Secondary Metabolites from Marine-Derived Bacteria with Antibiotic and Antibiofilm Activities against Drug-Resistant Pathogens.

The search for new antibiotics against drug-resistant microbes has been expanded to marine bacteria. Marine bacteria have been proven to be a prolific source of a myriad of novel compounds with potential biological activities. Therefore, this review highlights novel and bioactive compounds from marine bacteria reported during the period of January 2016 to December 2021. Published articles containing novel marine bacterial secondary metabolites that are active against drug-resistant pathogens were collected. Previously described compounds (prior to January 2016) are not included in this review. Unreported compounds during this period that exhibited activity against pathogenic microbes were discussed and compared in order to find the cue of the structure-bioactivity relationship. The results showed that Streptomyces are the most studied bacteria with undescribed bioactive compounds, followed by other genera in the Actinobacteria. We have categorized the structures of the compounds in the present review into four groups, based on their biosynthetic origins, as polyketide derivatives, amino acid derivatives, terpenoids, as well as compounds with mixed origin. These compounds were active against one or more drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), vancomycin-resistant Enterococci (VRE), multidrug-resistant Mycobacterium tuberculosis (MDR-TB), and amphotericin B-resistant Candida albicans. In addition, some of the compounds also showed activity against biofilm formation of the test bacteria. Some previously undescribed compounds, isolated from marine-derived bacteria during this period, could have a good potential as lead compounds for the development of drug candidates to overcome multidrug-resistant pathogens.

Quantification of 3-Hydroxypropyl Mercapturic Acid in the Urine of Patients with Breast Cancer to Monitor Cyclophosphamide Toxicity.

BACKGROUND: The alkylating agent cyclophosphamide is used in chemotherapy regimens for various type of cancer. However, cyclophosphamide may lead to toxic side effects on the bladder, namely hemorrhagic cystitis, which can cause hematuria, and, potentially, bladder cancer. These effects are caused by acrolein, a byproduct of cyclophosphamide metabolism. In this study, a method to quantify 3-hydroxypropyl mercapturic acid (3-HPMA) in urine was developed. 3-HPMA is a stable metabolite of acrolein that serves as biomarker of acrolein. METHODS: Urine samples were collected 4 hours after cyclophosphamide administration and analyzed to determine the risk of hematuria. 3-HPMA was analyzed by reverse-phase LC-MS/MS using a triple quadrupole electrospray ionization mass spectrometer in the positive-ion mode. The mobile phase was a 90:10 (vol/vol) mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Multiple reaction monitoring mode was used, with m/z 222.10 → 90.97 for 3-HPMA and 164.10 → 122.02 for the internal standard N-acetyl cysteine (NAC). Samples were prepared by acidification and dilution. RESULTS: The analytical method produced a linear response within the concentration range of 40-10,000 ng/mL. The method was validated in accordance with 2018 FDA guidelines and applied to quantify 3-HPMA in the urine of 40 patients with breast cancer. The measured concentrations ranged from 820.3 to 5596.1 ng/mg creatinine. Seven patients identified with hematuria had low 3-HPMA concentrations of 4445.824 ± 411.17 ng/mg creatinine, and 33 patients without hematuria had low 3-HPMA concentrations of 2419.4 ± 1171.8 ng/mg creatinine. CONCLUSIONS: The method was applicable for the quantification of 3-HPMA in human urine. Large variations in 3-HPMA concentrations were found in 40 patients with breast cancer treated with cyclophosphamide, with a significant difference (P < 0.05) observed between patients with hematuria and those without hematuria.

A Comparative Linear Interaction Energy and MM/PBSA Study on SIRT1-Ligand Binding Free Energy Calculation.

Binding free energy (ΔGbind) computation can play an important role in prioritizing compounds to be evaluated experimentally on their affinity for target proteins, yet fast and accurate ΔGbind calculation remains an elusive task. In this study, we compare the performance of two popular end-point methods, i.e., linear interaction energy (LIE) and molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA), with respect to their ability to correlate calculated binding affinities of 27 thieno[3,2-d]pyrimidine-6-carboxamide-derived sirtuin 1 (SIRT1) inhibitors with experimental data. Compared with the standard single-trajectory setup of MM/PBSA, our study elucidates that LIE allows to obtain direct ("absolute") values for SIRT1 binding free energies with lower compute requirements, while the accuracy in calculating relative values for ΔGbind is comparable (Pearson's r = 0.72 and 0.64 for LIE and MM/PBSA, respectively). We also investigate the potential of combining multiple docking poses in iterative LIE models and find that Boltzmann-like weighting of outcomes of simulations starting from different poses can retrieve appropriate binding orientations. In addition, we find that in this particular case study the LIE and MM/PBSA models can be optimized by neglecting the contributions from electrostatic and polar interactions to the ΔGbind calculations.

