Active Site Pocket Research Articles

Role of Gly197 in the structure and function of protein C

We previously demonstrated that heterozygous Gly197 to Arg mutation in PROC is associated with venous thrombosis due to the mutation abrogating both zymogenic and enzymatic activities of protein C and activated protein C (APC). In this study, we investigated the role of Gly197 on the structure and function of protein C by replacing it with Ala, Lys and Glu in separate constructs. Characterization of protein C mutants indicated their activation by thrombin is improved ~5–20-fold with the order of PC-G197K > PC-G197E > PC-G197A > PC-WT. Interestingly, the cofactor function of thrombomodulin (TM) in promoting the activation of zymogens by thrombin followed the reverse order of PC-WT > PC-G197A > PC-G197E > PC-G197K. The thrombin-generation inhibitory profiles of zymogens in a tissue factor-mediated thrombin generation assay using protein C-deficient plasma with or without supplementation with TM followed the same order of zymogen activation in the purified system. Evaluation of anticoagulant activities of APC derivatives by prothrombinase and aPTT assays revealed a normal activity for APC-G197A but dramatically impaired activity for the other two mutants. In the endothelial cell permeability assay, APC-G197A exhibited normal antiinflammatory activity, but the other two mutants were nearly inactive. These results suggest that Gly197 plays a key role in TM cofactor-dependent protein C activation by thrombin. It facilitates the recognition of protein C by thrombin in the presence of TM but impedes it in the absence of the cofactor. In APC, a small residue at this position is required for the proper folding/reactivity of the active-site pocket of the protease, a hypothesis supported by structural modeling.

Structural Insights into a Bifunctional Peptide Methionine Sulfoxide Reductase MsrA/B Fusion Protein from Helicobacter pylori.

Methionine sulfoxide reductase (Msr) is a family of enzymes that reduces oxidized methionine and plays an important role in the survival of bacteria under oxidative stress conditions. MsrA and MsrB exist in a fusion protein form (MsrAB) in some pathogenic bacteria, such as Helicobacter pylori (Hp), Streptococcus pneumoniae, and Treponema denticola. To understand the fused form instead of the separated enzyme at the molecular level, we determined the crystal structure of HpMsrABC44S/C318S at 2.2 Å, which showed that a linker region (Hpiloop, 193–205) between two domains interacted with each HpMsrA or HpMsrB domain via three salt bridges (E193-K107, D197-R103, and K200-D339). Two acetate molecules in the active site pocket showed an sp2 planar electron density map in the crystal structure, which interacted with the conserved residues in fusion MsrABs from the pathogen. Biochemical and kinetic analyses revealed that Hpiloop is required to increase the catalytic efficiency of HpMsrAB. Two salt bridge mutants (D193A and E199A) were located at the entrance or tailgate of Hpiloop. Therefore, the linker region of the MsrAB fusion enzyme plays a key role in the structural stability and catalytic efficiency and provides a better understanding of why MsrAB exists in a fused form.

In Silico Based Approach to Investigate Plant Lignans as Inhibitor Candidates for Estrogen Receptor in Breast Cancer

Background: In the last decades, growing evidence demonstrates interest in phytoestrogen intake to modulate targets in different types of cancer. Plant lignans have proven efficacious in blocking estrogen receptors of breast cancer cells. Among them, four phytoestrogen lignans: pinoresinol, matairesinol, lariciresinol, and secoisolariciresinol have been most studied. However, available studies have mostly dealt with the anti-cancer effects of groups of lignans in certain foods or plants and the effects of specific lignans, especially from a molecular interaction viewpoint, have been rarely addressed in the literature. Objective: We aimed to in silico predict pharmacological properties, binding ability and binding strength of pinoresinol, matairesinol, lariciresinol and secoisolariciresinol as possible inhibitors of estrogen receptor alpha which is the most important biomarker in breast cancer. Methods: Firstly, we evaluated the pharmacological properties of four lignans using SwissADME. Then we investigated the ligand-receptor interactions of these molecules as positively appraised ligands for ER-positive breast cancer targeted therapy using docking method. We finally compared the inhibitory effect possibility of the lignans against endoxifen which is the active metabolite of tamoxifen. Results: The best binding affinity of endoxifen, matairesinol, pinoresinol, lariciresinol and secoisolariciresinol were respectively -9.2, -7.5, -6.7, -6.7, -5.8 kcal/mol. In the meantime, matairesinol showed a minimum binding energy than other studied lignans in addition to the most similar interactions to endoxifen with conserved domain residues of the active site pocket in Leu:391, Ala:350, Met:421, and Phe:404. Conclusion: Among the studied lignans, matairesinol showed favorable pharmacokinetics and drug-likeliness properties, the least binding energy as well as the most common interactions in conserved residues of the active site pocket with estrogens. This makes it a molecule with low number of nonspecific interactions, better target selectivity, and hence fewer side effects. Thus, our results introduce matairesinol as a possibly effective anti-estrogen receptor inhibitor candidate.

