Efficiency of membrane fusion inhibitors on different hemagglutinin subtypes: insight from a molecular dynamics simulation perspective.

The challenge in vaccine development, along with drug resistance issues, has encouraged the search for new anti-influenza drugs targeting different viral proteins. Hemagglutinin (HA) glycoprotein, crucial in the viral replication cycle, has emerged as a promising therapeutic target. CBS1117 and JNJ4796 were reported to exhibit similar potencies against infectious group 1 influenza, which included H1 and H5 HAs; however, their potencies were significantly reduced against group 2 HA. This study aims to explore the molecular binding mechanisms and group specificity of these fusion inhibitors against both group 1 (H5) and group 2 (H3) HA influenza viruses using molecular dynamics simulations. CBS1117 and JNJ4796 exhibit stronger interactions with key residues within the H5 HA binding pocket compared to H3-ligand complexes. Hydrogen bonding and hydrophobic interactions involving residues, such as H381, Q401, T3251 (H5-CBS1117), T3181 (H5-JNJ4796), W212, I452, V482, and V522 predominantly contribute to stabilizing H5-ligand systems. In contrast, these interactions are notably weakened in H3-inhibitor complexes. Predicted protein-ligand binding free energies align with experimental data, indicating CBS1117 and JNJ4796's preference for heterosubtypic group 1 HA binding. Understanding the detailed atomistic mechanisms behind the varying potencies of these inhibitors against the two HA groups can significantly contribute to the development and optimization of effective HA fusion inhibitors. To accomplish this, the knowledge of the transition of HA from its pre- to post-fusion states, the molecular size of ligands, and their potential binding regions, could be carefully considered.Communicated by Ramaswamy H. Sarma.

Naturally occurring quercetin and myricetin as potent inhibitors for human ectonucleotide pyrophosphatase/phosphodiesterase 1.

Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation of several fundamental pathophysiological processes. In this study, Kyoto Green, a rapid chemical sensor of pyrophosphate, was employed to screen for effective ENPP1 inhibitors among five representative flavonoids (quercetin, myricetin, morin, kaempferol, and quercetin-3-glucoside), five nucleosides (adenosine, guanosine, inosine, uridine, and cytidine), and five deoxynucleosides (2'- and 3'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyuridine). Conventional colorimetric, fluorescence, and bioluminescence assays revealed that ENPP1 was effectively inhibited by quercetin (Ki ~ 4 nM) and myricetin (Ki ~ 32 nM) when ATP was used as a substrate at pH 7.4. In silico analysis indicated that the presence of a chromone scaffold, particularly one containing a hydroxyl group at the 3' position on the B ring, may promote binding to the active site pocket of ENPP1 and enhance inhibition. This study demonstrated that the naturally derived quercetin and myricetin could effectively inhibit ENPP1 enzymatic activity and may offer health benefits in arthritis management.

Molecular binding of different classes of organophosphates to methyl parathion hydrolase from Ochrobactrum species.

Methyl parathion hydrolase (MPH) is an enzyme of the metallo-ß-lactamase superfamily, which hydrolyses a wide range of organophosphates (OPs). Recently, MPH has attracted attention as a promising enzymatic bioremediator. The crystal structure of MPH enzyme shows a dimeric form, with each subunit containing a binuclear metal ion center. MPH also demonstrates metal ion-dependent selectivity patterns. The origins of these patterns remain unclear but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. We aimed to investigate and compare the binding of different OP pesticides to MPH with cobalt(II) metal ions. In this study, MPH was modeled from Ochrobactrum sp. with different OP pesticides bound, including methyl paraoxon and dichlorvos and profenofos. The docked structures for each substrate optimized by DFT calculation were selected and subjected to atomistic molecular dynamics simulations for 500 ns. It was found that alpha metal ions did not coordinate with all the pesticides. Rather, the pesticides coordinated with less buried beta metal ions. It was also observed that the coordination of beta metal ions was perturbed to accommodate the pesticides. The binding free energy calculations and structure-based pharmacophore model revealed that all the three substrates could bind well at the active site. However, profenofos exhibit a stronger binding affinity to MPH in comparison to the other two substrates. Therefore, our findings provide molecular insight on the binding of different OP pesticides which could help us design the enzyme for OP pesticides degradation.