(2E,5E)-2,5-Bis(4-hy-droxy-3-meth-oxy-benzyl-idene)cyclo-penta-none ethanol monosolvate.

In the title structure, C21H20O5·C2H5OH, the curcumine-type mol-ecule has a double E conformation for the two benzyl-idene double bonds [C=C = 1.342 (4) and 1.349 (4) Å] and is nearly planar with respect to the non-H atoms (r.m.s. deviation from planarity = 0.069 Å). The two phenolic OH groups form bifurcated hydrogen bonds with intra-molecular branches to adjacent meth-oxy O atoms and inter-molecular branches to either a neighbouring mol-ecule or an ethanol solvent mol-ecule. The ethanol O atom donates a hydrogen bond to the keto O atom. These hydrogen bonds link the constituents into layers parallel to (101) in the crystal structure.

Synthesis and in vitro Activity of Eugenyl Benzoate Derivatives as BCL-2 Inhibitor in Colorectal Cancer with QSAR and Molecular Docking Approach.

OBJECTIVE: This study is aimed to acquiring new compounds of Eugenyl benzoate (2-methoxy-4-(prop-2-en-1-yl)phenyl benzoate) derivatives that can inhibit HT29 colorectal cancer cells. METHODS: In this research, we used several chemical reactions to synthesize novel compounds, such as Esterification, Demethylation, Halohydrin, and Sharpless reaction. Cytotoxicity assays were performed to test the inhibitory activity of compounds against HT29 colon cancer cells. QSAR analysis were carried out to analyse the relationship of chemical structure of the novel compounds with their cytotoxic activity. RESULT: Ten novel compounds were successfully synthesized and tested in vitro against the HT29 cell. The IC50 of the novel compounds were between 26.56 µmol/ml - 286.81 µmol/ml which compound 4-[(2S)-2,3-dihydroxypropyl]-2-methoxyphenyl 2-hydroxybenzoate (9) showed as best active compound as BCL-2 inhibitors better than other synthesized compounds and Eugenol as well. QSAR analysis of the in vitro results gave a Log equation: 1/IC50 = -0.865-0.210 (LogP)2 + 1,264 (logP)-0.994 CMR (n = 10; r = 0.706; SE: 0.21; F = 0.497, sig = 7.86). The equation shows the log variable P and CMR affect IC50. The properties of hydrophobicity (log P) are more instrumental than the ones of steric (CMR). CONCLUSION: The active compound (9) given best activities as BCL-2 inhibitors than other eugenol derivatives. QSAR indicates the logP and CMR have effect on its colorectal cytotoxic activity which the hydrophobicity parameter (logP) plays more role than the steric parameter (CMR).

Identification of potential Indonesian marine invertebrate bioactive compounds as TMPRSS2 and SARS-CoV-2 Omicron spike protein inhibitors through computational screening.

The coronavirus pandemic led to the announcement of a worldwide health emergency. The SARS-CoV-2 Omicron variant, which swiftly spread worldwide, has fueled existing challenges. Appropriate medication is necessary to avoid severe SARS-CoV-2 disease. The human TMPRSS2 and SARS-CoV-2 Omicron spike protein, which are required for viral entry into the host phase, were identified as the target proteins through computational screening. Structure-based virtual screening; molecular docking; absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis; and molecular dynamics simulation were the methods applied for TMPRSS2 and spike protein inhibitors. Bioactive marine invertebrates from Indonesia were employed as test ligands. Camostat and nafamostat (co-crystal) were utilized as reference ligands against TMPRSS2, whereas mefloquine was used as a reference ligand against spike protein. Following a molecular docking and dynamics simulation, we found that acanthomanzamine C has remarkable effectiveness against TMPRSS2 and spike protein. Compared to camostat (-8.25 kcal/mol), nafamostat (-6.52 kcal/mol), and mefloquine (-6.34 kcal/mol), acanthomanzamine C binds to TMPRSS2 and spike protein with binding energies of -9.75 kcal/mol and -9.19 kcal/mol, respectively. Furthermore, slight variances in the MD simulation demonstrated consistent binding to TMPRSS2 and spike protein after the initial 50 ns. These results are highly valuable in the search for a treatment for SARS-CoV-2 infection.

