analgesic and anti-inflammatory actions via its receptor subtype 4 (sst4) located both at the periphery and the central nervous system. Therefore, sst4 agonists are promising novel drug candidates for neuropathic pain and neurogenic inflammation, but rational drug design was not possible due to the lack of knowledge about its 3-dimensional structure. We modeled the sst4 receptor structure, described its agonist binding properties, and characterized the binding of our novel small molecule sst4 agonists (4-phenetylamino-7H-pyrrolo[2,3-d]pyrimidine derivatives) using an in silico platform. In addition to the in silico binding data, somatostatin displacement by Compound 1 was demonstrated in the competitive binding assay on sst4-expressing cells. In vivo effects were investigated in rat models of neurogenic inflammation and chronic traumatic neuropathic pain. We defined high- and low-affinity binding pockets of sst4 for our ligands, binding of the highest affinity compounds were similar to that of the reference ligand J-2156. We showed potent G-protein activation with the highest potency of 10 nM EC50 value and highest efficacy of 342%. Oral administration of 100 µg/kg of 5 compounds significantly inhibited acute neurogenic plasma protein extravasation in the paw skin by 40-60%, one candidate abolished and 3 others diminished sciatic nerve-ligation induced neuropathic hyperalgesia by 28-62%. The in silico predictions on sst4-ligands were tested in biological systems. Low oral dose of our novel agonists inhibit neurogenic inflammation and neuropathic pain, which opens promising drug developmental perspectives for these unmet medical need conditions.

Incidence and prevalence of multiple sclerosis in Hungary based on record linkage of nationwide multiple healthcare administrative data.

OBJECTIVES: As there were only regional studies in Hungary about the prevalence of multiple sclerosis (MS), we aimed to estimate its epidemiological features using data of Hungary's single-payer health insurance system. METHODS: Pseudonymized database of claims reported by hospitals and outpatient services between 2004-2016 was analyzed and linked with an independent database of outpatient pharmacy refills between 2010-2016. We established an administrative case definition of MS and validated it on medical records of 309 consecutive patients. A subject was defined as MS-patient if received MS diagnosis (International Classification of Diseases, 10th edition, code G35) on three or more occasions at least in 2 calendar years and at least once documented by a neurologist. Patients were counted as incident cases in the year of the first submitted claim for MS. We allowed a 6-year-long run-in period, so only data between 2010-2015 are discussed. RESULTS: Sensitivity of the administrative case definition turned out to be 99%, while specificity was >99%. Crude prevalence of MS has increased from 109.3/100,000 in 2010 to 130.8/100,000 in 2015 (p-value = 0.000003). Crude incidence declined from 7.1/100,000 (2010) to 5.4/100,000 (2015) (p-value = 0.018). Direct standardization - based on European standard population and results of nationwide Hungarian census of 2011 - revealed that age standardized prevalence was 105.2/100,000 (2010), which has grown to 127.2/100,000 (2015) (p-value = 0.000001). Age standardized incidence rate declined from 6.7/100,000 (2010) to 5.1/100,000 (2015) (p-value = 0.016). The ratio of MS-patients receiving ≥1 prescription for disease modifying treatment increased from 0.19 (2010) to 0.29 (2015) (p-value = 0.0051). The female/male ratio of prevalent cases remained 2.6. DISCUSSION: The prevalence of MS in Hungary is higher than previously reported, the incidence rate is moderate. The prevalence is rising, the incidence rate shows decline. The proportion of patients receiving disease modifying treatment grows but was still around 30% in 2015.

Ndr/Lats Kinases Bind Specific Mob-Family Coactivators through a Conserved and Modular Interface.