Exploring Major Flavonoid Phytochemicals from Nelumbo nucifera Gaertn. as Potential Skin Anti-Aging Agents: In Silico and In Vitro Evaluations.

Nelumbo nucifera Gaertn., an aquatic medicinal plant (Nelumbonaceae family), has a history of use in traditional medicine across various regions. Our previous study demonstrated the skin anti-aging potential of its stamen ethanolic extract by effectively inhibiting collagenase and tyrosinase enzymes. While the major constituents of this extract are well documented, there is a lack of research on the individual compounds' abilities to inhibit skin aging enzymes. Therefore, this study aimed to evaluate the anti-aging potential of the primary flavonoids found in N. nucifera using both in silico and in vitro approaches. Our initial step involved molecular docking to identify compounds with the potential to inhibit collagenase, elastase, and tyrosinase. Among the seven flavonoids studied, kaempferol-3-O-robinobioside (Kae-3-Rob) emerged as the most promising candidate, exhibiting the highest docking scores for three skin aging-related enzymes. Subsequent enzyme-based inhibition assays confirmed that Kae-3-Rob displayed robust inhibitory activity against collagenase (58.24 ± 8.27%), elastase (26.29 ± 7.16%), and tyrosinase (69.84 ± 6.07%). Furthermore, we conducted extensive 200-ns molecular dynamics (MD) simulations, revealing the stability of the complexes formed between Kae-3-Rob and each enzyme along the MD simulation time. MM/PBSA-based binding free energy calculations indicated the considerably stronger binding affinity of Kae-3-Rob for collagenase and tyrosinase compared to elastase, which was related to the greater percentage of hydrogen bond occupations. These computational findings were consistent with the relatively high inhibitory activity of Kae-3-Rob against collagenase and tyrosinase observed in our in vitro experiment. In conclusion, the results obtained from this comprehensive study suggest that Kae-3-Rob, a key flavonoid from N. nucifera, holds significant potential as a source of bioactive compounds for anti-aging cosmeceutical and other phytopharmaceutical application.

Structural Dynamics of the Precatalytic State of Human Cytochrome c upon T28C, G34C, and A50C Mutations: A Molecular Dynamics Simulation Perspective.

The native structure of cytochrome c (cytc) contains hexacoordinate heme iron with His18 and Met80 residues ligated at the axial sites. Mutations of cytc at Ω-loops have been investigated in modulating the peroxidase activity and, hence, related to the initiation of the apoptotic pathway. Our previous experimental data reported on the peroxidase activity of the cysteine-directed mutants at different parts of the Ω-loop of human cytc (hCytc), that is, T28C, G34C, and A50C. In this work, we performed 1 µs molecular dynamics (MD) simulations to elucidate the detailed structural and dynamic changes upon these mutations, particularly at the proximal Ω-loop. The structures of hCytc were modeled in the hexacoordinated form, which was referred to as the "precatalytic state". The results showed that the structural features of the G34C mutant were more distinctive than those of other mutants. G34C mutation caused local destabilization and flexibility at the proximal Ω-loop (residues 12-28) and an extended distance between this Ω-loop region and heme iron. Besides, analysis of the orientation of the Arg38 side chain of the G34C mutant revealed the Arg38 conformer facing away from the heme iron. The obtained MD results also suggested structural diversity of the precatalytic states for the three hCytc mutants, specifically the effect of G34C mutation on the flexibility of the proximal Ω-loops. Therefore, our MD simulations combined with previous experimental data provide detailed insights into the structural basis of hCytc that could contribute to its pro-apoptotic function.

