Synthesis, in vitro Anti-HIV-1RT evaluation, molecular modeling, DFT and acute oral toxicity studies of some benzotriazole derivatives.

This study synthesized and evaluated a series of benzotriazole derivatives denoted 3(a-j) and 6(a-j) for their anti-HIV-1 RT activities compared to the standard drug efavirenz. Notably, compound 3 h, followed closely by 6 h, exhibited significant anti-HIV-1 RT efficacy relative to the standard drug. In vivo oral toxicity studies were conducted for the most active compound 3 h, confirming its nontoxic nature to ascertain the safety profile. By employing molecular docking techniques, we explored the potential interactions between the synthesized compounds (ligands) and a target biomolecule (protein)(PDB ID 1RT2) at the molecular level. We undertook the molecular dynamics study of 3 h, the most active compound, within the active binding pocket of the cocrystallized structure of HIV-1 RT (PDB ID 1RT2). We aimed to learn more about how biomolecular systems behave, interact, and change at the atomic or molecular level over time. Finally, the DFT-derived HOMO and LUMO orbitals, as well as analysis of the molecular electrostatic potential map, aid in discerning the reactivity characteristics of our molecule.

Design, preparation and biological evaluation of new Rociletinib-inspired analogs as irreversible EGFR inhibitors to treat non-small-cell-lung cancer.

Epidermal growth factor receptor (EGFR) kinase has been implicated in the uncontrolled cell growth associated with non-small cell lung cancer (NSCLC). This has prompted the development of 3 generations of EGFR inhibitors over the last 2 decades due to the rapid development of drug resistance issues caused by clinical mutations, including T790M, L858R and the double mutant T790M & L858R. In this work we report the design, preparation and biological assessment of new irreversible 2,4-diaminopyrimidine-based inhibitors of EGFR kinase. Twenty new compounds have been prepared and evaluated which incorporate a range of electrophilic moieties. These include acrylamide, 2-chloroacetamide and (2E)-3-phenylprop-2-enamide, to allow reaction with residue Cys797. In addition, more polar groups have been incorporated to provide a better balance of physical properties than clinical candidate Rociletinib. Inhibitory activities against EGFR wildtype (WT) and EGFR T790M & L858R have been evaluated along with cytotoxicity against EGFR-overexpressing (A549, A431) and normal cell lines (HepG2). Selectivity against JAK3 kinase as well as physicochemical properties determination (logD7.4 and phosphate buffer solubility) have been used to profile the compounds. We have identified 20, 21 and 23 as potent mutant EGFR inhibitors (≤20 nM), with comparable or better selectivity over WT EGFR, and lower activity at JAK3, than Osimertinib or Rociletinib. Compounds 21 displayed the best combination of EGFR mutant activity, JAK3 selectivity, cellular activity and physicochemical properties. Finally, kinetic studies on 21 were performed, confirming a covalent mechanism of action at EGFR.

Synthesis and characterization of molecularly imprinted polymer nanoparticles against porcine circovirus type 2 viral-like particles.

PCV2 is a significant epidemic agricultural pathogen that causes a variety of swine diseases. PCV2 infections have significant economic impact on the swine industry, making effective strategies for rapid detection of PCV2 in pigs essential. Herein, we report on the synthesis of the so-called nano-MIPs which can be utilized for molecular recognition of PCV2. The morphology and structure of nano-MIPs were characterized using scanning electron microscopy (SEM). Nano-MIPs are spherical with sizes around 120-150 nm. Binding experiments demonstrate that the fluorescence intensity of PCV2 samples decreases proportionally to increasing the concentration of nano-MIPs due to quenching, while non-imprinted polymer nanoparticles (nano-NIPs) do not affect the signal. The Stern-Volmer constant of nano-MIPs binding to PCV2 was 1.3 × 10-3 mL/µg, whereas nano-NIPs led to 7 × 10-5 mL/µg, i.e., 1.8 orders of magnitude lower. The detection limit for binding MIP particles to PCV2 by fluorescence measurements is 47 µg/mL. This affinity test allows for designing both direct and competitive quartz crystal microbalance (QCM) assays for PCV2 leading to QCM measurements. The QCM results show nano-MIPs binding to PCV2 immobilized on the sensor surface with appreciable reproducibility. QCM sensor characteristics reveal signal saturation above around 200 µg/mL at a response of - 354 Hz and an LOD of approximately 35 µg/mL. Nano-MIPs also show selectivity factors of 2-5 for CSFV and PRRSV probably because the three viruses have similar diameters around 50 nm.