Structural studies of RNase M5 reveal two-metal-ion supported two-step dsRNA cleavage for 5S rRNA maturation

ABSTRACT All species transcribe ribosomal RNA in an immature form that requires several enzymes for processing into mature rRNA. The number and types of enzymes utilized for these processes vary greatly between different species. In low G + C Gram-positive bacteria including Bacillus subtilis and Geobacillus stearothermophilus, the endoribonuclease (RNase) M5 performs the final step in 5S rRNA maturation, by removing the 3ʹ- and 5ʹ-extensions from precursor (pre) 5S rRNA. This cleavage activity requires initial complex formation between the pre-rRNA and a ribosomal protein, uL18, making the full M5 substrate a ribonucleoprotein particle (RNP). M5 contains a catalytic N-terminal Toprim domain and an RNA-binding C-terminal domain, respectively, shown to assist in processing and binding of the RNP. Here, we present structural data that show how two Mg2+ ions are accommodated in the active site pocket of the catalytic Toprim domain and investigate the importance of these ions for catalysis. We further perform solution studies that support the previously proposed 3ʹ-before-5ʹ order of removal of the pre-5S rRNA extensions and map the corresponding M5 structural rearrangements during catalysis.

Investigation of sphingosine kinase 1 inhibitory potential of cinchonine and colcemid targeting anticancer therapy

Sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate (S1P) signaling regulates numerous diseases such as cancer, diabetes, and inflammation-related ailments, rheumatoid arthritis, atherosclerosis, and multiple sclerosis. The importance of SphK1 in chemo-resistance has been extensively explored in breast, lung, colon, and hepatocellular carcinomas. SphK1 is considered an attractive drug target for the development of anticancer therapy. New drug molecules targeting the S1P signaling are required owing to its pleiotropic nature and association with multiple downstream targets. Here, we have investigated the binding affinity and SphK1 inhibitory potential of cinchonine and colcemid using a combined molecular docking and simulation studies followed by experimental analysis. These compounds bind to SphK1 with a significantly high affinity and subsequently inhibit kinase activity (IC50 7–9 μM). Further, MD simulation studies revealed that both cinchonine and colcemid bind to the residues at the active site pocket of SphK1 with several non-covalent interactions, which may be responsible for inhibiting its kinase activity. Besides, the binding of cinchonine and colcemid causes substantial conformational changes in the structure of SphK1. Taken together, cinchonine and colcemid may be implicated in designing potential drug molecules with improved affinity and specificity for SphK1 targeting anticancer therapy. Communicated by Ramaswamy H. Sarma

Structural Insights into a Novel Esterase from the East Pacific Rise and Its Improved Thermostability by a Semirational Design.

Lipolytic enzymes are essential biocatalysts in food processing as well as pharmaceutical and pesticide industries, catalyzing the cleavage of ester bonds in a variety of acyl chain substrates. Here, we report the crystal structure of an esterase from the deep-sea hydrothermal vent of the East Pacific Rise (EprEst). The X-ray structure of EprEst in complex with the ligand, acetate, has been determined at 2.03 Å resolution. The structure reveals a unique spatial arrangement and orientation of the helix cap domain and α/β hydrolase domain, which form a substrate pocket with preference for short-chain acyl groups. Molecular docking analysis further demonstrated that the active site pocket could accommodate p-nitrophenyl (pNP) carboxyl ligands of varying lengths (≤6 C atoms), with pNP-butyrate ester predicted to have the highest binding affinity. Additionally, the semirational design was conducted to improve the thermostability of EprEst by enzyme engineering based on the established structure and multiple sequence alignment. A mutation, K114P, introduced in the hinge region of the esterase, which displayed increased thermostability and enzyme activity. Collectively, the structural and functional data obtained herein could be used as basis for further protein engineering to ultimately expand the scope of industrial applications of marine-derived lipolytic enzymes.

