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  1. Fulltext Finding Lead Compounds for Dengue Antivirals from a Collection of Old Drugs through In Silico Target Prediction and Subsequent In Vitro Validation
    Abdullah ZL, Chee HY, Yusof R, Mohd Fauzi F
    ACS Omega, 2023 Sep 12;8(36):32483-32497.
    PMID: 37720780 DOI: 10.1021/acsomega.3c02607
    Dengue virus (DENV) infection is one of the most widely spread flavivirus infections. Despite the fatality it could cause, no antiviral treatment is currently available to treat the disease. Hence, this study aimed to repurpose old drugs as novel DENV NS3 inhibitors. Ligand-based (L-B) and proteochemometric (PCM) prediction models were built using 62,354 bioactivity data to screen for potential NS3 inhibitors. Selected drugs were then subjected to the foci forming unit reduction assay (FFURA) and protease inhibition assay. Finally, molecular docking was performed to validate these results. The in silico studies revealed that both models performed well in the internal and external validations. However, the L-B model showed better accuracy in the external validation in terms of its sensitivity (0.671). In the in vitro validation, all drugs (zileuton, trimethadione, and linalool) were able to moderately inhibit the viral activities at the highest concentration tested. Zileuton showed comparable results with linalool when tested at 2 mM against the DENV NS3 protease, with a reduction of protease activity at 17.89 and 18.42%, respectively. Two new compounds were also proposed through the combination of the selected drugs, which are ziltri (zilueton + trimethadione) and zilool (zileuton + linalool). The molecular docking study confirms the in vitro observations where all drugs and proposed compounds were able to achieve binding affinity ≥ -4.1 kcal/mol, with ziltri showing the highest affinity at -7.7 kcal/mol, surpassing the control, panduratin A. The occupation of both S1 and S2 subpockets of NS2B-NS3 may be essential and a reason for the lower binding energy shown by the proposed compounds compared to the screened drugs. Based on the results, this study provided five potential new lead compounds (ziltri, zilool, zileuton, linalool, and trimethadione) for DENV that could be modified further.
  2. Different serotypes of dengue viruses differently regulate the expression of the host cell antigen processing machinery
    Gan CS, Yusof R, Othman S
    Acta Trop, 2015 Sep;149:8-14.
    PMID: 25981524 DOI: 10.1016/j.actatropica.2015.05.005
    Dengue virus (DV) infection demonstrates an intriguing virus-induced intracellular membrane alteration that results in the augmentation of major histocompatibility complex (MHC) class I-restricted antigen presentation. As oppose to its biological function in attracting CD8(+) T-cells, this phenomenon appears to facilitate the immune evasion. However, the molecular events that attribute to the dysregulation of the antigen presenting mechanism (APM) by DV remain obscure. In this study, we aimed to characterize the host cell APM upon infection with all serotypes of whole DV. Cellular RNA were isolated from infected cells and the gene expressions of LMP2, LMP7, TAP1, TAP2, TAPBP, CALR, CANX, PDIA3, HLA-A and HLA-B were analyzed via quantitative PCR. The profiles of the gene expression were further validated. We showed that all four DV serotypes modulate host APM at the proteasomal level with DV2 showing the most prominent expression profile.
  3. Fulltext A Demonstration Of The Polymerase Chain Reaction Technique In Class Teaching
    Yusof, R., Abdul Rahman, P.S., Rahim, Z.H.A.
    Ann Dent, 1999;6(1):-.
    MyJurnal
    The application of PCR technique in genetic screening was demonstrated using the genetic materials from buccal cells of the students in the class. Two factors were taken into consideration when designing the experiments. The DNA region to be amplified should not be associated with any disease state. This is to eliminate any emotional and ethical problems associated with the experiments. In this practical, the presence and absence of a 38 bp sequence in the intron of COLIA2 gene were studied. The students were also shown on how to analyse the presence of homozygous and heterozygous alleles and the genetic variations that might be observed in the different ethnic groups of students. Another factor was the time taken to complete the experiment. Our experience showed that this experiment would take at least six hours to obtain and analyse the results. It is therefore suitable to be used in class teaching.