Molecularly Imprinted Nanoparticle Ensembles for Rapidly Identifying S. epidermidis.

Staphylococcus epidermidis (S. epidermidis) belongs to methicillin-resistant bacteria strains that cause severe disease in humans. Herein, molecularly imprinted polymer (MIP) nanoparticles resulting from solid-phase synthesis on entire cells were employed as a sensing material to identify the species. MIP nanoparticles revealed spherical shapes with diameters of approximately 70 nm to 200 nm in scanning electron microscopy (SEM), which atomic force microscopy (AFM) confirmed. The interaction between nanoparticles and bacteria was assessed using height image analysis in AFM. Selective binding between MIP nanoparticles and S. epidermidis leads to uneven surfaces on bacteria. The surface roughness of S. epidermidis cells was increased to approximately 6.3 ± 1.2 nm after binding to MIP nanoparticles from around 1 nm in the case of native cells. This binding behavior is selective: when exposing Escherichia coli and Bacillus subtilis to the same MIP nanoparticle solutions, one cannot observe binding. Fluorescence microscopy confirms both sensitivity and selectivity. Hence, the developed MIP nanoparticles are a promising approach to identify (pathogenic) bacteria species.

In Silico and In Vitro Study of Janus Kinases Inhibitors from Naphthoquinones.

Janus kinases (JAKs) are involved in numerous cellular signaling processes related to immune cell functions. JAK2 and JAK3 are associated with the pathogenesis of leukemia and common lymphoid-derived illnesses. JAK2/3 inhibitors could reduce the risk of various diseases by targeting this pathway. Herein, the naphthoquinones were experimentally and theoretically investigated to identify novel JAK2/3 inhibitors. Napabucasin and 2'-methyl napabucasin exhibited potent cell growth inhibition in TF1 (IC50 = 9.57 and 18.10 µM) and HEL (IC50 = 3.31 and 6.65 µM) erythroleukemia cell lines, and they significantly inhibited JAK2/3 kinase activity (in a nanomolar range) better than the known JAK inhibitor, tofacitinib. Flow cytometric analysis revealed that these two compounds induced apoptosis in TF1 cells in a time and dose-dependent manner. From the molecular dynamics study, both compounds formed hydrogen bonds with Y931 and L932 residues and hydrophobically contacted with the conserved hinge region, G loop, and catalytic loop of the JAK2. Our obtained results suggested that napabucasin and its methylated analog were potential candidates for further development of novel anticancer drug targeting JAKs.

Discovery of Novel EGFR Inhibitor Targeting Wild-Type and Mutant Forms of EGFR: In Silico and In Vitro Study.

Targeting L858R/T790M and L858R/T790M/C797S mutant EGFR is a critical challenge in developing EGFR tyrosine kinase inhibitors to overcome drug resistance in non-small cell lung cancer (NSCLC). The discovery of next-generation EGFR tyrosine kinase inhibitors (TKIs) is therefore necessary. To this end, a series of furopyridine derivatives were evaluated for their EGFR-based inhibition and antiproliferative activities using computational and biological approaches. We found that several compounds derived from virtual screening based on a molecular docking and solvated interaction energy (SIE) method showed the potential to suppress wild-type and mutant EGFR. The most promising PD13 displayed strong inhibitory activity against wild-type (IC50 of 11.64 ± 1.30 nM), L858R/T790M (IC50 of 10.51 ± 0.71 nM), which are more significant than known drugs. In addition, PD13 revealed a potent cytotoxic effect on A549 and H1975 cell lines with IC50 values of 18.09 ± 1.57 and 33.87 ± 0.86 µM, respectively. The 500-ns MD simulations indicated that PD13 formed a hydrogen bond with Met793 at the hinge region, thus creating excellent EGFR inhibitory activity. Moreover, the binding of PD13 in the hinge region of EGFR was the major determining factor in stabilizing the interactions via hydrogen bonds and van der Waals (vdW). Altogether, PD13 is a promising novel EGFR inhibitor that could be further clinically developed as fourth-generation EGFR-TKIs.