In Vitro and In Silico Study on the Molecular Encapsulation of α-Tocopherol in a Large-Ring Cyclodextrin.

α-tocopherol is the physiologically most active form of vitamin E, with numerous biological activities, such as significant antioxidant activity, anticancer capabilities, and anti-aging properties. However, its low water solubility has limited its potential use in the food, cosmetic, and pharmaceutical industries. One possible strategy for addressing this issue is the use of a supramolecular complex with large-ring cyclodextrins (LR-CDs). In this study, the phase solubility of the CD26/α-tocopherol complex was investigated to assess the possible ratios between host and guest in the solution phase. Next, the host-guest association of the CD26/α-tocopherol complex at different ratios of 1:2, 1:4, 1:6, 2:1, 4:1, and 6:1 was studied by all-atom molecular dynamics (MD) simulations. At 1:2 ratio, two α-tocopherol units interact spontaneously with CD26, forming an inclusion complex, as supported by the experimental data. In the 2:1 ratio, a single α-tocopherol unit was encapsulated by two CD26 molecules. In comparison, increasing the number of α-tocopherol or CD26 molecules above two led to self-aggregation and consequently limited the solubility of α-tocopherol. The computational and experimental results indicate that a 1:2 ratio could be the most suitable stoichiometry to use in the CD26/α-tocopherol complex to improve α-tocopherol solubility and stability in inclusion complex formation.

Insights into binding molecular mechanism of hemagglutinin H3N2 of influenza virus complexed with arbidol and its derivative: A molecular dynamics simulation perspective.

Recently, the H3N2 influenza outbreak has caused serious global public health concern for future control of the next influenza pandemic. Since using current anti-influenza drugs targeting neuraminidase (oseltamivir and zanamivir) and the proton M2 channel (amantadine and rimantadine) leads to drug resistance, it is essential to seek new anti-viral agents that act on additional viral targets. Hemagglutinin (HA), a glycoprotein embedded in the viral surface and playing a critical role in influenza the viral replication cycle has become an attractive target. This work investigates the molecular binding mechanism of HA H3N2 of influenza virus complexed with the fusion inhibitor, arbidol and its derivative (der-arbidol), by means of molecular dynamics simulation. The result showed that the arbidol derivative could form many and strong hydrogen bonds with the HA surrounding amino acids comprising GLU1032(1), LYS3071(1) and LYS3102(1) while arbidol makes this type of interaction with only LYS582(1). The introduction of hydroxyl group at the meta-position of the thiophenol ring was detected to replace the nearby water molecule, thus allowing the direct hydrogen bond formation between der-arbidol and GLU1032(1) of HA residue. Furthermore, the salt bridge networks established among residues GLU572(1)···ARG542(1)···GLU972(2) were considerably more stable in HA-Der-arbidol than that found in HA-Arbidol. The predicted protein-ligand binding free energies were in agreement with experimental data indicating that der-arbidol exhibits higher inhibitory potency against HA H3N2 of influenza virus. Detailed information could be useful for further designing and optimizing HA fusion inhibitors with improved efficiency.

Discovery of C-12 dithiocarbamate andrographolide analogues as inhibitors of SARS-CoV-2 main protease: In vitro and in silico studies.