  4. A B-myb--DREAM complex is not critical to regulate the G2/M genes in HPV-transformed cell lines
    Rashid NN, Yusof R, Watson RJ
    Anticancer Res, 2014 Nov;34(11):6557-63.
    PMID: 25368258
    It is well-established that HPV E7 proteins, encoded by human papillomavirus (HPV) genes, frequently associated with cervical cancers bind avidly to the retinoblastoma (RB) family of pocket proteins and disrupt their association with members of the E2F transcription factor family. Our previous study showed that the repressive p130-dimerization partner, RB-like, E2F and multi-vulval class (DREAM) complex was disrupted by HPV16 E7 proteins in order to maintain the viral replication in CaSki cells. However, we would like to address whether the activator B-myb-DREAM complex is critical in regulating the replication and mitosis phase since our previous study showed increased B-myb-DREAM expression in HPV-transformed cell lines when compared to control cells.
  5. Inhibitory effects of a peptide-fusion protein (Latarcin-PAP1-Thanatin) against chikungunya virus
    Rothan HA, Bahrani H, Shankar EM, Rahman NA, Yusof R
    Antiviral Res, 2014 Aug;108:173-80.
    PMID: 24929084 DOI: 10.1016/j.antiviral.2014.05.019
    Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.
  6. Mefenamic acid in combination with ribavirin shows significant effects in reducing chikungunya virus infection in vitro and in vivo
    Rothan HA, Bahrani H, Abdulrahman AY, Mohamed Z, Teoh TC, Othman S, et al.
    Antiviral Res, 2016 Mar;127:50-6.
    PMID: 26794398 DOI: 10.1016/j.antiviral.2016.01.006
    Chikungunya virus (CHIKV) infection is a persistent problem worldwide due to efficient adaptation of the viral vectors, Aedes aegypti and Aedes albopictus mosquitoes. Therefore, the absence of effective anti-CHIKV drugs to combat chikungunya outbreaks often leads to a significant impact on public health care. In this study, we investigated the antiviral activity of drugs that are used to alleviate infection symptoms, namely, the non-steroidal anti-inflammatory drugs (NSAIDs), on the premise that active compounds with potential antiviral and anti-inflammatory activities could be directly subjected for human use to treat CHIKV infections. Amongst the various NSAID compounds, Mefenamic acid (MEFE) and Meclofenamic acid (MECLO) showed considerable antiviral activity against viral replication individually or in combination with the common antiviral drug, Ribavirin (RIBA). The 50% effective concentration (EC50) was estimated to be 13 μM for MEFE, 18 μM for MECLO and 10 μM for RIBA, while MEFE + RIBA (1:1) exhibited an EC50 of 3 μM, and MECLO + RIBA (1:1) was 5 μM. Because MEFE is commercially available and its synthesis is easier compared with MECLO, MEFE was selected for further in vivo antiviral activity analysis. Treatment with MEFE + RIBA resulted in a significant reduction of hypertrophic effects by CHIKV on the mouse liver and spleen. Viral titre quantification in the blood of CHIKV-infected mice through the plaque formation assay revealed that treatment with MEFE + RIBA exhibited a 6.5-fold reduction compared with untreated controls. In conclusion, our study demonstrated that MEFE in combination with RIBA exhibited significant anti-CHIKV activity by impairing viral replication in vitro and in vivo. Indeed, this finding may lead to an even broader application of these combinatorial treatments against other viral infections.
  7. Fulltext Small molecule grp94 inhibitors block dengue and Zika virus replication
    Rothan HA, Zhong Y, Sanborn MA, Teoh TC, Ruan J, Yusof R, et al.
    Antiviral Res, 2019 11;171:104590.