Dimeric phosphorylation of glyoxalase I alters its symmetry and substrate binding mechanism: simulation studies

Glyoxalase I (GLO1) is a dimeric esterase of the glyoxalase system. Phosphorylation of the residue T106 has been found to inhibit GLO1 activity, and contribute to the onset of oxidative stress and cellular damage. This research uses multiple molecular dynamics simulations and automated docking of both GLO1 and dimerically phosphorylated GLO1 (p2-GLO1) to predict the initial structural differences induced by phosphorylation, and their interaction with the intermediate substrate Hemimercaptal. This research indicates that immediately following phosphorylation, GLO1 exhibits reduced sphericity, partly caused by outward splaying of the loop region surrounding T106. Phosphorylation induces enhanced concerted motions in the loop composed of residues immediately surrounding T106, which are correlated with motions at the active site pocket at the distant, opposite end of the dimer. These T106 region loop motions result in the distortion of the shape of the active site, and potentially alter its accessibility. Phosphorylation alters the manner in which GLO1 interacts with Hemimercaptal. For GLO1, Hemimercaptal is predicted to bind to T106, which we propose constitutes a novel, highly accessible ‘capture site’ responsible for initial contact with the substrate. In contrast, for p2-GLO1, Hemimercaptal is unable to bind favourably to (phosphorylated) position T106, suggesting that this proposed transient ‘capture site’ is abolished upon phosphorylation of GLO1. Hence, a novel physiological role is here proposed for the known essential GLO1 residue T106. These results may further contribute to understanding the inhibition mechanism of GLO1 upon phosphorylation. Communicated by Ramaswamy H. Sarma

Redesigning the Molecular Choreography to Prevent Hydroxylation in Germacradien-11-ol Synthase Catalysis.

Natural sesquiterpene synthases have evolved to make complex terpenoids by quenching reactive carbocations either by proton transfer or by hydroxylation (water capture), depending on their active site. Germacradien-11-ol synthase (Gd11olS) from Streptomyces coelicolor catalyzes the cyclization of farnesyl diphosphate (FDP) into the hydroxylated sesquiterpene germacradien-11-ol. Here, we combine experiment and simulation to guide the redesign of its active site pocket to avoid hydroxylation of the product. Molecular dynamics simulations indicate two regions between which water molecules can flow that are responsible for hydroxylation. Point mutations of selected residues result in variants that predominantly form a complex nonhydroxylated product, which we identify as isolepidozene. Our results indicate how these mutations subtly change the molecular choreography in the Gd11olS active site and thereby pave the way for the engineering of terpene synthases to make complex terpenoid products.

Molecular basis for high ligand sensitivity and selectivity of strigolactone receptors in Striga.

Seeds of the root parasitic plant Striga hermonthica can sense very low concentrations of strigolactones (SLs) exuded from host roots. The S. hermonthica hyposensitive to light (ShHTL) proteins are putative SL receptors, among which ShHTL7 reportedly confers sensitivity to picomolar levels of SL when expressed in Arabidopsis thaliana. However, the molecular mechanism underlying ShHTL7 sensitivity is unknown. Here we determined the ShHTL7 crystal structure and quantified its interactions with various SLs and key interacting proteins. We established that ShHTL7 has an active-site pocket with broad-spectrum response to different SLs and moderate affinity. However, in contrast to other ShHTLs, we observed particularly high affinity of ShHTL7 for F-box protein AtMAX2. Furthermore, ShHTL7 interacted with AtMAX2 and with transcriptional regulator AtSMAX1 in response to nanomolar SL concentration. ShHTL7 mutagenesis analyses identified surface residues that contribute to its high-affinity binding to AtMAX2 and residues in the ligand binding pocket that confer broad-spectrum response to SLs with various structures. Crucially, yeast-three hybrid experiments showed that AtMAX2 confers responsiveness of the ShHTL7-AtSMAX1 interaction to picomolar levels of SL in line with the previously reported physiological sensitivity. These findings highlight the key role of SL-induced MAX2-ShHTL7-SMAX1 complex formation in determining the sensitivity to SL. Moreover, these data suggest a strategy to screen for compounds that could promote suicidal seed germination at physiologically relevant levels.