A global crisis of coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted millions of people's lives throughout the world. In parallel to vaccine development, identifying potential antiviral agents against SARS-CoV-2 has become an urgent need to combat COVID-19. One of the most attractive drug targets for discovering anti-SARS-CoV-2 agents is the main protease (Mpro), which plays a pivotal role in the viral life cycle. This study aimed to elucidate a series of twenty-one 12-dithiocarbamate-14-deoxyandrographolide analogues as SARS-CoV-2 Mpro inhibitors using in vitro and in silico studies. These compounds were initially screened for the inhibitory activity toward SARS-CoV-2 Mpro by in vitro enzyme-based assay. We found that compounds 3 k, 3 l, 3 m and 3 t showed promising inhibitory activity against SARS-CoV-2 Mpro with >50% inhibition at 10 µM. Afterward, the binding mode of each compound in the active site of SARS-CoV-2 Mpro was explored by molecular docking. The optimum docked complexes were then chosen and subjected to molecular dynamic (MD) simulations. The MD results suggested that all studied complexes were stable along the simulation time, and most of the compounds could fit well with the SARS-CoV-2 Mpro active site, particularly at S1, S2 and S4 subsites. The per-residue decomposition free energy calculations indicated that the hot-spot residues essential for ligand binding were T25, H41, C44, S46, M49, C145, H163, M165, E166, L167, D187, R188, Q189 and T190. Therefore, the obtained information from the combined experimental and computational techniques could lead to further optimization of more specific and potent andrographolide analogues toward SARS-CoV-2 Mpro.

Exploring of paritaprevir and glecaprevir resistance due to A156T mutation of HCV NS3/4A protease: molecular dynamics simulation study.

Hepatitis C virus (HCV) NS3/4A serine protease is a promising drug target for the discovery of anti-HCV drugs. However, its amino acid mutations, particularly A156T, commonly lead to rapid emergence of drug resistance. Paritaprevir and glecaprevir, the newly FDA-approved HCV drugs, exhibit distinct resistance profiles against the A156T mutation of HCV NS3/4A serine protease. To illustrate their different molecular resistance mechanisms, molecular dynamics simulations and binding free energy calculations were carried out on the two compounds complexed with both wild-type (WT) and A156T variants of HCV NS3/4A protease. QM/MM-GBSA-based binding free energy calculations revealed that the binding affinities of paritaprevir and glecaprevir towards A156T NS3/4A were significantly reduced by ∼4 kcal/mol with respect to their WT complexes, which were in line with the experimental resistance folds. Moreover, the relatively weak intermolecular interactions with amino acids such as H57, R155, and T156 of NS3 protein, the steric effect and the destabilized protein binding surface, which is caused by the loss of salt bridge between R123 and D168, are the main contributions for the higher fold-loss in potency of glecaprevir due to A156T mutation. An insight into the difference of molecular mechanism of drug resistance against the A156T substitution among the two classes of serine protease inhibitors could be useful for further optimization of new generation HCV NS3/4A inhibitors with enhanced inhibitory potency.Communicated by Ramaswamy H. Sarma.

Molecular insights on the conformational dynamics of a P76C mutant of human cytochrome c and the enhancement on its peroxidase activity.

In apoptotic pathway, the interaction of Cytochrome c (Cytc) with cardiolipin in vivo is a key process to induce peroxidase activity of Cytc and trigger the release of Cytc in the inner mitochondria into cytosol. The peroxidase active form of Cytc occurs due to local conformational changes that support the opening of the heme crevice and the loss of an axial ligand between Met80 and heme Fe. Structural adjustments at the Ω-loop segments of Cytc are required for such process. To study the role of the distal Ω-loop segments comprising residues 71-85 in human Cytc (hCytc), we investigated a cysteine mutation at Pro76, one of the highly conserved residues in this loop. The effect of P76C mutant was explored by the combination of experimental characterizations and molecular dynamics (MD) simulations. The peroxidase activity of the P76C mutant was found to be significantly increased by ∼13 folds relative to the wild type. Experimental data on global denaturation, alkaline transition, heme bleaching, and spin-labeling Electron Spin Resonance were in good agreement with the enhancement of peroxidase activity. The MD results of hCytc in the hexacoordinate form suggest the important changes in P76C mutant occurred due to the unfolding at the central Ω-loop (residues 40-57), and the weakening of H-bond between Tyr67 and Met80. Whereas the experimental data implied that the P76C mutant tend to be in equilibrium between the pentacoordinate and hexacoordinate forms, the MD and experimental information are complementary and were used to support the mechanisms of peroxidase active form of hCytc.

Titania Nanosheet Generates Peroxynitrite-Dependent S-Nitrosylation and Enhances p53 Function in Lung Cancer Cells.

