Cooperative Weak Dispersive Interactions Actuate Catalysis in a Shape-Selective Abiological Racemase.

A simple abiological host-guest system demonstrates racemase activity with catalytic rate enhancements of 104 without employing traditional functional groups. Cooperative weak interactions enhanced through shape-complementarity between the catalyst active site and the reaction transition state drive this activity, as proposed by Pauling for enzymes. In analogy to the Jencks' concept of catalytic antibodies, it is shown that a hapten resembling the planar transition state of the bowl inversion acts as a potent inhibitor of this catalytic process. In contrast, no substrate/product inhibition is detected, and a relatively weak binding of the substrate is observed (Ka ≈ 102 M-1 at 293 K). This simple box-and-bowl system demonstrates that shape selectivity arising from cooperative dispersive forces suffices for the emergence of a catalytic system with an enzyme-like thermodynamic profile.

Two protein disulfide isomerase subgroups work synergistically in catalyzing oxidative protein folding.

Disulfide bonds play essential roles in the folding of secretory and plasma membrane proteins in the endoplasmic reticulum (ER). In eukaryotes, protein disulfide isomerase (PDI) is an enzyme catalyzing the disulfide bond formation and isomerization in substrates. The Arabidopsis (Arabidopsis thaliana) genome encodes diverse PDIs including structurally distinct subgroups PDI-L and PDI-M/S. It remains unclear how these AtPDIs function to catalyze the correct disulfide formation. We found that one Arabidopsis ER oxidoreductin-1 (Ero1), AtERO1, can interact with multiple PDIs. PDI-L members AtPDI2/5/6 mainly serve as an isomerase, while PDI-M/S members AtPDI9/10/11 are more efficient in accepting oxidizing equivalents from AtERO1 and catalyzing disulfide bond formation. Accordingly, the pdi9/10/11 triple mutant exhibited much stronger inhibition than pdi1/2/5/6 quadruple mutant under dithiothreitol treatment, which caused disruption of disulfide bonds in plant proteins. Furthermore, AtPDI2/5 work synergistically with PDI-M/S members in relaying disulfide bonds from AtERO1 to substrates. Our findings reveal the distinct but overlapping roles played by two structurally different AtPDI subgroups in oxidative protein folding in the ER.

Comprehensive analysis of human chorionic membrane extracts regulating mesenchymal stem cells during osteogenesis.

OBJECTIVE: Human chorionic membrane extracts (CMEs) from placenta are known to be a natural biomaterial for bone regeneration, with their excellent osteogenic efficacy on osteoblasts. However, little is known about the regulatory mechanism involved. METHODS AND RESULTS: We have shown the in vitro and in vivo bone-forming ability of CME using human osteoblasts and bone defect animal models, suggesting that CME greatly enhances osteogenesis by providing an osteoconductive environment for the osteogenesis of osteoblasts. Proteomic analysis revealed that CME contained several osteogenesis-related stimulators such as osteopontin, osteomodulin, Thy-1, netrin 4, retinol-binding protein and DJ-1. Additionally, 23 growth factors/growth factor-related proteins were found in CME, which may trigger mitogen-activated protein kinase (MAPK) signalling as a specific cellular signalling pathway for osteogenic differentiation. Microarray analysis showed four interaction networks (chemokine, Wnt signalling, angiogenesis and ossification), indicating the possibility that CME can promote osteogenic differentiation through a non-canonical Wnt-mediated CXCL signalling-dependent pathway. CONCLUSIONS: The results of this study showed the function and mechanism of action of CME during the osteogenesis of osteoblasts and highlighted a novel strategy for the use of CME as a biocompatible therapeutic material for bone regeneration.

Small Molecule CD38 Inhibitors: Synthesis of 8-Amino-N1-inosine 5'-monophosphate, Analogues and Early Structure-Activity Relationship.

Although a monoclonal antibody targeting the multifunctional ectoenzyme CD38 is an FDA-approved drug, few small molecule inhibitors exist for this enzyme that catalyzes inter alia the formation and metabolism of the N1-ribosylated, Ca2+-mobilizing, second messenger cyclic adenosine 5'-diphosphoribose (cADPR). N1-Inosine 5'-monophosphate (N1-IMP) is a fragment directly related to cADPR. 8-Substituted-N1-IMP derivatives, prepared by degradation of cyclic parent compounds, inhibit CD38-mediated cADPR hydrolysis more efficiently than related cyclic analogues, making them attractive for inhibitor development. We report a total synthesis of the N1-IMP scaffold from adenine and a small initial compound series that facilitated early delineation of structure-activity parameters, with analogues evaluated for inhibition of CD38-mediated hydrolysis of cADPR. The 5'-phosphate group proved essential for useful activity, but substitution of this group by a sulfonamide bioisostere was not fruitful. 8-NH2-N1-IMP is the most potent inhibitor (IC50 = 7.6 µM) and importantly HPLC studies showed this ligand to be cleaved at high CD38 concentrations, confirming its access to the CD38 catalytic machinery and demonstrating the potential of our fragment approach.