Exploring the Apoptotic-Induced Biochemical Mechanism of Traditional Thai Herb (Kerra™) Extract in HCT116 Cells Using a Label-Free Proteomics Approach.

Background and Objectives: Natural products have proven to be a valuable source for the discovery of new candidate drugs for cancer treatment. This study aims to investigate the potential therapeutic effects of "Kerra™", a natural extract derived from a mixture of nine medicinal plants mentioned in the ancient Thai scripture named the Takxila Scripture, on HCT116 cells. Materials and Methods: In this study, the effect of the Kerra™ extract on cancer cells was assessed through cell viability assays. Apoptotic activity was evaluated by examining the apoptosis characteristic features. A proteomics analysis was conducted to identify proteins and pathways associated with the extract's mechanism of action. The expression levels of apoptotic protein markers were measured to validate the extract's efficacy. Results: The Kerra™ extract demonstrated a dose-dependent inhibitory effect on the cells, with higher concentrations leading to decreased cell viability. Treatment with the extract for 72 h induced characteristic features of early and late apoptosis, as well as cell death. An LC-MS/MS analysis identified a total of 3406 proteins. The pathway analysis revealed that the Kerra™ extract stimulated apoptosis and cell death in colorectal cancer cell lines and suppressed cell proliferation in adenocarcinoma cell lines through the EIF2 signaling pathway. Upstream regulatory proteins, including cyclin-dependent kinase inhibitor 1A (CDKN1A) and MYC proto-oncogene, bHLH transcription factor (MYC), were identified. The expressions of caspase-8 and caspase-9 were significantly elevated by the Kerra™ extract compared to the chemotherapy drug Doxorubicin (Dox). Conclusions: These findings provide strong evidence for the ability of the Kerra™ extract to induce apoptosis in HCT116 colon cancer cells. The extract's efficacy was demonstrated by its dose-dependent inhibitory effect, induction of apoptotic activity, and modulation of key proteins involved in cell death and proliferation pathways. This study highlights the potential of Kerra™ as a promising therapeutic agent in cancer treatment.

The Inhibitory Effect of KerraTM, KSTM, and MinozaTM on Human Papillomavirus Infection and Cervical Cancer.

Background and Objectives: Cervical cancer is one of the most common types of frequently found cancers in Thailand. One of the causative agents is the infection of the high-risk human papillomavirus (HPV) type 16 and 18. Traditional medicines are rich sources of bioactive compounds which are a valuable source for the development of novel cancer therapies. In this study, the therapeutic effects of 3 traditional medicines, KerraTM, KSTM, and MinozaTM, were studied on HeLa and CaSki cells. Materials and Methods: The effects of KerraTM, KSTM, and MinozaTM on cancer cells were evaluated through cytotoxicity and cell death assays. The infection assay using HPV-16 pseudovirus was also carried out. Results: All traditional medicines efficiently suppressed cell growths of HeLa and CaSki, with KerraTM being the most potent anticancer agent followed by KSTM and MinozaTM. KerraTM at 158 µg/mL and 261 µg/mL significantly increases the percentage inhibition of the HPV-16 pseudovirus infection in a pre-attachment step in a dose-dependent manner, while KSTM at 261 µg/mL efficiently inhibited viral infection in both pre-attachment and adsorption steps. However, KerraTM, KSTM, and MinozaTM at subtoxic concentrations could not reduce the viral E6 mRNA expressions of HPV-16 and HPV-18. Cell death assay by acridine orange/ethidium bromide showed that KerraTM increased population of dead cells in dose-dependent manner in both CaSki and HeLa. The percentage of secondary necrosis in KerraTM-treated CaSki was higher than that of HeLa cells, while the percentage of late apoptotic cells in HeLa was higher than that of CaSki, indicating that HeLa was more susceptible to KerraTM than CaSki. For KSTM and MinozaTM, these extracts at 250 µg/mL promoted autophagy over cell death. At 500 µg/mL, the percentage of dead cells in KerraTM was higher than that of KSTM and MinozaTM. Conclusions: KerraTM is a potent traditional medicine for promoting cancer cell death. KerraTM is possibly useful in the prevention and treatment of cervical cancer. Further investigation will be carried out to gain a better understanding of the biochemical mechanism and the pharmacological activity underlying this effect.