Virtual screening of Indonesian herbal compounds as COVID-19 supportive therapy: machine learning and pharmacophore modeling approaches.

BACKGROUND: The number of COVID-19 cases continues to grow in Indonesia. This phenomenon motivates researchers to find alternative drugs that function for prevention or treatment. Due to the rich biodiversity of Indonesian medicinal plants, one alternative is to examine the potential of herbal medicines to support COVID therapy. This study aims to identify potential compound candidates in Indonesian herbal using a machine learning and pharmacophore modeling approaches. METHODS: We used three classification methods that had different decision-making processes: support vector machine (SVM), multilayer perceptron (MLP), and random forest (RF). For the pharmacophore modeling approach, we performed a structure-based analysis on the 3D structure of the main protease SARS-CoV-2 (3CLPro) and repurposed SARS, MERS, and SARS-CoV-2 drugs identified from the literature as datasets in the ligand-based method. Lastly, we used molecular docking to analyze the interactions between the 3CLpro and 14 hit compounds from the Indonesian Herbal Database (HerbalDB), with lopinavir as a positive control. RESULTS: From the molecular docking analysis, we found six potential compounds that may act as the main proteases of the SARS-CoV-2 inhibitor: hesperidin, kaempferol-3,4'-di-O-methyl ether (Ermanin); myricetin-3-glucoside, peonidin 3-(4'-arabinosylglucoside); quercetin 3-(2G-rhamnosylrutinoside); and rhamnetin 3-mannosyl-(1-2)-alloside. CONCLUSIONS: Our layered virtual screening with machine learning and pharmacophore modeling approaches provided a more objective and optimal virtual screening and avoided subjective decision making of the results. Herbal compounds from the screening, i.e. hesperidin, kaempferol-3,4'-di-O-methyl ether (Ermanin); myricetin-3-glucoside, peonidin 3-(4'-arabinosylglucoside); quercetin 3-(2G-rhamnosylrutinoside); and rhamnetin 3-mannosyl-(1-2)-alloside are potential antiviral candidates for SARS-CoV-2. Moringa oleifera and Psidium guajava that consist of those compounds, could be an alternative option as COVID-19 herbal preventions.

Virtual screening of dipeptidyl peptidase-4 inhibitors using quantitative structure-activity relationship-based artificial intelligence and molecular docking of hit compounds.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are becoming an essential drug in the treatment of type 2 diabetes mellitus; however, some classes of these drugs exert side effects, including joint pain and pancreatitis. Studies suggest that these side effects might be related to secondary inhibition of DPP-8 and DPP-9. In this study, we identified DPP-4-inhibitor hit compounds selective against DPP-8 and DPP-9. We built a virtual screening workflow using a quantitative structure-activity relationship (QSAR) strategy based on artificial intelligence to allow faster screening of millions of molecules for the DPP-4 target relative to other screening methods. Five regression machine learning algorithms and four classification machine learning algorithms were applied to build virtual screening workflows, with the QSAR model applied using support vector regression (R2pred 0.78) and the classification QSAR model using the random forest algorithm with 92.2% accuracy. Virtual screening results of > 10 million molecules obtained 2 716 hits compounds with a pIC50 value of > 7.5. Additionally, molecular docking results of several potential hit compounds for DPP-4, DPP-8, and DPP-9 identified CH0002 as showing high inhibitory potential against DPP-4 and low inhibitory potential for DPP-8 and DPP-9 enzymes. These results demonstrated the effectiveness of this technique for identifying DPP-4-inhibitor hit compounds selective for DPP-4 and against DPP-8 and DPP-9 and suggest its potential efficacy for applications to discover hit compounds of other targets.

The correlation between the level of 3-hydroxypropyl mercapturic acid, CYP2B6 polymorphisms, and hematuria occurrences after cyclophosphamide administration and its bioanalytical methods: A systematic review.