Direct Electrochemical Generation of Catalytically Competent Oxyferryl Species of Classes I and P Dye Decolorizing Peroxidases.

This work introduces a novel way to obtain catalytically competent oxyferryl species for two different dye-decolorizing peroxidases (DyPs) in the absence of H2O2 or any other peroxide by simply applying a reductive electrochemical potential under aerobic conditions. UV-vis and resonance Raman spectroscopies show that this method yields long-lived compounds II and I for the DyPs from Bacillus subtilis (BsDyP; Class I) and Pseudomonas putida (PpDyP; Class P), respectively. Both electrochemically generated high valent intermediates are able to oxidize ABTS at both acidic and alkaline pH. Interestingly, the electrocatalytic efficiencies obtained at pH 7.6 are very similar to the values recorded for regular catalytic ABTS/H2O2 assays at the optimal pH of the enzymes, ca. 3.7. These findings pave the way for the design of DyP-based electrocatalytic reactors operable in an extended pH range without the need of harmful reagents such as H2O2.

Efficient Degradation of Zearalenone by Dye-Decolorizing Peroxidase from Streptomyces thermocarboxydus Combining Catalytic Properties of Manganese Peroxidase and Laccase.

Ligninolytic enzymes, including laccase, manganese peroxidase, and dye-decolorizing peroxidase (DyP), have attracted much attention in the degradation of mycotoxins. Among these enzymes, the possible degradation pathway of mycotoxins catalyzed by DyP is not yet clear. Herein, a DyP-encoding gene, StDyP, from Streptomyces thermocarboxydus 41291 was identified, cloned, and expressed in Escherichia coli BL21/pG-Tf2. The recombinant StDyP was capable of catalyzing the oxidation of the peroxidase substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), phenolic lignin compounds 2,6-dimethylphenol, and guaiacol, non-phenolic lignin compound veratryl alcohol, Mn2+, as well as anthraquinone dye reactive blue 19. Moreover, StDyP was able to slightly degrade zearalenone (ZEN). Most importantly, we found that StDyP combined the catalytic properties of manganese peroxidase and laccase, and could significantly accelerate the enzymatic degradation of ZEN in the presence of their corresponding substrates Mn2+ and 1-hydroxybenzotriazole. Furthermore, the biological toxicities of the main degradation products 15-OH-ZEN and 13-OH-ZEN-quinone might be remarkably removed. These findings suggested that DyP might be a promising candidate for the efficient degradation of mycotoxins in food and feed.

Facile synthesis and characterization of chitosan/zinc oxide nanocomposite for enhanced antibacterial and photocatalytic activity.

In this report, chitosan/zinc oxide (CS/ZnO) nanocomposite was synthesized using Sida acuta and assessed their antibacterial and photocatalytic properties. The formation of CS/ZnO nanocomposite was preliminary confirmed by colour change and UV-visible spectroscopy. The crystalline peaks related to CS and ZnO in CS/ZnO nanocomposite were demonstrated by XRD. Morphological analysis through FE-SEM and TEM showed a rod like appearance for ZnO NPs and agglomerated grains with rod shaped morphology was observed for the CS/ZnO nanocomposite. The peaks around 400-800 cm-1 in the IR spectrum of nanocomposite indicated the vibrations of metal-oxygen (ZnO), whereas bands at 1659 cm-1 and 1546 cm-1 indicated the presence of amine groups, which confirms the CS in the synthesized CS/ZnO nanocomposite. The CS/ZnO nanocomposite exhibited remarkable growth inhibition activity against B. subtilis and E. coli with 22 ± 0.3 and 16.5 ± 0.5 mm zone of inhibitions. In addition, CS/ZnO nanocomposite treated cotton fabrics also exhibited antibacterial activity against B. subtilis and E. coli. Furthermore, the ZnO NPs and nanocomposite showed time depended photodegradation activity and revealed 76% and 91% decomposition of CR under sunlight irradiation. In conclusion, our study revealed that the functionalization of biopolymer CS to the inorganic ZnO enhances the bio and catalytic properties.