Ndr/Lats kinases bind Mob coactivator proteins to form complexes that are essential and evolutionarily conserved components of "Hippo" signaling pathways, which control cell proliferation and morphogenesis in eukaryotes. All Ndr/Lats kinases have a characteristic N-terminal regulatory (NTR) region that binds a specific Mob cofactor: Lats kinases associate with Mob1 proteins, and Ndr kinases associate with Mob2 proteins. To better understand the significance of the association of Mob protein with Ndr/Lats kinases and selective binding of Ndr and Lats to distinct Mob cofactors, we determined crystal structures of Saccharomyces cerevisiae Cbk1NTR-Mob2 and Dbf2NTR-Mob1 and experimentally assessed determinants of Mob cofactor binding and specificity. This allowed a significant improvement in the previously determined structure of Cbk1 kinase bound to Mob2, presently the only crystallographic model of a full length Ndr/Lats kinase complexed with a Mob cofactor. Our analysis indicates that the Ndr/LatsNTR-Mob interface provides a distinctive kinase regulation mechanism, in which the Mob cofactor organizes the Ndr/Lats NTR to interact with the AGC kinase C-terminal hydrophobic motif (HM), which is involved in allosteric regulation. The Mob-organized NTR appears to mediate association of the HM with an allosteric site on the N-terminal kinase lobe. We also found that Cbk1 and Dbf2 associated specifically with Mob2 and Mob1, respectively. Alteration of residues in the Cbk1 NTR allows association of the noncognate Mob cofactor, indicating that cofactor specificity is restricted by discrete sites rather than being broadly distributed. Overall, our analysis provides a new picture of the functional role of Mob association and indicates that the Ndr/LatsNTR-Mob interface is largely a common structural platform that mediates kinase-cofactor binding.

Systematic Exploration of Binding Modes of Ligands on Drug Targets.

Exploration of binding sites of ligands (drug candidates) on macromolecular targets is a central question of molecular design. Although there are experimental and theoretical methods available for the determination of atomic resolution structure of drug-target complexes, they are often limited to identify only the primary binding mode (site and conformation). Systematic exploration of multiple (allosteric or prerequisite) binding modes is a challenge for present methods. The Wrapper module of our new method, Wrap 'n' Shake, answers this challenge by a fast, computational blind docking approach. Beyond the primary (orthosteric) binding mode, Wrapper systematically produces all possible binding modes of a drug scanning the entire surface of the target. In several fast blind docking cycles, the entire surface of the target molecule is systematically wrapped in a monolayer of N ligand copies. The resulted target-ligandN complex structure can be used as it is, or important ligand binding modes can be further distinguished in molecular dynamics shakers. Wrapper has been tested on important protein targets of drug design projects on cellular signaling and cancer. Here, we provide a practical description of the application of Wrapper.

A Fragmenting Protocol with Explicit Hydration for Calculation of Binding Enthalpies of Target-Ligand Complexes at a Quantum Mechanical Level.

Optimization of the enthalpy component of binding thermodynamics of drug candidates is a successful pathway of rational molecular design. However, the large size and missing hydration structure of target-ligand complexes often hinder such optimizations with quantum mechanical (QM) methods. At the same time, QM calculations are often necessitated for proper handling of electronic effects. To overcome the above problems, and help the QM design of new drugs, a protocol is introduced for atomic level determination of hydration structure and extraction of structures of target-ligand complex interfaces. The protocol is a combination of a previously published program MobyWat, an engine for assigning explicit water positions, and Fragmenter, a new tool for optimal fragmentation of protein targets. The protocol fostered a series of fast calculations of ligand binding enthalpies at the semi-empirical QM level. Ligands of diverse chemistry ranging from small aromatic compounds up to a large peptide helix of a molecular weight of 3000 targeting a leukemia protein were selected for systematic investigations. Comparison of various combinations of implicit and explicit water models demonstrated that the presence of accurately predicted explicit water molecules in the complex interface considerably improved the agreement with experimental results. A single scaling factor was derived for conversion of QM reaction heats into binding enthalpy values. The factor links molecular structure with binding thermodynamics via QM calculations. The new protocol and scaling factor will help automated optimization of binding enthalpy in future molecular design projects.

Linking Individual Patient Data to Estimate Incidence and Prevalence of Parkinson's Disease by Comparing Reports of Neurological Services and Pharmacy Prescription Refills at a Nationwide Level.

Objectives: We set forth to estimate the number of those with Parkinson's disease (PD) in Hungary, a country with a single-payer health insurance system covering 10 million inhabitants. Methods: We analyzed all hospital and outpatient reports from neurological services and pharmacy reports of prescription refills. We cross-checked clinically administered diagnosis of PD with prescription refills of antiparkinsonian medications using record linkage. We used the ICD-10 code of G20 in any diagnostic category to find all cases with possible PD. For case certification those patients were considered to have PD who were recorded with G20 code in at least 2 calendar years. For a more conservative estimation we determined the number of those who also refilled antiparkinsonian medication. Results: Between 2010 and 2012 there were 46,383 subjects with certified PD by clinical criteria. Crude and age-standardized incidence were 49/100,000/year (95% CI: 45-53), and 56/100,000/year (95% CI: 51-60). Crude and age standardized prevalence rates were 404/100,000 (95% CI: 392-416) and 471/100,000 (95% CI: 456-485). Of all clinically certified PD patients 72% refilled antiparkinsonian medications. Discussion: The incidence and prevalence of PD in Hungary is higher than earlier estimates, which should be considered in organizing healthcare services for this patient group.

