Reactive Metabolites from Thiazole-Containing Drugs: Quantum Chemical Insights into Biotransformation and Toxicity.

Drugs containing thiazole and aminothiazole groups are known to generate reactive metabolites (RMs) catalyzed by cytochrome P450s (CYPs). These RMs can covalently modify essential cellular macromolecules and lead to toxicity and induce idiosyncratic adverse drug reactions. Molecular docking and quantum chemical hybrid DFT study were carried out to explore the molecular mechanisms involved in the biotransformation of thiazole (TZ) and aminothiazole (ATZ) groups leading to RM epoxide, S-oxide, N-oxide, and oxaziridine. The energy barrier required for the epoxidation is 13.63 kcal/mol, that is lower than that of S-oxidation, N-oxidation, and oxaziridine formation (14.56, 17.90, and 20.20, kcal/mol respectively). The presence of the amino group in ATZ further facilitates all the metabolic pathways, for example, the barrier for the epoxidation reaction is reduced by ∼2.5 kcal/mol. Some of the RMs/their isomers are highly electrophilic and tend to form covalent bonds with nucleophilic amino acids, finally leading to the formation of metabolic intermediate complexes (MICs). The energy profiles of these competitive pathways have also been explored.

Ubiquitin Ligase SMURF2 Interacts with Filovirus VP40 and Promotes Egress of VP40 VLPs.

Filoviruses Ebola (EBOV) and Marburg (MARV) are devastating high-priority pathogens capable of causing explosive outbreaks with high human mortality rates. The matrix proteins of EBOV and MARV, as well as eVP40 and mVP40, respectively, are the key viral proteins that drive virus assembly and egress and can bud independently from cells in the form of virus-like particles (VLPs). The matrix proteins utilize proline-rich Late (L) domain motifs (e.g., PPxY) to hijack specific host proteins that contain WW domains, such as the HECT family E3 ligases, to facilitate the last step of virus-cell separation. We identified E3 ubiquitin ligase Smad Ubiquitin Regulatory Factor 2 (SMURF2) as a novel interactor with VP40 that positively regulates VP40 VLP release. Our results show that eVP40 and mVP40 interact with the three WW domains of SMURF2 via their PPxY motifs. We provide evidence that the eVP40-SMURF2 interaction is functional as the expression of SMURF2 positively regulates VLP egress, while siRNA knockdown of endogenous SMURF2 decreases VLP budding compared to controls. In sum, our identification of novel interactor SMURF2 adds to the growing list of identified host proteins that can regulate PPxY-mediated egress of VP40 VLPs. A more comprehensive understanding of the modular interplay between filovirus VP40 and host proteins may lead to the development of new therapies to combat these deadly infections.

Angiomotin Counteracts the Negative Regulatory Effect of Host WWOX on Viral PPxY-Mediated Egress.

Filoviridae family members Ebola (EBOV) and Marburg (MARV) viruses and Arenaviridae family member Lassa virus (LASV) are emerging pathogens that can cause hemorrhagic fever and high rates of mortality in humans. A better understanding of the interplay between these viruses and the host will inform about the biology of these pathogens, and may lead to the identification of new targets for therapeutic development. Notably, expression of the filovirus VP40 and LASV Z matrix proteins alone drives assembly and egress of virus-like particles (VLPs). The conserved PPxY Late (L) domain motifs in the filovirus VP40 and LASV Z proteins play a key role in the budding process by mediating interactions with select host WW-domain containing proteins that then regulate virus egress and spread. To identify the full complement of host WW-domain interactors, we utilized WT and PPxY mutant peptides from EBOV and MARV VP40 and LASV Z proteins to screen an array of GST-WW-domain fusion proteins. We identified WW domain-containing oxidoreductase (WWOX) as a novel PPxY-dependent interactor, and we went on to show that full-length WWOX physically interacts with eVP40, mVP40 and LASV Z to negatively regulate egress of VLPs and of a live VSV/Ebola recombinant virus (M40). Interestingly, WWOX is a versatile host protein that regulates multiple signaling pathways and cellular processes via modular interactions between its WW-domains and PPxY motifs of select interacting partners, including host angiomotin (AMOT). Notably, we demonstrated recently that expression of endogenous AMOT not only positively regulates egress of VLPs, but also promotes egress and spread of live EBOV and MARV. Toward the mechanism of action, we show that the competitive and modular interplay among WWOX-AMOT-VP40/Z regulates VLP and M40 virus egress. Thus, WWOX is the newest member of an emerging group of host WW-domain interactors (e.g. BAG3; YAP/TAZ) that negatively regulate viral egress. These findings further highlight the complex interplay of virus-host PPxY/WW-domain interactions and their potential impact on the biology of both the virus and the host during infection.Author Summary Filoviruses (Ebola [EBOV] and Marburg [MARV]) and arenavirus (Lassa virus; LASV) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we identified host WW-domain containing protein WWOX as a novel interactor with VP40 and Z, and showed that WWOX inhibited budding of VP40/Z virus-like particles (VLPs) and live virus in a PPxY/WW-domain dependent manner. Our findings are important to the field as they expand the repertoire of host interactors found to regulate PPxY-mediated budding of RNA viruses, and further highlight the competitive interplay and modular virus-host interactions that impact both the virus lifecycle and the host cell.