Identification and Characterization of Two Structurally Related Dipeptides that Enhance Catalytic Efficiency of Neurolysin.

Neurolysin (Nln) is a recently recognized endogenous mechanism functioning to preserve the brain from ischemic injury. To further understand the pathophysiological function of this peptidase in stroke and other neurologic disorders, the present study was designed to identify small molecule activators of Nln. Using a computational approach, the structure of Nln was explored, which was followed by docking and in silico screening of ∼140,000 molecules from the National Cancer Institute Developmental Therapeutics Program database. Top ranking compounds were evaluated in an Nln enzymatic assay, and two hit histidine-dipeptides were further studied in detail. The identified dipeptides enhanced the rate of synthetic substrate hydrolysis by recombinant (human and rat) and mouse brain-purified Nln in a concentration-dependent manner (micromolar A50 and Amax ≥ 300%) but had negligible effect on activity of closely related peptidases. Both dipeptides also enhanced hydrolysis of Nln endogenous substrates neurotensin, angiotensin I, and bradykinin and increased efficiency of the synthetic substrate hydrolysis (Vmax/Km ratio) in a concentration-dependent manner. The dipeptides and competitive inhibitor dynorphin A (1-13) did not affect each other's affinity for Nln, suggesting differing nature of their respective binding sites. Lastly, drug affinity responsive target stability (DARTS) and differential scanning fluorimetry (DSF) assays confirmed concentration-dependent interaction of Nln with the activator molecule. This is the first study demonstrating that Nln activity can be enhanced by small molecules, although the peptidic nature and low potency of the activators limit their application. The identified dipeptides provide a chemical scaffold to develop high-potency, drug-like molecules as research tools and potential drug leads. SIGNIFICANCE STATEMENT: This study describes discovery of two molecules that selectively enhance activity of peptidase Nln-a newly recognized cerebroprotective mechanism in the poststroke brain. The identified molecules will serve as a chemical scaffold for development of drug-like molecules to further study Nln and may become lead structures for a new class of drugs. In addition, our conceptual and methodological framework and research findings might be used for other peptidases and enzymes, the activation of which bears therapeutic potential.

REPORT: IR study of degradation of acetaminophen by iron nano-structured catalyst.

Full degradation of acetaminophen (paracetamol) in aqueous solution was investigated at room temperature through heterogeneous iron nano-structured as catalyst in this article. Iron Nano-structured was prepared through simple hydrothermal processes using Iron oxide (Fe2O3) as precursor. The catalytic activity of as prepared Nano-catalyst (NC) was investigated in the degradation of the acetaminophen as an environmental pollutant, commonly called paracetamol, under different operating parameters like pH, dosages of acetaminophen and dose of NC. Remarkable differences in IR spectra were observed after reaction which showed complete degradation of 15 ppm of Acetaminophen using 0.1 g of nano-structured with the recovery of NC followed by its activity four times with full catalytic performance.

Reusable Nano-Zirconia-Catalyzed Synthesis of Benzimidazoles and Their Antibacterial and Antifungal Activities.

In this article, a zirconia-based nano-catalyst (Nano-ZrO2), with intermolecular C-N bond formation for the synthesis of various benzimidazole-fused heterocycles in a concise method is reported. The robustness of this reaction is demonstrated by the synthesis of a series of benzimidazole drugs in a one-pot method. All synthesized materials were characterized using 1HNMR, 13CNMR, and LC-MS spectroscopy as well as microanalysis data. Furthermore, the synthesis of nano-ZrO2 was processed using a standard hydrothermal technique in pure form. The crystal structure of nano-ZrO2 and phase purity were studied, and the crystallite size was calculated from XRD analysis using the Debye-Scherrer equation. Furthermore, the antimicrobial activity of the synthesized benzimidazole drugs was evaluated in terms of Gram-positive, Gram-negative, and antifungal activity, and the results were satisfactory.

Mussel-Inspired Immobilization of Photocatalysts with Synergistic Photocatalytic-Photothermal Performance for Water Remediation.