BACKGROUND: Cyclophosphamide (CPA) is a cytotoxic prodrug that needs to be metabolized by cytochrome P450 enzymes, like CYP2B6. Unfortunately, CYP2B6 is a very polymorphic enzyme and can cause a change in 3-hydroxypropyl mercapturic acid (3-HPMA), the most found CYP metabolite in urine levels. Change in 3-HPMA levels can also indicate the level change in its precursor, acrolein, which is responsible for the hematuria incidence after CPA administration.This review's purpose is to obtain a conclusion about the optimal 3-HPMA analysis method in urine after the administration of cyclophosphamide using liquid chromatography-tandem mass spectrometry (LC-MS/MS) through literature review from previous studies. Also, this review was written to examine the relationship between levels of 3-HPMA in urine, polymorphisms of CYP2B6 enzymes, and the incidence of hematuria after cyclophosphamide administration in cancer patients. METHODS: Major databases, such as Universitas Indonesia's library database ScienceDirect, PubMed/Medline, Frontiers Media, and Google Scholar database, were used to find both published and unpublished studies without a time limit until 2020. Studies on pharmacokinetics, pharmacodynamics, drug therapy monitoring of cyclophosphamide, bioanalysis, and polymerase chain reaction (PCR) published in Indonesian and English were included. Meanwhile, non-related studies or studies written in other languages besides Indonesian and English were excluded. Two independent reviewers screened the titles, abstracts, and full-text manuscripts. Data obtained from eligible sources were used to answer the purpose of this review in a narrative form. RESULTS: The authors found 436 related studies from various databases and websites. Then, the authors narrowed it down into 62 pieces of literature by removing the duplicates and reviewing the abstracts and full-text manuscripts. Out of 62 sources, the authors found 30 studies that explained 3-HPMA analysis using LC/MS-MS, CYP2B6 polymorphisms, and hematuria occurrences. The authors used those 30 studies to build a conclusion regarding the purpose of this study. We strengthened the results with some additional information from the other 32 eligible sources. CONCLUSIONS: The authors conclude that according to literature searches from previous studies, the optimal 3-HPMA analysis method in urine after cyclophosphamide administration using LC-MS/MS is using triple quadrupole LC-MS/MS; source of positive ion electrospray ionization (ESI); mobile phase combination of 0.1% formic acid in water (A) - 0.1% formic acid in acetonitrile (90:10 v/v) (B); the Acquity® BEH C18 column (2.1 × 100 mm; 1.7 µm); injection volume of 10 µl; flow rate of 0.2 ml/minute; gradient elution method. Detection was carried out using mass spectrometry with m/z ratio of 222.10 > 90 for 3-HPMA and m/z 164.10 > 122 for n-acetylcysteine (NAC). The optimum sample preparation method is acidification and dilution ratio of 1:5 v/v. Also, there is a relationship between 3-HPMA levels, CYP2B6 polymorphisms, and the occurrences of hematuria after the administration of cyclophosphamide, which is a type of CYP2B6 polymorph, namely CYP2B6∗6, can increase cyclophosphamide hydroxylation so that it can increase the levels of acrolein and 3-HPMA, as its metabolites, and risk of hematuria. ETHICS AND DISSEMINATION: This research does not use human participants, human data, or human tissue for being directly studied for the review. Therefore, ethics approval and consent to participate are not applicable. REGISTRATION: This research has not been registered yet.

In silico and in vitro identification of candidate SIRT1 activators from Indonesian medicinal plants compounds database.

Sirtuin 1 (SIRT1) is a class III family of protein histone deacetylases involved in NAD+-dependent deacetylation reactions. It has been suggested that SIRT1 activators may have a protective role against type 2 diabetes, the aging process, and inflammation. This study aimed to explore and identify medicinal plant compounds from Indonesian Herbal Database (HerbalDB) that might potentially become a candidate for SIRT1 activators through a combination of in silico and in vitro methods. Two pharmacophore models were developed using co-crystalized ligands that allosterically bind with SIRT1 similar to the putative ligands used by SIRT1 activators. Then, these were used for the virtual screening of HerbalDB. The identified compounds were subjected to molecular docking and 50 ns molecular dynamics simulation. Molecular dynamics simulation was analyzed using MM-GB(PB)SA methods. The compounds identified by these methods were tested in an in vitro study using a SIRT-Glo™ luminescence assay. Virtual screening using structure-based pharmacophores predicted that mulberrin as the best candidate SIRT1 activator. Virtual screening using ligand-based pharmacophores predicted that gartanin, quinidine, and quinine to be the best candidates as SIRT1 activators. The molecular docking studies showed the important residues involved were Ile223 and Ile227 at the allosteric region. The MM-GB(PB)SA calculations confirmed that mulberrin, gartanin, quinidine, quinine showed activity at allosteric region and their EC50 in vitro values are 2.10; 1.79; 1.71; 1.14 µM, respectively. Based on in silico and in vitro study results, mulberin, gartanin, quinidine, and quinine had good activity as SIRT1 activators.