    PMID: 31421166 DOI: 10.1016/j.antiviral.2019.104590
    Two major flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), cause severe health and economic burdens worldwide. Recently, genome-wide screenings have uncovered the importance of regulators of the Hrd1 ubiquitin ligase-mediated endoplasmic reticulum (ER)-associated degradation (ERAD) pathway for flavivirus replication in host cells. Here we report the identification of the compound Bardoxolone methyl (CDDO-me) as a potent inhibitor of the Hrd1 ubiquitin ligase-mediated ERAD, which possesses a broad-spectrum activity against both DENV and ZIKV. Cellular thermal shift assay (CETSA) suggested that CDDO-me binds to grp94, a key component of the Hrd1 pathway, at a low nanomolar concentration, whereas interaction was not detected with its paralog Hsp90. CDDO-me and the grp94 inhibitor PU-WS13 substantially suppressed DENV2 replication and the cytopathic effects caused by DENV and ZIKV infection. The antiviral activities of both compounds were demonstrated for all four DENV serotypes and four ZIKV strains in multiple human cell lines. This study defines grp94 as a crucial host factor for flavivirus replication and identified CDDO-me as a potent small molecule inhibitor of flavivirus infection. Inhibition of grp94 may contribute to the antiviral activity of CDDO-me. Further investigation of grp94 inhibitors may lead to a new class of broad-spectrum anti-flaviviral medications.
  8. Inhibitory effect of doxycycline against dengue virus replication in vitro
    Rothan HA, Mohamed Z, Paydar M, Rahman NA, Yusof R
    Arch Virol, 2014 Apr;159(4):711-8.
    PMID: 24142271 DOI: 10.1007/s00705-013-1880-7
    Doxycycline is an antibiotic derived from tetracycline that possesses antimicrobial and anti-inflammatory activities. Antiviral activity of doxycycline against dengue virus has been reported previously; however, its anti-dengue properties need further investigation. This study was conducted to determine the potential activity of doxycycline against dengue virus replication in vitro. Doxycycline inhibited the dengue virus serine protease (DENV2 NS2B-NS3pro) with an IC50 value of 52.3 ± 6.2 μM at 37 °C (normal human temperature) and 26.7 ± 5.3 μM at 40 °C (high fever temperature). The antiviral activity of doxycycline was first tested at different concentrations against DENV2 using a plaque-formation assay. The virus titter decreased significantly after applying doxycycline at levels lower than its 50 % cytotoxic concentration (CC50, 100 μM), showing concentration-dependent inhibition with a 50 % effective concentration (EC50) of approximately 50 μM. Doxycycline significantly inhibited viral entry and post-infection replication of the four dengue serotypes, with serotype-specific inhibition (high activity against DENV2 and DENV4 compared to DENV1 and DENV3). Collectively, these findings underline the need for further experimental and clinical studies on doxycycline, utilizing its anti-dengue and anti-inflammatory activities to attenuate the clinical symptoms of dengue virus infection.
  9. Isolation of Bifidobacteria infantis and its antagonistic activity against ETEC 0157 and Salmonella typhimurium S-285 in weaning foods
    Yusof RM, Haque F, Ismail M, Hassan Z
    Asia Pac J Clin Nutr, 2000 Jun;9(2):130-5.
    PMID: 24394399
    Probiotic organism Bifidobacteria was isolated from the faeces of breast-fed infants at Universiti Putra Malaysia. Trypticase phytone peptone yeast extract agar (TPY) was used as a selective media for the isolation. Morphological examination of the isolates indicated that Bifidobacteria was Gram-positive rods in nature, curved with characteristics of V and Y shapes. The organisms were non-catalase producing, non-nitrate reducing, non-motile, had an absence of indole and were unable to liquify gelatin. The ratios of acetic and lactic acids were determined using high performance liquid chromatography (HPLC). Using carbohydrate fermentation profile test API-CH-50 kits, 20 Bifidobacteria strains had been identified: they were the species of Bifidobacteria infantis and two different sub-species, mainly infantis and lacentis. Based on a wide zone of inhibition, three suitable strains of B. infantis, Bifi-11, Bifi-19 and Bifi-20, were tested in weaning foods for antimicrobial activity towards two human pathogens: E. coli-0157 (World Health Organization) and Salmonella typhimurium S-285. The pH, titratable acidity of weaning foods and total colony count for Bifidobacteria, enteropathogenic Escherichia coli and S. typhimurium were recorded at 3-h intervals for 30 h. It was found that after 9 h of incubation of weaning foods, the pH declined to < 3.6 from pH 6.0, whereas titratable acidity increased from 0.026 to 0.08%. It was indicated that Bifidobacteria inhibited E. coli better than did S typhimurium due to low pH. After 24 h of incubation, approximately 98% of E. coli was inhibited by Bifidobacteria. It is suggested that the inhibitory effect of Bifidobacteria strains in weaning foods towards the growth of enteropathogenic E. coli and S. typhimurium was solely due to low pH and the production of volatile acid components by the organism.