Metal nanomaterials can enhance the efficacy of current cancer therapies. Here, we show that Ti0.8O2 nanosheets cause cytotoxicity in several lung cancer cells but not in normal cells. The nanosheet-treated cells showed certain apoptosis characteristics. Protein analysis further indicated the activation of the p53-dependent death mechanism. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses revealed the cellular uptake of the nanosheets and the induction of cell morphological change. The nanosheets also exhibited a substantial apoptosis effect on drug-resistant metastatic primary lung cancer cells, and it was found that the potency of the nanosheets was dramatically higher than standard drugs. Ti0.8O2 nanosheets induce apoptosis through a molecular mechanism involving peroxynitrite (ONOO-) generation. As peroxynitrite is known to be a potent inducer of S-nitrosylation, we further found that the nanosheets mediated the S-nitrosylation of p53 at C182, resulting in higher protein-protein complex stability, and this was likely to induce the surrounding residues, located in the interface region, to bind more strongly to each other. Molecular dynamics analysis revealed that S-nitrosylation stabilized the p53 dimer with a ΔGbindresidue of <-1.5 kcal/mol. These results provide novel insight on the apoptosis induction effect of the nanosheets via a molecular mechanism involving S-nitrosylation of the p53 protein, emphasizing the mechanism of action of nanomaterials for cancer therapy.

Bibenzyl analogue DS-1 inhibits MDM2-mediated p53 degradation and sensitizes apoptosis in lung cancer cells.

BACKGROUND: Lung cancer is a leading fatal malignancy due to the high incidence of treatment failure. Dysfunction of the tumor suppressor p53 contributes to cancer initiation, progression, and therapeutic resistance. Targeting MDM2, a negative regulator of p53, has recently attracted interest in cancer drug research as it may restore tumor suppressive function. PURPOSE: The present study aimed to investigate the effect of 3,4-dihydroxy-5,4'-dimethoxybibenzyl (DS-1) on targeting MDM2 and restoring p53 function in lung cancer cells. METHODS: The efficacy of DS-1 alone or in combination with cisplatin in lung cancer cells was determined by MTT, nuclear staining, and annexin V/PI assay. The expression of apoptosis-related proteins was determined by western blot analysis. To evaluate the role of DS-1 on the stabilization and degradation of p53, cycloheximide chasing assay and immunoprecipitation were conducted, and the active form of p53 was investigated by immunofluorescent staining assay. To confirm and demonstrate the site interaction between DS-1 and the MDM2 protein, in silico computational analysis was performed. RESULTS: DS-1 exhibited a cytotoxic effect and sensitized lung cancer cells to cisplatin-induced apoptosis. DS-1 caused a significant increase in the cellular level of p53 protein, while the active form of p53 (phosphorylation at Ser15) was unaltered. DS-1 treatment in combination with cisplatin could enhance activated p-p53 (Ser15) and p53 downstream signaling (Bax, Bcl-2, and Akt), leading to a higher level of apoptosis. Immunoprecipitation analysis revealed that DS-1 decreased the p53-ubiquitin complex, a prerequisite step in p53 proteasomal degradation. Molecular docking simulation further evidenced that DS-1 interacts with MDM2 within the p53-binding domain by carbon-hydrogen bond interaction at Lys27, π-alkyl interactions at Ile37 and Leu30, and van der Waals interactions at Ile75, Val51, Val69, Phe67, Met38, Tyr43, Gly34, and Phe31. Treatment by DS-1 and cisplatin in patient-derivated primary lung cancer cells showed consistent effects by increasing cisplatin sensitivity. CONCLUSIONS: Our findings provide evidence that DS-1 is an MDM2 inhibitor and its underlying mechanism involves MDM2 binding and p53 induction, which may benefit the development of this compound for lung cancer treatment.

Structural Basis of Specific Glucoimidazole and Mannoimidazole Binding by Os3BGlu7.