Role of conserved arginine in HadA monooxygenase for 4-nitrophenol and 4-chlorophenol detoxification.

HadA monooxygenase is involved in the initial step of the biodegradation pathway of toxic nitrophenols and halogenated phenols. HadA catalyzes the O2 -dependent denitration of nitrophenols and dehalogenation of halogenated phenols via the hydroquinone pathway. Based on bioinformatics and structural analysis, Arg208 of HadA is located at the proper position for substrate stabilization. This arginine is conserved among hydroquinone pathway-specific enzymes for toxicant detoxification. In this study, the function of Arg208 in HadA was determined by a single-point mutation creating HadAArg208 variants. 4-Nitrophenol was mineralized by HadAArg208 variants that contain side chains as a positive charge and hydrogen-bond donor, whereas 4-chlorophenol strictly required a positively charged environment for detoxification. Transient kinetic results indicated that the biodetoxification ability of HadAArg208 variants was diminished due to the slowing down of denitration/dehalogenation. The substrate-binding mode and affinity energy were evaluated by molecular docking. The findings were consistent with the experimental results indicating that arginine is the most fit for both 4-nitrophenol and 4-chlorophenol binding, whereas the active mutants provide a weaker interaction correlated with their denitration/dehalogenation activities. Altogether, Arg208 plays a role in providing proper chemical interactions to the substrate for binding at an appropriate orientation in the active site of hydroquinone pathway-specific enzymes. In addition, it is proposed to stabilize nitro groups and halide ions that are released in denitration/dehalogenation reactions. This conserved arginine might be the essential feature for related biocatalysts, which could be fundamental knowledge regarding this enzyme family.

A novel cyclic NP1 reveals obstruction of EGFR kinase activity and attenuation of EGFR-driven cell lines.

Aberrations of the epidermal growth factor receptor (EGFR), for example, mutations and overexpression, play pivotal roles in various cellular functions, such as proliferation, migration, and cell differentiation. Approved small molecule-based inhibitors, including gefitinib and erlotinib, are used clinically to target the tyrosine kinase domain of EGFR (TK-EGFR). However, the severity of the side effects, off-target effects, and drug resistance is a concern. Cyclic peptides are a well-known peptide format with high stability and are promising molecules for drug development. Herein, the Ph.D.™-C7C phage display library was used to screen cyclic peptides against TK-EGFR. Biopanning, both with and without propagation methods, was performed to assess the highest capacity peptides using the enzymatic activity of TK-EGFR. Interestingly, NP1, a peptide selected during biopanning without propagation demonstrated an inhibitory effect against TK-EGFR at IC50 within the nanomolar range; this effect was better than that of P1 obtained using biopanning with propagation. Moreover, NP1 elicited EGFR with an affinity binding (KD ) value of 18.40 ± 5.50 µM by surface plasmon resonance (SPR). Introducing cell-penetrating peptides or Arginine-9 (Arg9) at the N-terminus of NP1 thus improves cell-penetrability and can lead to the inhibition of EGFR-driven cancer cell lines; however, it exhibits no hepatotoxicity. Furthermore, NP1 caused a decrease in phosphorylated EGFR after activation within cells. A docking model shows that NP1 interacted primarily with TK-EGFR via hydrogen bonding. Together, this suggests that NP1 is a novel EGFR peptide inhibitor candidate with specificity and selectivity toward TK-EGFR, and may be applied to targeted therapy.