  10. Fulltext Iodine status among pregnant women in rural Sabah, Malaysia
    Lim KK, Chan YY, Teh CH, Ismail H, Yusof R, Muhi J, et al.
    Asia Pac J Clin Nutr, 2017 8 15;26(5):861-866.
    PMID: 28802296 DOI: 10.6133/apjcn.092016.06
    BACKGROUND AND OBJECTIVES: In 2000, legislation on mandatory universal salt iodisation was enacted in Sabah, Malaysia, to reduce the incidence of iodine deficiency disorders among its population. To evaluate the iodine levels among pregnant women from selected rural divisions in Sabah 13 years after the enactment of the universal salt iodisation programme.

    METHODS AND STUDY DESIGN: This cross-sectional study was conducted from 1 May to 30 June, 2013, in three rural divisions of Sabah (the Interior, the West Coast, and Kudat). Data regarding domestic iodised salt use and iodine-containing supplement consumption were obtained from respondents through face-to-face interviews; goitre enlargement was examined through palpation and graded according to the World Health Organization classification. Spot urine samples were also obtained to assess urinary iodine levels by using an in-house modified micromethod.

    RESULTS: In total, 534 pregnant women participated. The prevalence of goitre was 1.0% (n=5), noted only in the West Coast and Kudat divisions. Although all pregnant women consumed iodised salt, overall median urinary iodine concentration was only 106 μg/L, indicating insufficient iodine intake, with nearly two-thirds of the women (60%) having a median urinary iodine concentrations of <150 μg/L.

    CONCLUSIONS: Pregnant women from the rural divisions in Sabah still exhibit iodine deficiency disorder despite the mandatory universal salt iodisation programme. Iodine supplementation programmes targeting pregnant women are warranted.

  11. Fulltext Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1
    Rothan HA, Han HC, Ramasamy TS, Othman S, Rahman NA, Yusof R
    BMC Infect Dis, 2012;12:314.
    PMID: 23171075 DOI: 10.1186/1471-2334-12-314
    Global resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern. Therefore, there is an urgent need for the development of successful drugs that are both economical and offer a long-lasting protection. The viral NS2B-NS3 serine protease (NS2B-NS3pro) is a promising target for the development of drug-like inhibitors, which are not available at the moment. In this study, we report retrocyclin-1 (RC-1) production in E. coli as a recombinant peptide to test against dengue NS2B-NS3pro.
  12. Fulltext Identification of natural antimicrobial agents to treat dengue infection: In vitro analysis of latarcin peptide activity against dengue virus
    Rothan HA, Bahrani H, Rahman NA, Yusof R
    BMC Microbiol, 2014;14:140.
    PMID: 24885331 DOI: 10.1186/1471-2180-14-140
    Although there have been considerable advances in the study of dengue virus, no vaccines or anti-dengue drugs are currently available for humans. Therefore, new approaches are necessary for the development of potent anti-dengue drugs. Natural antimicrobial peptides (AMPs) with potent antiviral activities are potential hits-to-leads for antiviral drug discovery. We performed this study to identify and characterise the inhibitory potential of the latarcin peptide (Ltc 1, SMWSGMWRRKLKKLRNALKKKLKGE) against dengue virus replication in infected cells.
  13. Fulltext An analytical approach to evaluate the performance of graphene and carbon nanotubes for NH3 gas sensor applications
    Akbari E, Arora VK, Enzevaee A, Ahmadi MT, Saeidmanesh M, Khaledian M, et al.
    Beilstein J Nanotechnol, 2014;5:726-34.