ß-Glucosidases and ß-mannosidases hydrolyze substrates that differ only in the epimer of the nonreducing terminal sugar moiety, but most such enzymes show a strong preference for one activity or the other. Rice Os3BGlu7 and Os7BGlu26 ß-glycosidases show a less strong preference, but Os3BGlu7 and Os7BGlu26 prefer glucosides and mannosides, respectively. Previous studies of crystal structures with glucoimidazole (GIm) and mannoimidazole (MIm) complexes and metadynamic simulations suggested that Os7BGlu26 hydrolyzes mannosides via the B2,5 transition state (TS) conformation preferred for mannosides and glucosides via their preferred 4H3/4E TS conformation. However, MIm is weakly bound by both enzymes. In the present study, we found that MIm was not bound in the active site of crystallized Os3BGlu7, but GIm was tightly bound in the -1 subsite in a 4H3/4E conformation via hydrogen bonds with the surrounding residues. One-microsecond molecular dynamics simulations showed that GIm was stably bound in the Os3BGlu7 active site and the glycone-binding site with little distortion. In contrast, MIm initialized in the B2,5 conformation rapidly relaxed to a E3/4H3 conformation and moved out into a position in the entrance of the active site, where it bound more stably despite making fewer interactions. The lack of MIm binding in the glycone site in protein crystals and simulations implies that the energy required to distort MIm to the B2,5 conformation for optimal active site residue interactions is sufficient to offset the energy of those interactions in Os3BGlu7. This balance between distortion and binding energy may also provide a rationale for glucosidase versus mannosidase specificity in plant ß-glycosidases.

Delivery of Alpha-Mangostin Using Cyclodextrins through a Biological Membrane: Molecular Dynamics Simulation.

α-Mangostin (MGS) exhibits various pharmacological activities, including antioxidant, anticancer, antibacterial, and anti-inflammatory properties. However, its low water solubility is the major obstacle for its use in pharmaceutical applications. To increase the water solubility of MGS, complex formation with beta-cyclodextrins (ßCDs), particularly with the native ßCD and/or its derivative 2,6-dimethyl-ß-CD (DMßCD) is a promising technique. Although there have been several reports on the adsorption of ßCDs on the lipid bilayer, the release of the MGS/ßCDs inclusion complex through the biological membrane remains unclear. In this present study, the release the MGS from the two different ßCDs (ßCD and DMßCD) across the lipid bilayer was investigated. Firstly, the adsorption of the free MGS, free ßCDs, and inclusion complex formation was studied by conventional molecular dynamics simulation. The MGS in complex with those two ßCDs was able to spontaneously release free MGS into the inner membrane. However, both MGS and DMßCD molecules potentially permeated into the deeper region of the interior membrane, whereas ßCD only adsorbed at the outer membrane surface. The interaction between secondary rim of ßCD and the 1-palmitoeyl-2-oleoyl-glycero-3-phosphocholine (POPC) phosphate groups showed the highest number of hydrogen bonds (up to 14) corresponding to the favorable location of ßCD on the POPC membrane. Additionally, the findings suggested that electrostatic energy was the main driving force for ßCD adsorption on the POPC membrane, while van der Waals interactions played a predominant role in DMßCD adsorption. The release profile of MGS from the ßCDs pocket across the lipid bilayer exhibited two energy minima along the reaction coordinate associated with the permeation of the MGS molecule into the deeper region of the POPC membrane.

Why Are Lopinavir and Ritonavir Effective against the Newly Emerged Coronavirus 2019? Atomistic Insights into the Inhibitory Mechanisms.