A novel nanobody as therapeutics target for EGFR-positive colorectal cancer therapy: exploring the effects of the nanobody on SW480 cells using proteomics approach.

BACKGROUND: The epidermal growth factor receptor (EGFR) overexpression is found in metastatic colorectal cancer (mCRC). Targeted molecular therapies such as monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKI) are becoming more precise, targeting specifically for cancer therapeutics. However, there are adverse effects of currently available anti-EGFR drugs, including drug-resistant and side effects. Nanobodies can overcome these limitations. Our previous study has found that cell-penetrable nanobodies targeted at EGFR-tyrosine kinase were significantly reduced EGFR-positive lung cancer cells viability and proliferation. The aim of the present study was to determine the effect of cell-penetrable nanobody (R9VH36) on cell viability and proteomic profile in EGFR-positive human colorectal cancer cell lines. METHODS: The human colorectal carcinoma cell line (SW480) was treated with R9VH36, compared with gefitinib. Cell viability was monitored using the MTT cell viability assay. The proteomic profiling was analyzed by LC-MS/MS . RESULTS: The half-maximal inhibitory concentration (IC50) values determined for R9VH36 and gefitinib against SW480 were 527 ± 0.03 nM and 13.31 ± 0.02 µM, respectively. Moreover, both the gefitinib-treated group and nanobody-treated group had completely different proteome profiles. A total 6626 differentially expressed proteins were identified. PCA analysis revealed different proteome profiling in R9VH36 experiment. There were 8 proteins in R9VH36 that significantly exhibited opposite expression directions when compared to gefitinib. These proteins are involved in DNA-damage checkpoint processes. CONCLUSION: The proteomics explored those 6,626 proteins had different expressions between R9VH36 and gefitinib. There were 8 proteins in R9VH36 exhibited opposite expression direction when comparing to gefitinib. Our findings suggest that R9VH36 has the potential to be an alternative remedy for treating EGFR-positive colon cancer.

Discovery of potent antiproliferative agents from selected oxygen heterocycles as EGFR tyrosine kinase inhibitors from the U.S. National Cancer Institute database by in silico screening and bioactivity evaluation.

A similarity search was conducted on the U.S. Enhanced National Cancer Institute Database Browser 2.2 to find structures related to 1,5-dihydroxy-9H-xanthen-9-one, a previously established EGFR-TK inhibitor. Compounds were virtually screened and selected for bioactivity testing revealed 5 candidates, mostly displayed stronger antiproliferative activities than erlotinib with IC50 values between 0.95 and 17.71 µM against overexpressed EGFR-TK cancer cell lines: A431 and HeLa. NSC107228 displayed the strongest antiproliferative effects with IC50 values of 2.84 and 0.95 µM against A431 and HeLa cancer cell lines, respectively. Three compounds, NSC81111, NSC381467 and NSC114126 inhibited EGFR-TK with IC50 values between 0.15 and 30.18 nM. NSC81111 was the best inhibitor with IC50 = 0.15 nM. Molecular docking analysis of the 3 compounds predicted hydrogen bonding and hydrophobic interactions with key residues were important for the bioactivities observed. Furthermore, calculations of the physicochemical properties suggest the compounds are drug-like and are potentially active orally.

Dual role of azo compounds in inhibiting Plasmodium falciparum adenosine deaminase and hemozoin biocrystallization.