    PMID: 24991510 DOI: 10.3762/bjnano.5.85
    Carbon, in its variety of allotropes, especially graphene and carbon nanotubes (CNTs), holds great potential for applications in variety of sensors because of dangling π-bonds that can react with chemical elements. In spite of their excellent features, carbon nanotubes (CNTs) and graphene have not been fully exploited in the development of the nanoelectronic industry mainly because of poor understanding of the band structure of these allotropes. A mathematical model is proposed with a clear purpose to acquire an analytical understanding of the field-effect-transistor (FET) based gas detection mechanism. The conductance change in the CNT/graphene channel resulting from the chemical reaction between the gas and channel surface molecules is emphasized. NH3 has been used as the prototype gas to be detected by the nanosensor and the corresponding current-voltage (I-V) characteristics of the FET-based sensor are studied. A graphene-based gas sensor model is also developed. The results from graphene and CNT models are compared with the experimental data. A satisfactory agreement, within the uncertainties of the experiments, is obtained. Graphene-based gas sensor exhibits higher conductivity compared to that of CNT-based counterpart for similar ambient conditions.
  14. Inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, Boesenbergia rotunda (L.), towards dengue-2 virus NS3 protease
    Kiat TS, Pippen R, Yusof R, Ibrahim H, Khalid N, Rahman NA
    Bioorg Med Chem Lett, 2006 Jun 15;16(12):3337-40.
    PMID: 16621533
    Boesenbergia rotunda (L.) cyclohexenyl chalcone derivatives, 4-hydroxypanduratin A and panduratin A, showed good competitive inhibitory activities towards dengue 2 virus NS3 protease with the Ki values of 21 and 25 microM, respectively, whilst those of pinostrobin and cardamonin were observed to be non-competitive. NMR and GCMS spectroscopic data formed the basis of assignment of structures of the six compounds isolated.
  15. Polymethacrylate coated electrospun PHB fibers: An exquisite outlook for fabrication of paper-based biosensors
    Hosseini S, Azari P, Farahmand E, Gan SN, Rothan HA, Yusof R, et al.
    Biosens Bioelectron, 2015 Jul 15;69:257-64.
    PMID: 25765434 DOI: 10.1016/j.bios.2015.02.034
    Electrospun polyhydroxybutyrate (PHB) fibers were dip-coated by polymethyl methacrylate-co-methacrylic acid, poly(MMA-co-MAA), which was synthesized in different molar ratios of the monomers via free-radical polymerization. Fabricated platfrom was employed for immobilization of the dengue antibody and subsequent detection of dengue enveloped virus in enzyme-linked immunosorbent assay (ELISA). There is a major advantage for combination of electrospun fibers and copolymers. Fiber structre of electrospun PHB provides large specific surface area available for biomolecular interaction. In addition, polymer coated parts of the platform inherited the premanent presence of surface carboxyl (-COOH) groups from MAA segments of the copolymer which can be effectively used for covalent and physical protein immobilization. By tuning the concentration of MAA monomers in polymerization reaction the concentration of surface -COOH groups can be carefully controlled. Therefore two different techniques have been used for immobilization of the dengue antibody aimed for dengue detection: physical attachment of dengue antibodies to the surface and covalent immobilization of antibodies through carbodiimide chemistry. In that perspective, several different characterization techniques were employed to investigate the new polymeric fiber platform such as scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle (WCA) measurement and UV-vis titration. Regardless of the immobilization techniques, substantially higher signal intensity was recorded from developed platform in comparison to the conventional ELISA assay.
  16. Biosensing enhancement of dengue virus using microballoon mixers on centrifugal microfluidic platforms
    Aeinehvand MM, Ibrahim F, Harun SW, Djordjevic I, Hosseini S, Rothan HA, et al.
    Biosens Bioelectron, 2015 May 15;67:424-30.
    PMID: 25220800 DOI: 10.1016/j.bios.2014.08.076
    Dengue is the current leading cause of death among children in several Latin American and Asian countries. Due to poverty in areas where the disease is prevalent and the high cost of conventional diagnostic systems, low cost devices are needed to reduce the burden caused by dengue infection. Centrifugal microfluidic platforms are an alternative solution to reduce costs and increase the availability of a rapid diagnostic system. The rate of chemical reactions in such devices often depends on the efficiency of the mixing techniques employed in their microfluidic networks. This paper introduces a micromixer that operates by the expansion and contraction of a microballoon to produce a consistent periodical 3D reciprocating flow. We established that microballoons reduced mixing time of 12 μl liquids from 170 min, for diffusional mixing, to less than 23 s. We have also tested the effect of the microballoon mixers on the detection of the dengue virus. The results indicate that employing a microballoon mixer enhances the detection sensitivity of the dengue virus by nearly one order of magnitude compared to the conventional ELISA method.