A covalent p97/VCP ATPase inhibitor can overcome resistance to CB-5083 and NMS-873 in colorectal cancer cells.

Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins involved in tumorigenesis and virus infections. Multiple p97 inhibitors identified from previous high-throughput screening studies are thiol-reactive compounds targeting Cys522 in the D2 ATP-binding domain. Thus, these findings suggest a potential strategy to develop covalent p97 inhibitors. We first used purified p97 to assay several known covalent kinase inhibitors to determine if they can inhibit ATPase activity. We evaluated their selectivity using our dual reporter cells that can distinguish p97 dependent and independent degradation. We selected a β-nitrostyrene scaffold to further study the structure-activity relationship. In addition, we used p97 structures to design and synthesize analogues of pyrazolo[3,4-d]pyrimidine (PP). We incorporated electrophiles into a PP-like compound 17 (4-amino-1-tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18 (N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent selectivity in an invitro ATPase activity assay: IC50 of 0.6μM, 300μM, and 100μM for wild type p97, yeast Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified and shown to increase IC50 values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with Cysteine decreased the IC50 by 10-fold. The molecular modeling suggested the hydrogen bonds and hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA. Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was inhibited by PPA with IC50 of 2.7μM, 6.1μM, and 3.4μM, respectively. In addition, PPA is able to inhibit proliferation of two HCT116cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future exploration to improve the potency of p97 inhibitors with different mechanisms of action.

Symmetry-related residues as promising hotspots for the evolution of de novo oligomeric enzymes.

Directed evolution has provided us with great opportunities and prospects in the synthesis of tailor-made proteins. It, however, often requires at least mid to high throughput screening, necessitating more effective strategies for laboratory evolution. We herein demonstrate that protein symmetry can be a versatile criterion for searching for promising hotspots for the directed evolution of de novo oligomeric enzymes. The randomization of symmetry-related residues located at the rotational axes of artificial metallo-β-lactamase yields drastic effects on catalytic activities, whereas that of non-symmetry-related, yet, proximal residues to the active site results in negligible perturbations. Structural and biochemical analysis of the positive hits indicates that seemingly trivial mutations at symmetry-related spots yield significant alterations in overall structures, metal-coordination geometry, and chemical environments of active sites. Our work implicates that numerous artificially designed and natural oligomeric proteins might have evolutionary advantages of propagating beneficial mutations using their global symmetry.

Exploration of Theoretical and Antioxidant Mimetic Activities of Binuclear Copper(II) and Nickel(II) Coordination Complexes

Three dinuclear copper(II) and nickel(II) complexes viz. [Cu2(La)(py)2]·(ClO4)2 (1), [Ni2(Lb)(ImH)2]·(ClO4)2 (2) and [Ni2(Lc)(H2O)2]·(ClO4)2 (3) were designed and synthesized using disulfide and auxiliary ligands as functional models for antioxidant superoxide dismutase enzyme (where, H2La-H2Lc = disulfide ligands bearing S-S bond, py = pyridine, ImH = imidazole). The disulfide ligands were synthesized by condensation reaction of 2-(2-(2-aminophenyl)disulfanyl)benzeneamine with 3-bromo-2-hydroxy-5-nitrobenzaldehyde; 5-bromo-2-hydroxybenzaldehyde and 3,5-dichloro- 2-hydroxybenzaldehyde, respectively. All the compounds were characterized by various spectrometry methods. The molecular docking study was performed against penicillin binding protein 4 (PBP4) active site pocket and E. coli DNA Gyrase B ATPase domain to evaluate the possible mechanism of antibacterial property of disulfide ligands. The results revealed that H2La-H2Lc possess higher binding affinity for active site of PBP4 and Gyrase B ATPase domain. The present study also demonstrated that disulfide compounds have potential to inhibit the PBP4 and Gyrase B ATPase domain and can be developed as lead compounds. In addition, antioxidant superoxide dismutase (SOD) activity is also discussed of these complexes. The antioxidant superoxide measurements show that complexes behave as superoxide mimic in alkaline nitro blue tetrazolium chloride (NBT) assay.

