Der p 2 is a major dust mite allergen and >80% of mite allergic individuals have specific IgE to this allergen. Although it is well characterized in terms of allergenicity, there is still some ambiguity in terms of its biological function. Three-dimensional structural analysis of Der p 2 and its close homologues indicate the presence of a hydrophobic cavity which can potentially bind to lipid molecules. In this study, we aimed to identify the potential ligand of Der p 2. Using a liposome pulldown assay, we show that recombinant Der p 2 binds to liposomes prepared with exogenous cholesterol in a dose dependent fashion. Next, an ELISA based assay using immobilized lipids was used to study binding specificities of other lipid molecules. Cholesterol was the preferred ligand of Der p 2 among 11 different lipids tested. Two homologues of Der p 2, Der f 2 and Der f 22 also bound to cholesterol. Further, using liquid chromatography-mass spectrometry (LC-MS), we confirmed that cholesterol is the natural ligand of Der p 2. Three amino acid residues of Der p 2, V104, V106 and V110 are possible cholesterol binding sites, as alanine mutations of these residues showed a significant decrease in binding (p
Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins.
Protein-protein interaction plays an essential role in almost all cellular processes and biological functions. Coupling molecular dynamics (MD) simulations and nanoparticle tracking analysis (NTA) assay offered a simple, rapid, and direct approach in monitoring the protein-protein binding process and predicting the binding affinity. Our case study of designed ankyrin repeats proteins (DARPins)-AnkGAG1D4 and the single point mutated AnkGAG1D4-Y56A for HIV-1 capsid protein (CA) were investigated. As reported, AnkGAG1D4 bound with CA for inhibitory activity; however, it lost its inhibitory strength when tyrosine at residue 56 AnkGAG1D4, the most key residue was replaced by alanine (AnkGAG1D4-Y56A). Through NTA, the binding of DARPins and CA was measured by monitoring the increment of the hydrodynamic radius of the AnkGAG1D4-gold conjugated nanoparticles (AnkGAG1D4-GNP) and AnkGAG1D4-Y56A-GNP upon interaction with CA in buffer solution. The size of the AnkGAG1D4-GNP increased when it interacted with CA but not AnkGAG1D4-Y56A-GNP. In addition, a much higher binding free energy (∆GB) of AnkGAG1D4-Y56A (-31 kcal/mol) obtained from MD further suggested affinity for CA completely reduced compared to AnkGAG1D4 (-60 kcal/mol). The possible mechanism of the protein-protein binding was explored in detail by decomposing the binding free energy for crucial residues identification and hydrogen bond analysis.
The inhibition of carbohydrate-hydrolyzing enzymes in human digestive organs is crucial in controlling blood sugar levels, which is important in treating type 2 diabetes. In the current study, pahangensin A (1), a bis-labdanic diterpene characterized previously in the rhizomes of Alpinia pahangensis Ridl., was identified as an active dual inhibitor for α-amylase (IC50 =114.80 μm) and α-glucosidase (IC50 =153.87 μm). This is the first report on the dual α-amylase and α-glucosidase inhibitory activities of a bis-labdanic diterpene. The Lineweaver-Burk plots of compound 1 indicate that it is a mixed-type inhibitor with regard to both enzymes. Based on molecular docking studies, compound 1 docked in a non-active site of both enzymes. The dual inhibitory activity of compound 1 makes it a suitable natural alternative in the treatment of type 2 diabetes.
The α-amylase acts as attractive target to treat type-2 diabetes mellitus. Therefore in discovering a small molecule as α-amylase inhibitor, we have synthesized benzofuran carbohydrazide analogs (1-25), characterized through different spectroscopic techniques such as 1HNMR and EI-MS. All screened analog shows good α-amylase inhibitory potentials with IC50 value ranging between 1.078±0.19 and 2.926±0.05µM when compared with acarbose having IC50=0.62±0.22µM. Only nine analogs among the series such as analogs 3, 5, 7, 8, 10, 12, 21, 23 and 24 exhibit good inhibitory potential with IC50 values 1.644±0.128, 1.078±0.19, 1.245±0.25, 1.843±0.19, 1.350±0.24, 1.629±0.015, 1.353±0.232, 1.359±0.119 and 1.488±0.07µM when compare with standard drug acarbose. All other analogs showed good to moderate α-amylase inhibitory potentials. The SAR study was conducted on the basis of substituent difference at the phenyl ring. The binding interaction between analogs and active site of enzyme was confirmed by docking studies.