Towards Unraveling the Histone Code by Fragment Blind Docking.

Histones serve as protein spools for winding the DNA in the nucleosome. High variability of their post-translational modifications result in a unique code system often responsible for the pathomechanisms of epigenetics-based diseases. Decoding is performed by reader proteins via complex formation with the N-terminal peptide tails of histones. Determination of structures of histone-reader complexes would be a key to unravel the histone code and the design of new drugs. However, the large number of possible histone complex variations imposes a true challenge for experimental structure determination techniques. Calculation of such complexes is difficult due to considerable size and flexibility of peptides and the shallow binding surfaces of the readers. Moreover, location of the binding sites is often unknown, which requires a blind docking search over the entire surface of the target protein. To accelerate the work in this field, a new approach is presented for prediction of the structure of histone H3 peptide tails docked to their targets. Using a fragmenting protocol and a systematic blind docking method, a collection of well-positioned fragments of the H3 peptide is produced. After linking the fragments, reconstitution of anchoring regions of the target-bound H3 peptide conformations was possible. As a first attempt of combination of blind and fragment docking approaches, our new method is named fragment blind docking (FBD).

Interaction of the mycotoxin metabolite dihydrocitrinone with serum albumin.

Citrinin (CIT) is a nephrotoxic mycotoxin produced by Penicillium, Monascus, and Aspergillus species. CIT appears as a contaminant in cereals, cereal-based products, fruits, nuts, and spices. During the biotransformation of CIT, its major urinary metabolite dihydrocitrinone (DHC) is formed. Albumin interacts with several compounds (including mycotoxins) affecting their tissue distribution and elimination. CIT-albumin interaction is known; however, the complex formation of DHC with albumin has not been reported previously. In this study, we aimed to investigate the interaction of DHC with albumin, employing fluorescence spectroscopy, circular dichroism, and molecular modeling studies. Furthermore, species differences and thermodynamics of the interaction as well as the effects of albumin on the acute in vitro toxicity of DHC and CIT were also tested. Our main observations/conclusions are as follows: (1) Fluorescence signal of DHC is strongly enhanced by albumin. (2) Formation of DHC-albumin complexes is supported by both fluorescence spectroscopic and circular dichroism studies. (3) DHC forms similarly stable complexes with human albumin (K~105 L/mol) as CIT. (4) DHC-albumin interaction did not show significant species differences (tested with human, bovine, porcine, and rat albumins). (5) Based on modeling studies and investigations with site markers, DHC occupies the Heme binding site (subdomain IB) on human albumin. (6) The presence of albumin significantly decreased the acute in vitro cytotoxic effects of both DHC and CIT on MDCK cell line.

Interaction of 2'R-ochratoxin A with Serum Albumins: Binding Site, Effects of Site Markers, Thermodynamics, Species Differences of Albumin-binding, and Influence of Albumin on Its Toxicity in MDCK Cells.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin. Roasting of OTA-contaminated coffee results in the formation of 2'R-ochratoxin A (2'R-OTA), which appears in the blood of coffee drinkers. Human serum albumin (HSA) binds 2'R-OTA (and OTA) with high affinity; therefore, albumin may influence the tissue uptake and elimination of ochratoxins. We aimed to investigate the binding site of 2'R-OTA (verses OTA) in HSA and the displacing effects of site markers to explore which molecules can interfere with its albumin-binding. Affinity of 2'R-OTA toward albumins from various species (human, bovine, porcine and rat) was tested to evaluate the interspecies differences regarding 2'R-OTA-albumin interaction. Thermodynamic studies were performed to give a deeper insight into the molecular background of the complex formation. Besides fluorescence spectroscopic and modeling studies, effects of HSA, and fetal bovine serum on the cytotoxicity of 2'R-OTA and OTA were tested in MDCK kidney cell line in order to demonstrate the influence of albumin-binding on the cellular uptake of ochratoxins. Site markers displaced more effectively 2'R-OTA than OTA from HSA. Fluorescence and binding constants of 2'R-OTA-albumin and OTA-albumin complexes showed different tendencies. Albumin significantly decreased the cytotoxicity of ochratoxins. 2'R-OTA, even at sub-toxic concentrations, increased the toxic action of OTA.