Review of Angiotensin-converting Enzyme Inhibitory Assay: Rapid Method in Drug Discovery of Herbal Plants.

The renin-angiotensin-aldosterone system is a signaling pathway which responsible in the blood pressure regulation. Angiotensin-converting enzyme (ACE) is one of the key elements responsible for the hypertensive mechanism. It converts angiotensin-I to angiotensin-II. The discovery history of the ACE inhibitory activity assay method has been through a long stage for decades and development continues until today. The ACE inhibitory activity has become an effective screening method in the search for new antihypertensive agents from herbal plants. Some of in vitro assay methods were used to examine the activity of ACE inhibitors based on the substrate usage, such as; Cushman and Cheung Method using a substrate hippuryl-histidyl-leucine (HHL), Holmquist method using a substrate furanacryloyl-tripeptide, Elbl and Wagner method using a substrate benzoil-[l-14C] glicyl-L-histidine-L-leucine, Carmel and Yaron method using a substrate o-aminobenzoylglycyl-p-nitrophenylalanilproline, and Lam method using 3-hydroxybutyrylglycyl-glycyl-glycine as substrate. Several different methods to measure the results of enzymatic reactions or separating substrate with products, including spectrophotometric, fluorometric, high-performance liquid chromatography, electrophoresis, and radiochemistry. Application of the test method for screening the ACE inhibitors activity and investigation of active compounds from natural products can be done easily with this method, it is very helpful in research because the results obtained are simple, accurate, and rapid.

In Silico Approach to Finding New Active Compounds from Histone Deacetylase (HDAC) Family.

BACKGROUND: Histone Deacetylase (HDAC) enzymes in the human body play an important role in the transcriptional regulation of gene expression. In the last decade, HDAC inhibitors and activators have been explored and have become known as therapeutic agents for many diseases such as osteodystrophy, neurogenerative disorders, cardiomyopathy, cancer, and diabetes. In recent years, the development of HDAC inhibitors or activators to obtain new potent lead compounds has been conducted using in vitro, in vivo, and in silico methods. Some HDAC family inhibitors and activators have been discovered. But some compounds have limitations such as not selectively binding to one of the HDAC variants. METHODS: At present, through bioinformation, HDAC family sequences have been revealed, and some in silico methods such as molecular modelling (homology modelling and pharmacophore modelling), virtual screening, and molecular dynamics are widely used to find and develop new potent and selective compounds. RESULTS: The main utilization of molecular modelling in this work is intended to complete the HDAC structure that partially lacks data regarding its amino acid monomer. Virtual screening methods are helpful in finding the best binding affinity of the test compounds. By molecular dynamic simulation, the temperature, time, and pressure can be adjusted to analyze the hydrogen bond. CONCLUSION: Combining these in silico approaches will be a more effective and efficient solution in finding new lead compounds for HDAC drug discovery research in the future.

Expression, purification, crystallization and preliminary crystallographic analysis of human Rad GTPase.

Human Rad is a new member of the Ras GTPase superfamily and is overexpressed in human skeletal muscle of individuals with type II diabetes. The GTPase core domain was overexpressed in Escherichia coli and purified for crystallization. Crystals were obtained at 293 K by vapour diffusion using a crystallization robot. The crystals were found to belong to space group P2(1), with unit-cell parameters a = 52.2, b = 58.6, c = 53.4 A, beta = 97.9 degrees , and contained two Rad molecules in the crystallographic asymmetric unit. A diffraction data set was collected to a resolution of 1.8 A using synchrotron radiation at SPring-8.

Virtual Screening of Indonesian Herbal Database as HIV-1 Protease Inhibitor.

HIV-1 (Human immunodeficiency virus type 1)׳s infection is considered as one of most harmful disease known by human, the survivability rate of the host reduced significantly when it developed into AIDS. HIV drug resistance is one of the main problems of its treatment and several drug designs have been done to find new leads compound as the cure. In this study, in silico virtual screening approach was used to find lead molecules from the library or database of natural compounds as HIV-1 protease inhibitor. Virtual screening against Indonesian Herbal Database with AutoDock was performed on HIV-1 protease. From the virtual screening, top ten compounds obtained were 8-Hydroxyapigenin 8-(2",4"-disulfatoglucuronide), Isoscutellarein 4'-methyl ether, Amaranthin, Torvanol A, Ursonic acid, 5-Carboxypyranocyanidin 3-O-(6"-O-malonyl-beta-glucopyranoside), Oleoside, Jacoumaric acid, Platanic acid and 5-Carboxypyranocyanidin 3-O-beta-glucopyranoside.