Interests in Acinetobacter haemolyticus lipases are showing an increasing trend concomitant with growth of the enzyme industry and the widening search for novel enzymes and applications. Here, we present a structural model that reveals the key catalytic residues of lipase KV1 from A. haemolyticus. Homology modeling of the lipase structure was based on the structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus as the template, which has a sequence that is 58% identical to that of lipase KV1. The lipase KV1 model is comprised of a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three structurally conserved active-site residues, Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the pH 8 modeled lipase KV1 active sites revealed an aromatic platform responsible for the substrate recognition and preference toward tributyrin. The resulting binding modes from the docking simulation correlated well with the experimentally determined hydrolysis pattern, for which pH 8 and tributyrin being the optimum pH and preferred substrate. The results reported herein provide useful insights into future structure-based tailoring of lipase KV1 to modulate its catalytic activity.
The potential of N. lappacheum and N. mutabile seed as a source of α-amylase inhibitor peptides was explored based on the local traditional practice of using the seed. Different gastro-digestive enzymes (i.e. pepsin or chymotrypsin) or a sequential digestion were used to extract the peptides. The effects of digestion time and enzyme to substrate (E:S) ratio on the α-amylase inhibitory activity were investigated. Results showed that chymotrypsin was effective in producing the inhibitor peptides from rambutan seed protein at E:S ratio 1:20 for 1 h, whereas pepsin was more effective for pulasan seed protein under the same condition. A total of 20 and 31 novel inhibitor peptides were identified, respectively. These peptides could bind with the subsites of α-amylase (i.e. Trp58, Trp59, Tyr62, Asp96, Arg195, Asp197, Glu233, His299, Asp300, and His305) and formed a sliding barrier that preventing the formation of enzyme/substrate intermediate leading to lower α-amylase activity.
Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2(1)2(1)2(1) and P2(1), respectively, and diffract to approximately 2.5 A resolution. All of the crystals are suitable for structural studies, which are currently ongoing.
Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF) α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL) death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKβ) and the Nuclear factor κB (NF-κB) component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKβ kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis.
Aedes aegypti, Aedes albopictus and Culex quinquefasciatus are vectors of dengue fever and West Nile virus diseases. This study was conducted to determine the toxicity, mechanism of action and the binding interaction of three active phenylpropanoids from Piper sarmentosum (Piperaceae) toward late 3rd or early 4th larvae of above vectors. A bioassay guided-fractionation on the hexane extract from the roots of Piper sarmentosum led to the isolation and identification of three active phenylpropanoids; asaricin 1, isoasarone 2 and trans-asarone 3. The current study involved evaluation of the toxicity and acetylcholinesterase (AChE) inhibition of these compounds against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus larvae. Asaricin 1 and isoasarone 2 were highly potent against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus larvae causing up to 100% mortality at ≤ 15 μg/mL concentration. The ovicidal activity of asaricin 1, isoasarone 2 and trans-asarone 3 were evaluated through egg hatching. Asaricin 1 and isoasarone 2 showed potent ovicidal activity. Ovicidal activity for both compounds was up to 95% at 25μg/mL. Asaricin 1 and isoasarone 2 showed strong inhibition on acetylcholinesterase with relative IC50 values of 0.73 to 1.87 μg/mL respectively. These findings coupled with the high AChE inhibition may suggest that asaricin 1 and isoasarone 2 are neuron toxic compounds toward Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. Further computational docking with Autodock Vina elaborates the possible interaction of asaricin 1 and isoasarone 2 with three possible binding sites of AChE which includes catalytic triads (CAS: S238, E367, H480), the peripheral sites (PAS: E72, W271) and anionic binding site (W83). The binding affinity of asaricin 1 and isoasarone 2 were relatively strong with asaricin 1 showed a higher binding affinity in the anionic pocket.
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya infection in humans. Despite the widespread distribution of CHIKV, no antiviral medication or vaccine is available against this virus. Therefore, it is crucial to find an effective compound to combat CHIKV. We aimed to predict the possible interactions between non-structural protein 3 (nsP) of CHIKV as one of the most important viral elements in CHIKV intracellular replication and 3 potential flavonoids using a computational approach. The 3-dimensional structure of nsP3 was retrieved from the Protein Data Bank, prepared and, using AutoDock Vina, docked with baicalin, naringenin and quercetagetin as ligands. The first-rated ligand with the strongest binding affinity towards the targeted protein was determined based on the minimum binding energy. Further analysis was conducted to identify both the active site of the protein that reacts with the tested ligands and all of the existing intermolecular bonds. Compared to the other ligands, baicalin was identified as the most potential inhibitor of viral activity by showing the best binding affinity (-9.8 kcal/mol). Baicalin can be considered a good candidate for further evaluation as a potentially efficient antiviral against CHIKV.