Interactions of zearalenone and its reduced metabolites α-zearalenol and ß-zearalenol with serum albumins: species differences, binding sites, and thermodynamics.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species. ZEN mainly appears in cereals and related foodstuffs, causing reproductive disorders in animals, due to its xenoestrogenic effects. The main reduced metabolites of ZEN are α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL). Similarly to ZEN, ZELs can also activate estrogen receptors; moreover, α-ZEL is the most potent endocrine disruptor among these three compounds. Serum albumin is the most abundant plasma protein in the circulation; it affects the tissue distribution and elimination of several drugs and xenobiotics. Although ZEN binds to albumin with high affinity, albumin-binding of α-ZEL and ß-ZEL has not been investigated. In this study, the complex formation of ZEN, α-ZEL, and ß-ZEL with human (HSA), bovine (BSA), porcine (PSA), and rat serum albumins (RSA) was investigated by fluorescence spectroscopy, affinity chromatography, thermodynamic studies, and molecular modeling. Our main observations are as follows: (1) ZEN binds with higher affinity to albumins than α-ZEL and ß-ZEL. (2) The low binding affinity of ß-ZEL toward albumin may result from its different binding position or binding site. (3) The binding constants of the mycotoxin-albumin complexes significantly vary with the species. (4) From the thermodynamic point of view, the formation of ZEN-HSA and ZEN-RSA complexes are similar, while the formation of ZEN-BSA and ZEN-PSA complexes are markedly different. These results suggest that the toxicological relevance of ZEN-albumin and ZEL-albumin interactions may also be species-dependent.

Pharmacokinetic interaction of diosmetin and silibinin with other drugs: Inhibition of CYP2C9-mediated biotransformation and displacement from serum albumin.

Diosmin and silibinin (SIL) are polyphenolic compounds which are the active components of several drugs and dietary supplements. After the oral administration of diosmin (flavonoid glycoside), only its aglycone diosmetin (DIO) reaches the systemic circulation. Both DIO and SIL form complexes with serum albumin and are able to inhibit several cytochrome P450 enzymes. Therefore, it is reasonable to hypothesize that these polyphenols may displace some drugs from serum albumin and inhibit their biotransformation, potentially leading to the disruption of drug therapy. In this study, the inhibitory action of DIO and SIL on CYP2C9-catalyzed metabolism of diclofenac to 4'-hydroxydiclofenac was examined, using warfarin as a positive control. Furthermore, interaction of DIO and SIL with human and bovine serum albumins as well as the displacement of warfarin from albumin by DIO and SIL were tested, employing steady-state fluorescence spectroscopy, fluorescence anisotropy, ultrafiltration, and molecular modeling. It is demonstrated that DIO and SIL are potent inhibitors of CYP2C9 enzyme and are able to displace the Site I ligand warfarin from human serum albumin. Because DIO and SIL may interfere with the pharmacokinetics of several drugs through both ways, we need to consider the potentially hazardous consequences of the consumption of diosmin or SIL together with other drugs.

Analysis of the influence of simulation parameters on biomolecule-linked water networks.

Advancement of computational molecular dynamics allows rapid calculation of large biomolecular systems in their water surroundings. New approaches of prediction of hydration networks of biomolecular surfaces and complex interfaces are also based on molecular dynamics (MD). Calculations with explicit solvent models can trace thousands of water molecules individually on a real time scale, yielding information on their mobility, and predicting their networking with biomolecular solutes and other water partners. Here, we investigate the effect of key parameters of molecular dynamics simulations on the quality of such predictions. Accordingly, systematic scans on temperature, pressure, force field, explicit water model and thermodynamic ensemble are performed. Explanations of optimal parameter values are provided using structural examples and analyses of the corresponding hydration networks.

Systematic exploration of multiple drug binding sites.