Virtual screening of potentially endocrine-disrupting chemicals against nuclear receptors and its application to identify PPARγ-bound fatty acids.

Nuclear receptors (NRs) are key regulators of energy homeostasis, body development, and sexual reproduction. Xenobiotics binding to NRs may disrupt natural hormonal systems and induce undesired adverse effects in the body. However, many chemicals of concerns have limited or no experimental data on their potential or lack-of-potential endocrine-disrupting effects. Here, we propose a virtual screening method based on molecular docking for predicting potential endocrine-disrupting chemicals (EDCs) that bind to NRs. For 12 NRs, we systematically analyzed how multiple crystal structures can be used to distinguish actives and inactives found in previous high-throughput experiments. Our method is based on (i) consensus docking scores from multiple structures at a single functional state (agonist-bound or antagonist-bound), (ii) multiple functional states (agonist-bound and antagonist-bound), and (iii) multiple pockets (orthosteric site and alternative sites) of these NRs. We found that the consensus enrichment from multiple structures is better than or comparable to the best enrichment from a single structure. The discriminating power of this consensus strategy was further enhanced by a chemical similarity-weighted scoring scheme, yielding better or comparable enrichment for all studied NRs. Applying this optimized method, we screened 252 fatty acids against peroxisome proliferator-activated receptor gamma (PPARγ) and successfully identified 3 previously unknown fatty acids with Kd = 100-250 µM including two furan fatty acids: furannonanoic acid (FNA) and furanundecanoic acid (FUA), and one cyclopropane fatty acid: phytomonic acid (PTA). These results suggested that the proposed method can be used to rapidly screen and prioritize potential EDCs for further experimental evaluations.

Mechanistic studies on the drug metabolism and toxicity originating from cytochromes P450.

Cytochromes P450 are oxidizing enzymes; a few families of cytochromes P450 are implicated in drug metabolism. These enzymatic reactions involve many processes including (i) prodrug to drug conversion, (ii) easy excretion of drug, (iii) generation of reactive metabolites, many of which cause toxicity. In this review, the fundamental biochemical mechanisms associated with the conversion of drugs into the useful or toxic metabolites have been discussed. The mechanisms can be established with the help of many experimental methods like mass spectral analysis, NMR and in vitro analysis etc. Computational methods provide detailed atomic level information, which is generally not available from experimental studies. Thus, the in silico efforts in elucidating the molecular mechanisms are complementary to the known experimental methods and are often clearer (especially in providing 3D information about the metabolites and their reactions). Quantum chemical methods and molecular docking become especially very useful. This review includes five case studies, which explain how the atomic level details were obtained to explore the reaction mechanisms of drug metabolism by cytochromes P450.

Modular mimicry and engagement of the Hippo pathway by Marburg virus VP40: Implications for filovirus biology and budding.

Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies.

Exploring PfDHFR reaction surface: A combined molecular dynamics and QM/MM analysis.

The substrate to the enzyme PfDHFR (Plasmodium falciparum Dihydrofolate Reductase) is a small molecule dihydrofolate (DHF), it gets converted to tetrahydrofolate (THF) in the active site of the enzyme. The PfDHFR reaction surface involves the protonation of DHF to DHFP as an initial step before the catalytic conversion. The binding affinities of all these species (DHF, DHFP and THF) contribute to the mechanism of DHFR catalytic action. Molecular dynamics (MD) simulations and Quantum Mechanics/Molecular Mechanics (QM/MM) analysis were performed to evaluate the binding affinity and molecular recognition interactions of the substrate DHF/DHFP and the product THF, in the active site of wild-type PfDHFR (wtPfDHFR). The binding affinities of the cofactor NADPH/NADP+ were also estimated in all the three complexes. The molecular dynamics (MD) simulations of the substrate, product and cofactor in the cavities of wtPfDHFR revealed the variation of the atomic level interactions during the course of the catalytic conversion. It was found that the DHFP binds very strongly to the PfDHFR active site and pulls the cofactor NADPH closer to itself. The QM/MM analysis revealed that the binding energy of DHFP (-59.82 kcal/mol) and NADPH (-100.24 kcal/mol) in DHFP-wtPfDHFR complex, is higher in comparison to the binding energy of DHF (-38.67 kcal/mol) and NADPH (-77.53 kcal/mol) in DHF-wtPfDHFR complex and the binding energy of THF (-30.72 kcal/mol) and NADP+ (-73.72 kcal/mol) in THF-wtPfDHFR complex. The hydride ion donor-acceptor distance (DAD) analysis was also carried out. This combined MD and QM/MM analysis revealed that the protonation of DHF increases the proximity between the substrate and the cofactor, thus facilitates the reaction profile of PfDHFR.