Structure-based redesign of the bacterial prolidase active-site pocket for efficient enhancement of methyl-parathion hydrolysis

Mutagenesis at four residues surrounding the active-site pocket of an old bacterial prolidase scaffold led to a 10 000-fold increase in methyl-parathion hydrolysis and broadening substrate specificity against organophosphorus compounds.

Fragment-Based Drug Design of Selective HDAC6 Inhibitors.

Medicinal chemistry society has enough arguments to justify the usage of fragment-based drug design (FBDD) methodologies for the identification of lead compounds. Since the FDA approval of three kinase inhibitors-vemurafenib, venetoclax, and erdafitinib,FBDD has become a challenging alternative to high-throughput screening methods in drug discovery. The following protocol presents in silico drug design of selective histone deacetylase 6 (HDAC6) inhibitors through a fragment-based approach. To date, structural motifs that are important for HDAC inhibitory activity and selectivity are described as: surface recognition group (CAP group), aliphatic or aromatic linker, and zinc-binding group (ZBG). The main idea of this FBDD method is to identify novel and target-selective CAP groups by virtual scanning of publicly available fragment databases. Template structure used to search for novel heterocyclic and carbocyclic fragments is 1,8-naphthalimide (CAP group of scriptaid, a potent HDAC inhibitor). Herein, the design of HDAC6 inhibitors is based on linking the identified fragments with the aliphatic or aromatic linker and hydroxamic acid (ZBG) moiety. Final selection of potential selective HDAC6 inhibitors is based on combined structure-based (molecular docking) and ligand-based (three-dimensional quantitative structure-activity relationships, 3D-QSAR) techniques. Designed compounds are docked in the active site pockets of human HDAC1 and HDAC6 isoforms, and their docking conformations used to predict their HDAC inhibitory and selectivity profiles through two developed 3D-QSAR models (describing HDAC1 and HDAC6 inhibitory activities).

Evaluation of the combined activity of benzimidazole arylhydrazones as new anti-Parkinsonian agents: monoamine oxidase-B inhibition, neuroprotection and oxidative stress modulation.

Neuroprotective drugs and selective monoamine oxidase inhibitors can slow down the progression and improve symptoms of Parkinson's disease (PD). Since there is an implication of oxidative stress in the pathophysiological mechanisms of the disease, the compounds possessing an ability to reduce the oxidative stress are prime candidates for neuroprotection. Thereby our current study is focused on the development of new multi-target PD drugs capable of inhibiting the activity of monoamine oxidase-B while exerting neuroprotective and antioxidant properties. A small series of benzimidazole derivatives containing hydroxy and methoxy arylhydrazone fragments has been synthesized and the neurotoxicity of the compounds has been evaluated in vitro on neuroblastoma SH-SY5Y cells and on isolated rat brain synaptosomes by measuring the cell viability and the levels of reduced glutathione and a good safety profile has been shown. The 2-hydroxy-4-methoxy substituted arylhydrazone 7 was the least toxic on neuronal SH-SY5Y cells and showed the lowest neurotoxicity in rat brain synaptosomes. The neuroprotective properties of the test compounds were further assessed using two models: H2O2-induced oxidative stress on SH-SY5Y cells and 6-hydroxydopamine-induced neurotoxicity in rat brain synaptosomes. Compound 7 showed more pronounced neuroprotective activity on SH-SY5Y cells, compared to the referent melatonin and rasagiline. It also preserved the synaptosomal viability and the reduced glutathione levels; the effects were stronger than those of rasagiline and comparable to melatonin. All the tested compounds were capable to inhibit human monoamine oxidase-B enzyme to a significant extent, however, compound 7 exerted the most prominent inhibitory activity, similar to selegiline and rasagiline. The carried out molecular docking studies revealed that the activity is related to the appropriate molecular structure enabling the ligand to enter deeper in the narrow and highly lipophylic active site pocket of the human monoamine oxidase-B and has a favoring interaction with the key amino acid residues Tyr326 and Cys172. Since much scientific evidence points out the implication of iron dyshomeostasis in PD, the compounds were tested to reduce the ferrous iron induced oxidative molecular damage on biologically important molecules in an in vitro lecithin containing model system. All the investigated compounds denoted protection effect, stronger than the one of the referent melatonin. In order to support the assignments of the significant neuroprotective and antioxidant pharmacological activities, the radical-scavenging mechanisms of the most promising compound 7 were evaluated using DFT methods. It was found that the most probable free radicals scavenging mechanism in nonpolar phase is the hydrogen atom transfer from the amide group of compound 7, while in polar medium the process is expected to occur by a proton transfer. The current study outlines a perspective leading structure, bearing the potential for a new anti-PD drug. All performed procedures were approved by the Institutional Animal Care Committee of the Medical University of Sofia (Bulgarian Agency for Food Safety with Permission № 190, approved on February 6, 2020).