Synthesis and COX-2 Inhibitory Activity of 4-[(E)-2-(4-Oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethenyl]benzene-1-sulfonamide and Its Analogs.

Some novel 3-phenyl-2-[(E)-2-phenylethenyl]-3,4-dihydroquinazolin-4-one derivatives possessing para-sulfonamides groups on the phenyl ring of the 2-phenylethenyl moiety have been synthesized and their COX-2 inhibitory activity evaluated. The stuctures of the synthesized compounds were confirmed  on the basis of  FT-IR, 1H-NMR, 13C-NMR and mass spectral data. The COX-2 inhibition screening assay revealed that 4-[(E)-2-{3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl}ethenyl]benzene-1-sulfonamide had a  maximum COX-2 inhibition (47.1%), at a concentration of 20 µM.

Virtual screening of Indonesian herbal database as HIV-1 reverse transcriptase inhibitor.

HIV-1 (Human immunodeficiency virus type 1) is a member of retrovirus family that could infect human and causing AIDS disease. AIDS epidemic is one of most destructive diseases in modern era. There were more than 33 million people infected by HIV until 2010. Various studies have been widely employed to design drugs that target the essential enzymes of HIV-1 that is, reverse transcriptase, protease and integrase. In this study, in silico virtual screening approach is used to find lead molecules from the library or database of natural compounds as HIV-1 reverse transcriptase inhibitor. Virtual screening against Indonesian Herbal Database using AutoDock4 performed on HIV-1 reverse transcriptase. From the virtual screening, top ten compounds were mulberrin, plucheoside A, vitexilactone, brucine N-oxide, cyanidin 3-arabinoside, alpha-mangostin, guaijaverin, erycristagallin, morusin and sanggenol N.

Virtual screening of 2,3-disubstituted-4(3H)-quinazolinones possessing benzenesulfonamide moiety for COX-2 inhibitor.

COX inhibitors which selectively inhibits the inducible COX-2 is an oenzyme that causes inflammation. They are clinically effective anti-inflammatory agents with less gastrointestinal and renal toxicity. However, they lack anti-thrombotic activity and hence lead to increased incidences of adverse cardiovascular trombotic events such as myocardial infarction. Therefore, there is still a need to develop better therapeutic effect and tolerability COX-2 inhibitor. The majority of COX-2 inhibitors are diaryl heterocycles. For optimum COX-2 selectivity and inhibitory potency a -SO(3)CH(3) or a- SO(2)NH(2) substituent at the para-position of phenyl ring was essential. A wide variety of heterocycles can serve as central ring system of the diaryl heterocycles structures. We report the screening of various 2,3-disubstituted-4(3H)-quinazolinones possessing benzenesulfonamide moiety, directly or indirectly bound to the ring system, using the Protein-Ligand ANT System (PLANTS) docking software against the COX-2 enzyme. Various molecular structures of ligands were docked and scored to identify structurally similar ligands to SC-558 (reference ligand) in binding interaction to COX-2 binding site. The results show that 2,3-disubstituted-4(3H)-quinazolinones possess pbenzenesulfonamide moiety at C-2, and phenyl moiety at N-3 binds directly or indirectly to the ring system with high binding affinity. The docked ligand has orientations similar to that observed with SC-558 satisfying Lipinski's rule of five.

Crystal structure of human Rad GTPase of the RGK-family.

Rad (Ras associated with diabetes) is an RGK-family small GTPase that is over-expressed in the skeletal muscle of humans with type II diabetes. Unlike other small GTPases, RGK family members including Rad lack several conserved residues in the GTPase domain. Here, we report the crystal structure of the GTPase domain of human Rad in the GDP-bound form at 1.8 A resolution. The structure revealed unexpected disordered structures of both switches I and II. We showed that the conformational flexibility of both switches is caused by non-conservative substitutions in the G2 and G3 motifs forming the switch cores together with other substitutions in the structural elements interacting with the switches. Glycine-rich sequences of the switches would also contribute to the flexibility. Switch I lacks the conserved phenylalanine that makes non-polar interactions with the guanine base in H-Ras. Instead, water-mediated hydrogen bonding interactions were observed in Rad. The GDP molecule is located at the same position as in H-Ras and adopts a similar conformation as that bound in H-Ras. This similarity seems to be endowed by the conserved hydrogen bonding interactions with the guanine base-recognition loops and the magnesium ion that has a typical octahedral coordination shell identical to that in H-Ras.