Protein-ligand (GOLD) docking of the NCI compounds into the ligand-binding site of Plasmodium falciparum adenosine deaminase (PfADA) identified three most active azo compounds containing 4-[(4-hydroxy-2-oxo-1H-quinolin-3-yl) moiety. These compounds showed IC50 of 3.7-15.4 µM against PfADA, as well as inhibited the growth of P. falciparum strains 3D7 (chloroquine (CQ)-sensitive) and K1 (CQ-resistant) with IC50 of 1.8-3.1 and 1.7-3.6 µM, respectively. The identified compounds have structures similar to the backbone structure (4-N-(7-chloroquinolin-4-yl)) in CQ, and NSC45545 could mimic CQ by inhibiting the bioformation of hemozoin in parasitic food vacuole. The amount of in situ hemozoin in the ring-stage parasite was determined using a combination of synchrotron transmission Fourier transform infrared microspectroscopy and Principal Component Analysis. Stretching of the C-O bond of hemozoin propionate group measured at 1220-1210 cm-1 in untreated intraerythrocytic P. falciparum strains 3D7 and K1 was disappeared following treatment with 1.85 and 1.74 µM NSC45545, similar to those treated with 0.02 and 0.13 µM CQ, respectively. These findings indicate a novel dual function of 4-[(4-hydroxy-2-oxo-1H-quinolin-3-yl) azo compounds in inhibiting both PfADA and in situ hemozoin biocrystallization. These lead compounds hold promise for further development of new antimalarial therapeutics that could delay the onset of parasitic drug resistance.

Mass spectrometry and synchrotron-FTIR microspectroscopy reveal the anti-inflammatory activity of Bua Bok extracts.

INTRODUCTION: Bua Bok or Centella asiatica (CA) is an Asian vegetable with anti-inflammatory benefits. Asiaticoside, asiatic acid, madecassoside and madecassic have been characterised as major active ingredients with a wide range of pharmacological advantages. In manufacturing processes, high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS) are used to routinely determine the active compounds in raw materials. OBJECTIVES: This research aims to explore anti-inflammatory properties, characterise metabolites and observe the biochemical changes of the inflammatory induced macrophages after pretreatment with the potential extracted fractions. METHODS: Bua Bok leaf extracts were prepared. Macrophages were pretreated with non-toxic fractions to determine the anti-inflammatory action. Tentative metabolites of effective fractions were identified by LC-MS. Synchrotron fourier-transform infrared (S-FTIR) microspectroscopy was utilised to observe the biochemical change of the lipopolysaccharide (LPS)-induced cells after pretreatment with potential fractions. RESULTS: Fractions of ethyl acetate, 30% and 100% ethanol highly increased the nitrile scavenging and suppressed the function of phospholipase A2 . Fractions of 70% and 100% ethanol strongly decreased nitric oxide production. The comparison of 39 chemical compounds was presented. The change of proteins was improved after pretreatment of macrophages with fraction 70% ethanol. Fraction of 100% ethanol revealed the lipid accumulation was lower than 70% ethanol and diclofenac. CONCLUSION: While the anti-inflammatory actions of 70% and 100% ethanol were similar. S-FTIR expressed they inhibited inflammatory response with the distinct features of biomolecules. The S-FTIR, LC-MS and biological assay confidently provided the efficient strategies to inform the advantage of herbal extract on cellular organisation instead of a single compound.

Quinoxalinones as A Novel Inhibitor Scaffold for EGFR (L858R/T790M/C797S) Tyrosine Kinase: Molecular Docking, Biological Evaluations, and Computational Insights.