Vanillin enones as selective inhibitors of the cancer associated carbonic anhydrase isoforms IX and XII. The out of the active site pocket for the design of selective inhibitors?

New C-glycosides and α,β-unsaturated ketones incorporating the 4-hydroxy-3-methoxyphenyl (vanillin) moiety as inhibitors of carbonic anhydrase (CA, EC 4.2.1.1) isoforms have been investigated. The inhibition profile of these compounds is presented against four human CA (hCA) isozymes, comprising hCAs I and II (cytosolic, ubiquitous enzymes) and hCAs IX and XII (tumour associated isozymes). Docking analysis of the inhibitors within the active sites of these enzymes has been performed and is discussed, showing that the observed selectivity could be explained in terms of an alternative pocket out of the CA active site where some of these compounds may bind. Several derivatives were identified as selective inhibitors of the tumour-associated hCA IX and XII. Their discovery might be a step in the strategy for finding an effective non-sulfonamide CA inhibitor useful in therapy/diagnosis of hypoxic tumours or other pathologies in which CA isoforms are involved.

Antiviral activity of glycyrrhizic acid conjugates with amino acid esters against Zika virus

Zika virus (ZIKV) is a new pathogenic flavivirus transmitted by mosquitoes Aedes spp. ZIKV infection is accompanied by serious neurological complications and is especially dangerous for pregnant women, in which it can lead to congenital malformations of the fetus and microcephaly in neonates. Currently, there are no licensed vaccines or specific post-infectious therapies for ZIKV infection. This report is devoted to the study of glycyrrhizic acid (GL) derivatives as ZIKV inhibitors. The inhibitory assays on the cytopathic effect (CPE) and viral infectivity of ZIKV in three different human cell lines revealed that the conjugation of GL with amino acids and their esters (methyl, ethyl) is influenced by the antiviral activity of the compounds. GL conjugates with Glu(OMe)-OMe 11, Glu(OH)-OMe 12, Asp(OMe)-OMe 13, TyrOMe 14, LeuOEt 15, and PheOEt 16 with free COOH groups in the triterpene moiety were active against ZIKV. The most active compounds 13 and 14 have IC50 values of 0.23 μM and 0.09 μM against low doses (MOI = 0.05) of ZIKV strain PRVABC59, 1.20 μM and 0.74 μM against high doses (MOI = 10) of ZIKV strain Natal RGN single-round infectious particles, respectively. The lead compound was 14 with a high selectivity index (SI < 500). Compound 13 showed a higher inhibitory effect on the early stage (entry) of ZIKV replication than compound 14, and was less potent than compound 14 at the post-entry stage, consistent with the docking models. Compounds 13 and 14 also had a strong interaction with the active site pocket of NS5 MTase. Compounds 13 and 14 are recommended for expanded antiviral studies against ZIKV infection.

Baicalein, 7,8-Dihydroxyflavone and Myricetin as Potent Inhibitors of Human Ornithine Decarboxylase.