The serious problem of pharmaceutical and personal care product pollution places great pressure on aquatic environments and human health. Herein, a novel coating photocatalyst was synthesized by adhering Ag-AgCl/WO3/g-C3N4 (AWC) nanoparticles on a polydopamine (PDA)-modified melamine sponge (MS) through a facile layer-by-layer assembly method to degrade trimethoprim (TMP). The formed PDA coating was used for the anchoring of nanoparticles, photothermal conversion, and hydrophilic modification. TMP (99.9%; 4 mg/L) was removed in 90 min by the photocatalyst coating (AWC/PDA/MS) under visible light via a synergistic photocatalytic-photothermal performance route. The stability and reusability of the AWC/PDA/MS have been proved by cyclic experiments, in which the removal efficiency of TMP was still more than 90% after five consecutive cycles with a very little mass loss. Quantitative structure-activity relationship analysis revealed that the ecotoxicities of the generated intermediates were lower than those of TMP. Furthermore, the solution matrix effects on the photocatalytic removal efficiency were investigated, and the results revealed that the AWC/PDA/MS still maintained excellent photocatalytic degradation efficiency in several actual water and simulated water matrices. This work develops recyclable photocatalysts for the potential application in the field of water remediation.

Natural microbial polysaccharides as effective factors for modification of the catalytic properties of fungal cellobiose dehydrogenase.

Polysaccharides are biopolymers composed of simple sugars like glucose, galactose, mannose, fructose, etc. The major natural sources for the production of polysaccharides include plants and microorganisms. In the present work, four bacterial and two fungal polysaccharides (PS or EPS) were used for the modification and preservation of Pycnoporus sanguineus cellobiose dehydrogenase (CDH) activity. It was found that the presence of polysaccharide preparations clearly enhanced the stability of cellobiose dehydrogenase compared to the control value (4 °C). The highest stabilization effect was observed for CDH modified with Rh110EPS. Changes in the optimum pH in the samples of CDH incubated with the chosen polysaccharide modifiers were evidenced as well. The most significant effect was observed for Rh24EPS and Cu139PS (pH 3.5). Cyclic voltammetry used for the analysis of electrochemical parameters of modified CDH showed the highest peak values after 30 days of incubation with polysaccharides at 4 °C. In summary, natural polysaccharides seem to be an effective biotechnological tool for the modification of CDH activity to increase the possibilities of its practical applications in many fields of industry.

Product Inhibition and the Catalytic Destruction of a Nerve Agent Simulant by Zirconium-Based Metal-Organic Frameworks.

Rapid degradation/destruction of chemical warfare agents, especially ones containing a phosphorous-fluorine bond, is of notable interest due to their extreme toxicity and typically rapid rate of human incapacitation. Recent studies of the hydrolytic destruction of a key nerve agent simulant, dimethyl 4-nitrophenylphosphate (DMNP), catalyzed by Zr6-based metal-organic frameworks (MOFs), have suggested deactivation of the active sites due to inhibition by the products as the reaction progresses. In this study, the interactions of two MOFs, NU-1000 and MOF-808, and two hydrolysis products, dimethyl phosphate (DMP) and ethyl methyl phosphonate (EMP), from the hydrolysis of the simulant (DMNP) and nerve agent ethyl methylphosphonofluoridate (EMPF), resembling the hydrolysis degradation product of the G-series nerve agent, Sarin (GB), have been investigated to deconvolute the effect of product inhibition from other effects on catalytic activity. Kinetic studies via in situ nuclear magnetic resonance spectroscopy indicated substantial product inhibition upon catalyst activity after several tens to several thousand turnovers, depending on specific conditions. Apparent product binding constants were obtained by fitting initial reaction rates at pH 7.0 and pH 10.5 to a Langmuir-Freundlich binding/adsorption model. For the fits, varying amounts/concentrations of candidate inhibitors were introduced before the start of catalytic hydrolysis. The derived binding constants proved suitable for quantitatively describing product inhibition effects upon reaction rates over the extended time course of simulant hydrolysis by aqua-ligand-bearing hexa-zirconium(IV) nodes.

Synthesis of new chitosan Schiff base and its Fe2O3 nanocomposite: Evaluation of methyl orange removal and antibacterial activity.

New chitosan Schiff base (3EtO-4OH/Chit) and its 3EtO-4OH/Chit/Fe2O3 nanocomposite were synthesized and characterized by FTIR, 1H NMR, XRD, TGA, DSC and SEM. The result confirmed the preparation of 3EtO-4OH/Chit and its 3EtO-4OH/Chit/Fe2O3 nanocomposite. The efficiency of the prepared catalysts was studied for the methyl orange (MO) removal from aqueous solution. The effect of adsorbent dose and contact time on the removal of dye has been studied. Their antibacterial activities were considered against two Gram positive (S. aureus and B. cereus) and two Gram negative (E. coli and P. aeruginosa) bacteria and the results showed that the activity of the 3EtO-4OH/Chit/Fe2O3 is excellent and is more than chitosan and 3EtO-4OH/Chit. Thermogravimetry studies shows that the weight loss stages and the residual value at 600 °C are different for the two compounds. DSC curve of the title compounds 3EtO-4OH/Chit and 3EtO-4OH/Chit/Fe2O3 is different from each other. The reason for this difference could be due to the presence of iron oxide nanoparticles in 3EtO-4OH/Chit/Fe2O3.