Combating acquired drug resistance of EGFR tyrosine kinase (TK) is a great challenge and an urgent necessity in the management of non-small cell lung cancers. The advanced EGFR (L858R/T790M/C797S) triple mutation has been recently reported, and there have been no specific drugs approved for this strain. Therefore, our research aimed to search for effective agents that could impede the function of EGFR (L858R/T790M/C797S) TK by the integration of in silico and in vitro approaches. Our in-house quinoxalinone-containing compounds were screened through molecular docking and their biological activity was then verified by enzyme- and cell-based assay. We found that the four quinoxalinone-containing compounds including CPD4, CPD15, CPD16, and CPD21 were promising to be novel EGFR (L858R/T790M/C797S) TK inhibitors. The IC50 values measured by the enzyme-based assay were 3.04 ± 1.24 nM; 6.50 ± 3.02 nM,10.50 ± 1.10 nM; and 3.81 ± 1.80 nM, respectively, which are at a similar level to a reference drug; osimertinib (8.93 ± 3.01 nM). Besides that, they displayed cytotoxic effects on a lung cancer cell line (H1975) with IC50 values in the range of 3.47 to 79.43 µM. In this proposed study, we found that all screened compounds could interact with M793 at the hinge regions and two mutated residues including M790 and S797; which may be the main reason supporting the inhibitory activity in vitro. The structural dynamics revealed that the screened compounds have sufficient non-native contacts with surrounding amino acids and could be well-buried in the binding site's cleft. In addition, all predicted physicochemical parameters were favorable to be drug-like based on Lipinski's rule of five, and no extreme violation of toxicity features was found. Altogether, this study proposes a novel EGFR (L858R/T790M/C797S) TK inhibitor scaffold and provides a detailed understanding of compounds' recognition and susceptibility at the molecular level.

Biophysical Characterization of Novel DNA Aptamers against K103N/Y181C Double Mutant HIV-1 Reverse Transcriptase.

The human immunodeficiency virus type-1 Reverse Transcriptase (HIV-1 RT) plays a pivotal role in essential viral replication and is the main target for antiviral therapy. The anti-HIV-1 RT drugs address resistance-associated mutations. This research focused on isolating the potential specific DNA aptamers against K103N/Y181C double mutant HIV-1 RT. Five DNA aptamers showed low IC50 values against both the KY-mutant HIV-1 RT and wildtype (WT) HIV-1 RT. The kinetic binding affinity forms surface plasmon resonance of both KY-mutant and WT HIV-1 RTs in the range of 0.06-2 µM and 0.15-2 µM, respectively. Among these aptamers, the KY44 aptamer was chosen to study the interaction of HIV-1 RTs-DNA aptamer complex by NMR experiments. The NMR results indicate that the aptamer could interact with both WT and KY-mutant HIV-1 RT at the NNRTI drug binding pocket by inducing a chemical shift at methionine residues. Furthermore, KY44 could inhibit pseudo-HIV particle infection in HEK293 cells with nearly 80% inhibition and showed low cytotoxicity on HEK293 cells. These together indicated that the KY44 aptamer could be a potential inhibitor of both WT and KY-mutant HIV-RT.

Characterization of New DNA Aptamers for Anti-HIV-1 Reverse Transcriptase.

HIV-1 RT is a necessary enzyme for retroviral replication, which is the main target for antiviral therapy against AIDS. Effective anti-HIV-1 RT drugs are divided into two groups; nucleoside inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI), which inhibit DNA polymerase. In this study, new DNA aptamers were isolated as anti-HIV-1 RT inhibitors. The selected DNA aptamer (WT62) presented with high affinity and inhibition against wild-type (WT) HIV-1 RT and gave a KD value of 75.10±0.29 nM and an IC50 value of 84.81±8.54 nM. Moreover, WT62 decreased the DNA polymerase function of K103 N/Y181 C double mutant (KY) HIV-1 RT by around 80 %. Furthermore, the ITC results showed that this aptamer has small binding enthalpies with both WT and KY HIV-1 RTs through which the complex might form a hydrophobic interaction or noncovalent bonding. The NMR result also suggested that the WT62 aptamer could bind with both WT and KY mutant HIV-1 RTs at the connection domain.