Background: Human ornithine decarboxylase (ODC) is a well-known oncogene, and the discovery of ODC enzyme inhibitors is a beneficial strategy for cancer therapy and prevention. Methods: We examined the inhibitory effects of a variety of flavone and flavonol derivatives on ODC enzymatic activity, and performed in silico molecular docking of baicalein, 7,8-dihydroxyflavone and myricetin to the whole dimer of human ODC to investigate the possible binding site of these compounds on ODC. We also examined the cytotoxic effects of these compounds with cell-based studies. Results: Baicalein, 7,8-dihydroxyflavone and myricetin exhibited significant ODC suppression activity with IC50 values of 0.88 µM, 2.54 µM, and 7.3 µM, respectively, which were much lower than that of the active-site irreversible inhibitor α-DL-difluoromethylornithine (IC50, the half maximal inhibitory concentration, of approximately 100 µM). Kinetic studies and molecular docking simulations suggested that baicalein, and 7,8-dihydroxyflavone act as noncompetitive inhibitors that are hydrogen-bonded to the region near the active site pocket in the dimer interface of the enzyme. Baicalein and myricetin suppress cell growth and induce cellular apoptosis, and both of these compounds suppress the ODC-evoked anti-apoptosis of cells. Conclusions: Therefore, we suggest that the flavone or flavonol derivatives baicalein, 7,8-dihydroxyflavone, and myricetin are potent chemopreventive and chemotherapeutic agents that target ODC.

Structural Modeling of Wild and Mutant Forms of Human Plasma Platelet Activating Factor-Acetyl Hydrolase Enzyme.

PurposeTo investigate the structural features of wild and mutant forms of the pPAF-AH enzyme that are responsible for coronary artery disease.MethodsMutant variants of human pPAF-AH having either V279F, Q281R, or both were modelled and evaluated for stereo chemical and structural correctness. The 3D coordinates of substrate PAF were retrieved from the PubChem database was solvated and minimized on Discovery Studio, and docked to the wild and mutant enzyme models. The top docked pose complex was refined by MD simulation.ResultspPAF-AH model comprises of 420 amino acids in a α/β-hydrolase fold that contains a substrate-binding hydrophobic channel with an active site pocket having a catalytic triad of Ser273, Asp296 and His351. Mutations at positions 279 and 281 are opposite one another on the middle of 12 residues long H5 helix that forms the hydrophobic core of the enzyme. V279F causes a tilt on the axis of the mutation bearing helix to avoid steric clashes with the hydrophobic residues on the β-sheets adjacent to it, inducing subtle conformational changes on the H5-β8 loop, β8 sheet, and the loop bearing Asp296. A cascade of conformational changes induces a change in the orientation of His351 resulting in loss of hydrogen bonded interaction with catalytic Ser273. Q281R causes a shortening of H5 and β8, which induces conformational changes of the loops bearing Ser273 and Asp296, respectively. Simultaneous conformational changes of secondary structural elements result in the flipping of His351 causing a break in the catalytic triad. Also, there is a compromise in the substrate-binding area and volume in the mutants resulting in loss of binding to its substrate.ConclusionMutant enzymes show changes at the site of the mutation, secondary motif conformations and global structural conformations that adversely affect the active site, decrease substrate channel volume and decrease stability, thereby affecting enzymatic function.

Structural and Biochemical Characterization of the Two Drosophila Low Molecular Weight-Protein Tyrosine Phosphatases DARP and Primo-1.

The Drosophila genome contains four low molecular weight-protein tyrosine phosphatase (LMW-PTP) members: Primo-1, Primo-2, CG14297, and CG31469. The lack of intensive biochemical analysis has limited our understanding of these proteins. Primo-1 and CG31469 were previously classified as pseudophosphatases, but CG31469 was also suggested to be a putative protein arginine phosphatase. Herein, we present the crystal structures of CG31469 and Primo-1, which are the first Drosophila LMW-PTP structures. Structural analysis showed that the two proteins adopt the typical LMW-PTP fold and have a canonically arranged P-loop. Intriguingly, while Primo-1 is presumed to be a canonical LMW-PTP, CG31469 is unique as it contains a threonine residue at the fifth position of the P-loop motif instead of highly conserved isoleucine and a characteristically narrow active site pocket, which should facilitate the accommodation of phosphoarginine. Subsequent biochemical analysis revealed that Primo-1 and CG31469 are enzymatically active on phosphotyrosine and phosphoarginine, respectively, refuting their classification as pseudophosphatases. Collectively, we provide structural and biochemical data on two Drosophila proteins: Primo-1, the canonical LMW-PTP protein, and CG31469, the first investigated eukaryotic protein arginine phosphatase. We named CG31469 as DARP, which stands for Drosophila ARginine Phosphatase.