Since the emergence of a novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported from Wuhan, China, neither a specific vaccine nor an antiviral drug against SARS-CoV-2 has become available. However, a combination of two HIV-1 protease inhibitors, lopinavir and ritonavir, has been found to be effective against SARS-CoV, and both drugs could bind well to the SARS-CoV 3C-like protease (SARS-CoV 3CLpro). In this work, molecular complexation between each inhibitor and SARS-CoV-2 3CLpro was studied using all-atom molecular dynamics simulations, free energy calculations, and pair interaction energy analyses based on MM/PB(GB)SA and FMO-MP2/PCM/6-31G* methods. Both anti-HIV drugs interacted well with the residues at the active site of SARS-CoV-2 3CLpro. Ritonavir showed a somewhat higher number atomic contacts, a somewhat higher binding efficiency, and a somewhat higher number of key binding residues compared to lopinavir, which correspond with the slightly lower water accessibility at the 3CLpro active site. In addition, only ritonavir could interact with the oxyanion hole residues N142 and G143 via the formation of two hydrogen bonds. The interactions in terms of electrostatics, dispersion, and charge transfer played an important role in the drug binding. The obtained results demonstrated how repurposed anti-HIV drugs could be used to combat COVID-19.

Structure-Activity Relationships and Molecular Docking Analysis of Mcl-1 Targeting Renieramycin T Analogues in Patient-derived Lung Cancer Cells.

Myeloid cell leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2) proteins are promising targets for cancer therapy. Here, we investigated the structure-activity relationships (SARs) and performed molecular docking analysis of renieramycin T (RT) and its analogues and identified the critical functional groups of Mcl-1 targeting. RT have a potent anti-cancer activity against several lung cancer cells and drug-resistant primary cancer cells. RT mediated apoptosis through Mcl-1 suppression and it also reduced the level of Bcl-2 in primary cells. For SAR study, five analogues of RT were synthesized and tested for their anti-cancer and Mcl-1- and Bcl-2-targeting effects. Only two of them (TM-(-)-18 and TM-(-)-4a) exerted anti-cancer activities with the loss of Mcl-1 and partly reduced Bcl-2, while the other analogues had no such effects. Specific cyanide and benzene ring parts of RT's structure were identified to be critical for its Mcl-1-targeting activity. Computational molecular docking indicated that RT, TM-(-)-18, and TM-(-)-4a bound to Mcl-1 with high affinity, whereas TM-(-)-45, a compound with a benzene ring but no cyanide for comparison, showed the lowest binding affinity. As Mcl-1 helps cancer cells evading apoptosis, these data encourage further development of RT compounds as well as the design of novel drugs for treating Mcl-1-driven cancers.

Understanding of the drug resistance mechanism of hepatitis C virus NS3/4A to paritaprevir due to D168N/Y mutations: A molecular dynamics simulation perspective.

Hepatitis C virus (HCV) NS3/4A protease is an attractive target for the development of antiviral therapy. However, the evolution of antiviral drug resistance is a major problem for treatment of HCV infected patients. Understanding of drug-resistance mechanisms at molecular level is therefore very important for the guidance of further design of antiviral drugs with high efficiency and specificity. Paritaprevir is a potent inhibitor against HCV NS3/4A protease genotype 1a. Unfortunately, this compound is highly susceptible to the substitution at D168 in the protease. In this work, molecular dynamics simulations of paritaprevir complexed with wild-type (WT) and two mutated strains (D168 N and D168Y) were carried out. Due to such mutations, the inhibitor-protein hydrogen bonding between them was weakened and the salt-bridge network among residues R123, R155 and D168 responsible for inhibitor binding was disrupted. Moreover, the per-residue free energy decomposition suggested that the main contributions from key residues such as Q80, V132, K136, G137 and R155 were lost in the D168 N/Y mutations. These lead to a lower binding affinity of paritaprevir for D168 N/Y variants of the HCV NS3/4A protease, consistent with the experimental data. This detailed information could be useful for further design of high potency anti-HCV NS3/4A inhibitors.

Binding recognition of substrates in NS2B/NS3 serine protease of Zika virus revealed by molecular dynamics simulations.