BACKGROUND: Targets with multiple (prerequisite or allosteric) binding sites have an increasing importance in drug design. Experimental determination of atomic resolution structures of ligands weakly bound to multiple binding sites is often challenging. Blind docking has been widely used for fast mapping of the entire target surface for multiple binding sites. Reliability of blind docking is limited by approximations of hydration models, simplified handling of molecular flexibility, and imperfect search algorithms. RESULTS: To overcome such limitations, the present study introduces Wrap 'n' Shake (WnS), an atomic resolution method that systematically "wraps" the entire target into a monolayer of ligand molecules. Functional binding sites are extracted by a rapid molecular dynamics shaker. WnS is tested on biologically important systems such as mitogen-activated protein, tyrosine-protein kinases, key players of cellular signaling, and farnesyl pyrophosphate synthase, a target of antitumor agents.

Dynamic changes in binding interaction networks of sex steroids establish their non-classical effects.

Non-classical signaling in the intracellular second messenger system plays a pivotal role in the cytoprotective effect of estradiol. Estrogen receptor is a common target of sex steroids and important in mediating estradiol-induced neuroprotection. Whereas the mechanism of genomic effects of sex steroids is fairly understood, their non-classical effects have not been elucidated completely. We use real time molecular dynamics calculations to uncover the interaction network of estradiol and activator estren. Besides steroid interactions, we also investigate the co-activation of the receptor. We show how steroid binding to the alternative binding site of the non-classical action is facilitated by the presence of a steroid in the classical binding site and the absence of the co-activator peptide. Uncovering such dynamic mechanisms behind steroid action will help the structure-based design of new drugs with non-classical responses and cytoprotective potential.

Investigation of Non-Covalent Interactions of Aflatoxins (B1, B2, G1, G2, and M1) with Serum Albumin.

Aflatoxins are widely spread mycotoxins produced mainly by Aspergillus species. Consumption of aflatoxin-contaminated foods and drinks causes serious health risks for people worldwide. It is well-known that the reactive epoxide metabolite of aflatoxin B1 (AFB1) forms covalent adducts with serum albumin. However, non-covalent interactions of aflatoxins with human serum albumin (HSA) are poorly characterized. Thus, in this study the complex formation of aflatoxins was examined with HSA applying spectroscopic and molecular modelling studies. Our results demonstrate that aflatoxins form stable complexes with HSA as reflected by binding constants between 2.1 × 104 and 4.5 × 104 dm³/mol. A binding free energy value of -26.90 kJ mol-1 suggests a spontaneous binding process between AFB1 and HSA at room-temperature, while the positive entropy change of 55.1 JK-1 mol-1 indicates a partial decomposition of the solvation shells of the interacting molecules. Modeling studies and investigations with site markers suggest that Sudlow's Site I of subdomain IIA is the high affinity binding site of aflatoxins on HSA. Interaction of AFB1 with bovine, porcine, and rat serum albumins was also investigated. Similar stabilities of the examined AFB1-albumin complexes were observed suggesting the low species differences of the albumin-binding of aflatoxins.

Interaction of mycotoxin zearalenone with human serum albumin.

Zearalenone (ZEN) is a mycotoxin produced mainly by Fusarium species. Fungal contamination of cereals and plants can result in the formation of ZEN, leading to its presence in different foods, animal feeds, and drinks. Because ZEN is an endocrine disruptor, it causes reproductive disorders in farm animals and hyperoestrogenic syndromes in humans. Despite toxicokinetic properties of ZEN were studied in more species, we have no information regarding the interaction of ZEN with serum albumin. Since albumin commonly plays an important role in the toxicokinetics of different toxins, interaction of ZEN with albumin has of high biological importance. Therefore the interaction of ZEN with human serum albumin (HSA) was investigated using spectroscopic methods, ultrafiltration, and molecular modeling studies. Fluorescence spectroscopic studies demonstrate that ZEN forms complex with HSA. Binding constant (K) of ZEN-HSA complex was quantified with fluorescence quenching technique. The determined binding constant (logK=5.1) reflects the strong interaction of ZEN with albumin suggesting the potential biological importance of ZEN-HSA complex formation. Based on the results of the investigations with site markers as well as docking studies, ZEN occupies a non-conventional binding site on HSA. Considering the above listed observations, we should keep in mind this interaction if we would like to precisely understand the toxicokinetic behavior of ZEN.