NBS1, also known as NBN, plays an important role in maintaining genomic stability. Interestingly, rs2735383 G > C, located in a microRNA binding site in the 3'-untranslated region (UTR) of NBS1, was shown to be associated with increased susceptibility to lung and colorectal cancer. However, the relation between rs2735383 and susceptibility to breast cancer is not yet clear. Therefore, we genotyped rs2735383 in 1,170 familial non-BRCA1/2 breast cancer cases and 1,077 controls using PCR-based restriction fragment length polymorphism (RFLP-PCR) analysis, but found no association between rs2735383CC and breast cancer risk (OR = 1.214, 95% CI = 0.936-1.574, P = 0.144). Because we could not exclude a small effect size due to a limited sample size, we further analyzed imputed rs2735383 genotypes (r2 > 0.999) of 47,640 breast cancer cases and 46,656 controls from the Breast Cancer Association Consortium (BCAC). However, rs2735383CC was not associated with overall breast cancer risk in European (OR = 1.014, 95% CI = 0.969-1.060, P = 0.556) nor in Asian women (OR = 0.998, 95% CI = 0.905-1.100, P = 0.961). Subgroup analyses by age, age at menarche, age at menopause, menopausal status, number of pregnancies, breast feeding, family history and receptor status also did not reveal a significant association. This study therefore does not support the involvement of the genotype at NBS1 rs2735383 in breast cancer susceptibility.
Diabetes mellitus (DM) a metabolic disorder characterized by high blood sugar levels causing damage to various organs over time. Current anti-diabetic drugs have limitations and side effects, prompting a search for new inhibitors targeting the α-amylase enzyme. This study aims to discover such inhibitors from thirty isoxazole derivatives of usnic acid using in silico approaches. The potential inhibitory effects of compounds were investigated using ADMET, molecular docking, molecular dynamic simulation, principal component analysis and density functional theory studies. ADMET analysis exhibited a wide range of physicochemical, pharmacokinetic, and drug-like qualities with no significant side effects which were then investigated using molecular docking experiment to determine the lead compound with the best binding affinity for the α-amylase enzyme. All compounds showed good binding affinity against α-amylase enzyme (-7.9 to -9.2 kcal/mol) where compound-13 showed the best binding affinity of -9.2 kcal/mol forming hydrogen bonds with Leu162, Tyr62, Glu233 and Asp300 amino acids. Furthermore, the binding posture and the stability of the compound-13-α-amylase enzyme complex was confirmed by molecular dynamic simulation experiment. Moreover, compound-13 showed binding energy value of -27.92 ± 5.61 kcal/mol, which indicated it could be an α-amylase inhibitor. Additionally, the reactivity of compound-13 was further confirmed by density functional theory analysis. The above findings suggest compound-13 to be a potential α-amylase inhibitor in DM. And setting the stage for further in vitro and in vivo experimental validation.
Increased reports of oseltamivir (OTV)-resistant strains of the influenza virus, such as the H274Y mutation on its neuraminidase (NA), have created some cause for concern. Many studies have been conducted in the attempt to uncover the mechanism of OTV resistance in H274Y NA. However, most of the reported studies on H274Y focused only on the drug-bound system, so the direct effects of the mutation on NA itself prior to drug binding still remain unclear. Therefore, molecular dynamics simulations of NA in apo form, followed by principal component analysis and interaction energy calculations, were performed to investigate the structural changes of the NA binding site as a result of the H274Y mutation. It was observed that the disruption of the NA binding site due to the H274Y mutation was initiated by the repulsive effect of Y274 on the 250-loop, which in turn altered the hydrogen-bonding network around residue 274. The rotated W295 side chain caused the upward movement of the 340-loop. Consequently, sliding box docking results suggested that the binding pathway of OTV was compromised because of the disruption of this binding site. This study also highlighted the importance of the functional group at C6 of the sialic acid mimicry. It is hoped that these results will improve the understanding of OTV resistance and shed some light on the design of a novel anti-influenza drug.
Cytochrome P450s (CYPs) are important heme-containing proteins, well known for their monooxygenase reaction. The human cytochrome P450 4X1 (CYP4X1) is categorized as "orphan" CYP because of its unknown function. In recent studies it is found that this enzyme is expressed in neurovascular functions of the brain. Also, various studies have found the expression and activity of orphan human cytochrome P450 4X1 in cancer. It is found to be a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4X1 remain unclear.