Zika virus (ZIKV) has become a global public health concern. The recent epidemiological data has revealed a possible association of ZIKV infection with more serious complications, particularly for Guillain-Barré syndrome in adults and microcephaly in newborn children. Till now, there is no vaccine or effective drug commercially available to combat with ZIKV infection. An attractive drug target for the ZIKV treatment is the NS2B/NS3 serine protease, which is essential for viral polyprotein processing. Herein, classical molecular dynamics (MD) simulations were performed on the ZIKV NS2B/NS3 serine protease in complex with four peptide substrates to investigate the binding recognition and protein-substrate interactions. The obtained results indicate that the P1 and P2 positions of the substrate play a significant role in binding with the protease enzyme, while the P3 and P4 positions show a minor contribution in binding interaction. Moreover, the binding free energy calculation based on the MM/PBSA method suggests that among the four similar peptide substrates, the peptide Ac-D-RKOR-ACC displays the strongest binding affinity towards the ZIKV protease due to the high energy contribution at the S2 subsite particularly for the NS3 residue D75 with the P2(O) residue of this substrate, which is in line with the experimental data. Thus, the information derived from MD simulations presented here would be useful for the design of potent protease inhibitors.

The reaction mechanism of Zika virus NS2B/NS3 serine protease inhibition by dipeptidyl aldehyde: a QM/MM study.

Zika virus (ZIKV) infection has become a global public health problem, associated with microcephaly in newborns and Guillain-Barré syndrome in adults. Currently, there are no commercially available anti-ZIKV drugs. The viral protease NS2B/NS3, which is involved in viral replication and maturation, is a potential drug target. Peptidomimetic aldehyde inhibitors bind covalently to the catalytic S135 of the NS3 protease. Here, we apply hybrid quantum mechanics/molecular mechanics (QM/MM) free-energy simulations at the PDDG-PM3/ff14SB level to investigate the inhibition mechanism of the ZIKV protease by a dipeptidyl aldehyde inhibitor (acyl-KR-aldehyde). The results show that proton transfer from the catalytic S135 to H51 occurs in concert with nucleophilic addition on the aldehyde warhead by S135. The anionic covalent complex between the dipeptidyl aldehyde and the ZIKV protease is analogous to the tetrahedral intermediate for substrate hydrolysis. Spontaneous protonation by H51 forms the hemiacetal. In addition, we use correlated ab initio QM/MM potential energy path calculations at levels up to LCCSD(T)/(aug)-cc-pVTZ to obtain accurate potential energy profiles of the reaction, which also support a concerted mechanism. These results provide detailed insight into the mechanism of ZIKV protease inhibition by a peptidyl aldehyde inhibitor, which will guide in the design of inhibitors.

Low susceptibility of asunaprevir towards R155K and D168A point mutations in HCV NS3/4A protease: A molecular dynamics simulation.

Hepatitis C has become an important health problem that requires expensive treatment and leads to liver tumorigenesis. Hepatitis C virus (HCV), which is the main cause of hepatitis C, has a high mutation rate due to the lack of proofreading activity of the RNA polymerase enzyme. The NS3/4A serine protease is an important target for anti-HCV drug discovery and development because of its crucial role in the cleavage of the polypeptides involved in viral replication. In the present study, all-atom molecular dynamics simulation was performed to elucidate the effect of the single point mutations R155K and D168A in the HCV genotype 1 NS3/4A protease on the structural dynamics, molecular interactions and susceptibility of asunaprevir (ASV), a second-generation NS3/4A protease inhibitor. Principal component analysis indicated that these two mutations converted the direction of motion of residues 123, 155 and 168 in the binding pocket to significantly point outwards from ASV, resulting in a loss of the hydrogen bond network of residues R123···R155···D168. The free energy calculations based on different semiempirical QM/MM-GBSA methods revealed that the binding affinity of ASV with the two mutant forms of the NS3/4A protease was significantly decreased in the order of wild-type < R155K < D168A. This work provided useful structural information regarding the atomistic understanding of acquired drug resistance against ASV caused by the R155K and D168A mutations.