Biotransformation of Isoniazid by Cytochromes P450: Analyzing the Molecular Mechanism using Density Functional Theory.

Hydrazide group (-C(O)-NH-NH2) is considered as a structural alert in the drug discovery process because the biotransformation chemistry of this group leads to the generation of toxic radical intermediates. The most important antitubercular drug isoniazid (INH) carries the hydrazide group. The toxicity of INH has been attributed to the protein adduct formation involving isonicotinoyl radical. However, the structures of reactive metabolites (RMs) and metabolite intermediate complexes (MICs), as well as the reaction mechanism for the formation and fate of RMs/MICs, have not been established. This report provides a detailed account of the biotransformation of INH by cytochromes using quantum chemical (QC) methods. Two cycles of cytochrome catalysis are involved in the formation of the most important RM, isonicotinoyl radical. The first cycle requires ∼11 kcal/mol barrier on the oxidation pathway involving the formation of the RM isonicotinoyldiazene. The second cycle involves a barrier of ∼7 kcal/mol for the activation of the diazene intermediate leading to isonicotinic acid via three reaction steps: (i) N-H bond activation, (ii) loss of N2 molecule, and (iii) rebound of isonicotinoyl radical. The RMs on the pathway (diazene, isonicotinoyl radical, N-hydroxy diazene) can react with the porphyrin ring/the amino acids of the cytochrome leading to many MICs (at least nine varieties), which can cause mechanism based inhibition and drug-drug interactions. This QC, molecular docking, and QM/MM study explored all the above reaction pathways and established the 3D structures of the RMs and MICs.

Copper(ii)-catalyzed Chan-Lam cross-coupling: chemoselective N-arylation of aminophenols.

Copper(ii)-catalyzed boronic acid promoted chemoselective N-arylation of unprotected aminophenols has been developed. Selective N-arylation of 3-aminophenol is achieved with a Cu(OAc)2/AgOAc combination in MeOH at rt, whereas the chemoselective N-arylated products of 4-aminophenol can be obtained with a Cu(OAc)2/Cs2CO3 system and benzoic acid as an additive. These ligand-free conditions and "open-flask" chemistry are robust and compatible with a wide range of functional groups. The mechanistic investigation for this selective N-arylation has been studied by considering Density Functional Theory (DFT) calculations.

Toxicity Originating from Thiophene Containing Drugs: Exploring the Mechanism using Quantum Chemical Methods.

Drug metabolism of thiophene containing substrates by cytochrome P450s (CYP450) leads to toxic side effects, for example, nephrotoxicity (suprofen, ticlopidine), hepatotoxicity (tienilic acid), thrombotic thrombocytopenic purpura (clopidogrel), and aplastic anemia (ticlopidine). The origin of toxicity in these cases has been attributed to two different CYP450 mediated metabolic reactions: S-oxidation and epoxidation. In this work, the molecular level details of the bioinorganic chemistry associated with the generation of these competitive reactions are reported. Density functional theory was utilized (i) to explore the molecular mechanism for S-oxidation and epoxidation using the radical cationic center Cpd I [(iron(IV)-oxo-heme porphine system with SH(-) as the axial ligand, to mimic CYP450s] as the model oxidant, (ii) to establish the 3D structures of the reactants, transition states, and products on both the metabolic pathways, and (iii) to examine the potential energy (PE) profile for both the pathways to determine the energetically preferred toxic metabolite formation. The energy barrier required for S-oxidation was observed to be 14.75 kcal/mol as compared to that of the epoxidation reaction (13.23 kcal/mol) on the doublet PE surface of Cpd I. The formation of the epoxide metabolite was found to be highly exothermic (-23.24 kcal/mol), as compared to S-oxidation (-8.08 kcal/mol). Hence, on a relative scale the epoxidation process was observed to be thermodynamically and kinetically more favorable. The energy profiles associated with the reactions of the S-oxide and epoxide toxic metabolites were also explored. This study helps in understanding the CYP450-catalyzed toxic reactions of drugs containing the thiophene ring at the atomic level.

TEMPO-Promoted Domino Heck-Suzuki Arylation: Diastereoselective Cis-Diarylation of Glycals and Pseudoglycals.

A palladium-catalyzed regio- and diastereoselective diarylation of glycals and pseudoglycals, which is a kind of Heck-Suzuki arylation, is described. A wide range of arylboronic acids reacted under these conditions smoothly. Selectivity was C1-C2(α,α) in the case of glycals but C2-C3(ß,ß) for pseudoglycals. Quantum chemical analysis has been carried out to establish the reaction mechanism, which may involve Pd(II)/Pd(O). TEMPO plays a key role in the formation of diaryl glycoside due to its radical nature.