  17. Deep eutectic solvents (DES) mediated extraction of pectin from Averrhoa bilimbi: Optimization and characterization studies
    Shafie MH, Yusof R, Gan CY
    Carbohydr Polym, 2019 Jul 15;216:303-311.
    PMID: 31047070 DOI: 10.1016/j.carbpol.2019.04.007
    The Box-Behnken design was applied to optimize the extraction of pectin from Averrhoa bilimbi (ABP) using deep eutectic solvents (DES). The four variables of extraction were percentage of DES (X1), extraction time (X2), temperature (X3), and molar ratio of DES components (X4). The quadratic regression equation was established as a predicted model with R2 value of 0.9375. The optimal condition was X1 = 3.74% (w/v), X2 = 2.5 h, X3 = 80 °C, and X4 = 1:1. No significant difference between the predicted (14.70%) and experimental (14.44%) maximum yield of sample was noted. Characterization of physico-chemical properties characterization of ABP was performed. The main components of ABP were galacturonic acids, arabinoses, and xyloses. ABP also showed good functional properties such as water holding capacity (3.70 g/g), oil holding capacity (2.40 g/g), and foaming capacity (133.33%). The results also showed that ABP exhibited free radical scavenging activity (41.46%) and ferric reducing antioxidant power (1.15 mM).
  18. Fulltext Infected Baerveldt Glaucoma Drainage Device by Aspergillus niger
    Salim NL, Azhany Y, Abdul Rahman Z, Yusof R, Liza-Sharmini AT
    Case Rep Ophthalmol Med, 2015;2015:249419.
    PMID: 26064735 DOI: 10.1155/2015/249419
    Fungal endophthalmitis is rare but may complicate glaucoma drainage device surgery. Management is challenging as the symptoms and signs may be subtle at initial presentation and the visual prognosis is usually poor due to its resistant nature to treatment. At present there is lesser experience with intravitreal injection of voriconazole as compared to Amphotericin B. We present a case of successfully treated Aspergillus endophthalmitis following Baerveldt glaucoma drainage device implantation with intravitreal and topical voriconazole.
  19. Dengue envelope domain III-peptide binding analysis via tryptophan fluorescence quenching assay
    Baharuddin A, Amir Hassan A, Othman R, Xu Y, Huang M, Ario Tejo B, et al.
    Chem Pharm Bull (Tokyo), 2014;62(10):947-55.
    PMID: 25273053
    In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λmax for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.
  20. Rational discovery of dengue type 2 non-competitive inhibitors
    Heh CH, Othman R, Buckle MJ, Sharifuddin Y, Yusof R, Rahman NA
    Chem Biol Drug Des, 2013 Jul;82(1):1-11.
    PMID: 23421589 DOI: 10.1111/cbdd.12122
    Various works have been carried out in developing therapeutics against dengue. However, to date, no effective vaccine or anti-dengue agent has yet been discovered. The development of protease inhibitors is considered as a promising option, but most previous works have involved competitive inhibition. In this study, we focused on rational discovery of potential anti-dengue agents based on non-competitive inhibition of DEN-2 NS2B/NS3 protease. A homology model of the DEN-2 NS2B/NS3 protease (using West Nile Virus NS2B/NS3 protease complex, 2FP7, as the template) was used as the target, and pinostrobin, a flavanone, was used as the standard ligand. Virtual screening was performed involving a total of 13 341 small compounds, with the backbone structures of chalcone, flavanone, and flavone, available in the ZINC database. Ranking of the resulting compounds yielded compounds with higher binding affinities compared with the standard ligand. Inhibition assay of the selected top-ranking compounds against DEN-2 NS2B/NS3 proteolytic activity resulted in significantly better inhibition compared with the standard and correlated well with in silico results. In conclusion, via this rational discovery technique, better inhibitors were identified. This method can be used in further work to discover lead compounds for anti-dengue agents.
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