Comparative sequence analysis of the accessory and nucleocapsid genes of feline coronavirus strains isolated from cats diagnosed with effusive feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a lethal infectious disease of domestic cats caused by feline coronavirus (FCoV) infection. Feline infectious peritonitis virus (FIPV) is a mutant type of FCoV that is characterized by causing fibrinous serositis with effusions in the pleural and abdominal cavities (wet form) and/or granulomatous-necrotizing inflammatory lesions in several organs (dry form). There have been numerous studies on FIP worldwide, whereas information about this disease in Thailand is still limited. Most studies involving molecular surveillance and evaluation of FCoV field strains have examined the genetic diversity of the spike and accessory ORF3c coding regions. Of these, the S gene is more divergent and is responsible for the two FCoV serotypes, while ORF3c harbors mutations that result either in early termination or destruction of the protein. In this study, we investigated the genetic diversity and genetic relationships among the current Thai and global FCoV strains in the accessory and nucleocapsid genes using a virus-specific PCR method. Comparative sequence analysis suggested that the Thai FCoV isolates were most closely related to strains reported in the Netherlands, the USA, and China. In the ORF3ab sequences, some Thai strains were more than 99% identical to the DF-2 prototype strain. Truncation of the 3a gene product was found in Thai FCoV strains of group 2. Amino acid deletions were observed in the N, ORF3c, and ORF7b proteins of Thai FCoV sequences. The accessory gene sequence divergence may be responsible for driving the periodic emergence and continued persistence of FCoVs in Thai domestic cat populations. Our findings provide updated information about the molecular characteristics of the accessory and nucleocapsid genes of FCoV strains in circulation that were not previously documented in this country.

Biosensors for the detection of organophosphate exposure by a new diethyl thiophosphate-specific aptamer.

OBJECTIVE: An aptamer specifically binding to diethyl thiophosphate (DETP) was constructed and incorporated in an optical sensor and electrochemical techniques to enable the specific measurement of DETP as a metabolite and a biomarker of organophosphate exposure. RESULTS: A DETP-bound aptamer was selected from the library using capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). A colorimetric method revealed that the aptamer had the highest affinity for DETP, with a mean Kd value (± SD) of 0.103 ± 0.014 µM. The docking results and changes in resistance showed that the selectivity of the aptamer for DETP was higher than that for the similar structures of dithiophosphate (DEDTP) and diethyl phosphate (DEP). The altered amplitude of cyclic voltammetry showed a linear range of DETP detection covering 0.0001-10 µg/ml with a limit of detection of 0.007 µg/ml. The recovery value of a real sample of pH 7 was 97.2%. CONCLUSIONS: The current method showed great promise in using the DETP-specific aptamer to detect the exposure history to organophosphates by measuring their metabolites, although degradation of organophosphate parent compounds might occur.

Probing the Anti-Cancer Potency of Sulfated Galactans on Cholangiocarcinoma Cells Using Synchrotron FTIR Microspectroscopy, Molecular Docking, and In Vitro Studies.

Sulfated galactans (SG) isolated from red alga Gracilaria fisheri have been reported to inhibit the growth of cholangiocarcinoma (CCA) cells, which was similar to the epidermal growth factor receptor (EGFR)-targeted drug, cetuximab. Herein, we studied the anti-cancer potency of SG compared to cetuximab. Biological studies demonstrated SG and cetuximab had similar inhibition mechanisms in CCA cells by down-regulating EGFR/ERK pathway, and the combined treatment induced a greater inhibition effect. The molecular docking study revealed that SG binds to the dimerization domain of EGFR, and this was confirmed by dimerization assay, which showed that SG inhibited ligand-induced EGFR dimer formation. Synchrotron FTIR microspectroscopy was employed to examine alterations in cellular macromolecules after drug treatment. The SR-FTIR-MS elicited similar spectral signatures of SG and cetuximab, pointing towards the bands of RNA/DNA, lipids, and amide I vibrations, which were inconsistent with the changes of signaling proteins in CCA cells after drug treatment. Thus, this study demonstrates the underlined anti-cancer mechanism of SG by interfering with EGFR dimerization. In addition, we reveal that FTIR signature spectra offer a useful tool for screening anti-cancer drugs' effect.