Some chalcones have been designed and synthesized using Claisen-Schmidt reactions as inhibitors of the ferredoxin and ferredoxin-NADP+ reductase interaction to pursue a new selective antimalaria agent. The synthesized compounds exhibited inhibition interactions between PfFd-PfFNR in the range of 10.94%-50%. The three strongest inhibition activities were shown by (E)-1-(4-aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (50%), (E)-1-(4-aminophenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (38.16%), and (E)-1-(4-aminophenyl)-3-(2,3-dimethoxyphenyl)prop-2-en-1-one (31.58%). From the docking experiments we established that the amino group of the methoxyamino chlacone derivatives plays an important role in the inhibition activity by electrostatic interaction through salt bridges and that it forms more stable and better affinity complexes with FNR than with Fd.
GalNAc-T1, a key candidate of GalNac-transferases genes family that is involved in mucin-type O-linked glycosylation pathway, is expressed in most biological tissues and cell types. Despite the reported association of GalNAc-T1 gene mutations with human disease susceptibility, the comprehensive computational analysis of coding, noncoding and regulatory SNPs, and their functional impacts on protein level, still remains unknown. Therefore, sequence- and structure-based computational tools were employed to screen the entire listed coding SNPs of GalNAc-T1 gene in order to identify and characterize them. Our concordant in silico analysis by SIFT, PolyPhen-2, PANTHER-cSNP, and SNPeffect tools, identified the potential nsSNPs (S143P, G258V, and Y414D variants) from 18 nsSNPs of GalNAc-T1. Additionally, 2 regulatory SNPs (rs72964406 and #x26; rs34304568) were also identified in GalNAc-T1 by using FastSNP tool. Using multiple computational approaches, we have systematically classified the functional mutations in regulatory and coding regions that can modify expression and function of GalNAc-T1 enzyme. These genetic variants can further assist in better understanding the wide range of disease susceptibility associated with the mucin-based cell signalling and pathogenic binding, and may help to develop novel therapeutic elements for associated diseases.
One pot, three-component reaction of 1-acryloyl-3,5-bisarylmethylidenepiperidin-4-ones with isatin and sarcosine in molar ratios of 1:1:1 and 1:2:2 furnished to mono- and bis-spiropyrrolidine heterocyclic hybrids comprising functionalized piperidine, pyrrolidine and oxindole structural motifs. Both mono and bis-spiropyrrolidines displayed good inhibitory activity against acetylcholinesterase (AChE) with IC₅₀ values of 2.36-9.43 μM. For butyrylcholinesterase (BChE), mono-cycloadducts in series 8 with IC₅₀ values of lower than 10 μM displayed better inhibitory activities than their bis-cycloadduct analogs in series 9 with IC₅₀ values of 7.44-19.12 μM. The cycloadducts 9j and 8e were found to be the most potent AChE and BChE inhibitors with IC₅₀ values of 2.35 and 3.21 μM, respectively. Compound 9j was found to be competitive inhibitor of AChE while compound 8e was a mixed-mode inhibitor of BChE with calculated Ki values of 2.01 and 6.76 μM, respectively. Molecular docking on Torpedo californica AChE and human BChE showed good correlation between IC₅₀ values and free binding energy values of the synthesized compounds docked into the active site of the enzymes.
The dengue virus is the most significant arthropod-borne human pathogen, and an increasing number of cases have been reported over the last few decades. Currently neither vaccines nor drugs against the dengue virus are available. NS5 methyltransferase (MTase), which is located on the surface of the dengue virus and assists in viral attachment to the host cell, is a promising antiviral target. In order to search for novel inhibitors of NS5 MTase, we performed a computer-aided virtual screening of more than 5 million commercially available chemical compounds using two approaches: i) structure-based screening using the crystal structure of NS5 MTase and ii) ligand-based screening using active ligands of NS5 MTase. Structure-based screening was performed using the LIDAEUS (LIgand Discovery At Edinburgh UniverSity) program. The ligand-based screening was carried out using the EDULISS (EDinburgh University LIgand Selection System) program.
Hemagglutinin (HA) protein plays an important role in binding the influenza virus to infected cells and therefore mediates infection. Deposited HA sequences of 86 Asian strains of influenza A (H1N1) viruses during the first outbreak were obtained from the NCBI database and compared. Interaction of the HA protein of influenza A (H1N1) virus with the human sialic acid receptor was also studied using bioinformatics. Overall, not more than three single-point amino acid variants/changes were observed in the HA protein region of influenza A (H1N1) virus from Asian countries when a selected group sequence comparison was made. The bioinformatics study showed that the HA protein of influenza A (H1N1) binds to the sialic acid receptor in human airway receptors, possibly key to air-borne infection in humans.