Histone Parylation factor 1 contributes to the inhibition of PARP1 by cancer drugs.

Poly-(ADP-ribose) polymerase 1 and 2 (PARP1 and PARP2) are key enzymes in the DNA damage response. Four different inhibitors (PARPi) are currently in the clinic for treatment of ovarian and breast cancer. Recently, histone PARylation Factor 1 (HPF1) has been shown to play an essential role in the PARP1- and PARP2-dependent poly-(ADP-ribosylation) (PARylation) of histones, by forming a complex with both enzymes and altering their catalytic properties. Given the proximity of HPF1 to the inhibitor binding site both PARPs, we hypothesized that HPF1 may modulate the affinity of inhibitors toward PARP1 and/or PARP2. Here we demonstrate that HPF1 significantly increases the affinity for a PARP1 - DNA complex of some PARPi (i.e., olaparib), but not others (i.e., veliparib). This effect of HPF1 on the binding affinity of Olaparib also holds true for the more physiologically relevant PARP1 - nucleosome complex but does not extend to PARP2. Our results have important implications for the interpretation of PARP inhibition by current PARPi as well as for the design and analysis of the next generation of clinically relevant PARP inhibitors.

Modulating catalytic activity of human topoisomerase II α enzyme by fluorescent gold nanoclusters.

Precise monitoring of the enzyme activity by a suitable modulator is one of the very fundamental aspects of drug designing that provides the opportunity to overcome the challenges of several diseases. Herein, inhibition of human Topoisomerase IIα enzyme which serves as a potential target site for several anti-cancer drugs is demonstrated by using ultra-small size gold nanoclusters (Au NCs) with the dimension comparable with size of the active site of the enzyme. Molecular dynamics simulation results demonstrate that the Au NCs strongly interact with the human Topo IIα enzyme at its active site or allosteric site depending on forms of enzyme. Additionally, binding energy and interaction profile provides the molecular basis of understanding of interactions of ultra-small size Au NCs and human Topo IIα enzyme. Enthalpy change (ΔH) and binding constant (K) are measured based on a sequential binding model of the Au NCs with the enzyme as demonstrated by the ITC study. Moreover, the in-vitro inhibition study of the catalytic activity of the enzyme and gel electrophoresis indicates that the ultra-small size Au NCs may be used as a potent inhibitor of human Topo IIα enzyme.

Preparation of highly diffusible porous cross-linked lipase B from Candida antarctica conjugates: Advances in mass transfer and application in transesterification of 5-Hydroxymethylfurfural.

The present work pronounces the three phase partitioning (TPP)-facilitated preparation of porous cross-linked Candida antarctica lipase B (CaLB) aggregates (pCLEAs) for 5-Hydroxymethylfurfural (HMF) esters synthesis. CLEAs and pCLEAs of CaLB were prepared with eupergit as the support under the optimized conditions of pH 8.0, eupergit/protein ratio of 3.0:1.0, 50 mM cross-linker concentration and 3.3 mg/mL BSA concentration in 4 h. The optimum starch concentration for pCLEAs was 0.20%, m/v. The maximum biocatalytic load was 650 U/g (CLEAs) and 721 U/g (pCLEAs), and the immobilized biocatalysts were stable over a pH range of 6.0-9.0 and temperature range of (40-60)°C. The BET surface area of CLEAs and pCLEAs were 21.3 and 29.1 m2/g, respectively, and the catalytic efficiency of pCLEAs was 2.2-fold higher than that of CLEAs. Subsequently, the pCLEAs of CaLB were utilized for the manufacturing of industrially significant HMF esters. Under the optimized transesterification conditions, HMF conversion with pCLEAs CaLB was 1.41- and 1.25-fold higher than with free and CLEAs CaLB, respectively. The pCLEAs were reused upto 8 consecutive transesterification cycles and the produced HMF esters reduced the surface tension of water from 72 mN/m to 32.6 mN/m, proving its potential application as surface-active compounds.

Catalytic patch with redox Cr/CeO2 nanozyme of noninvasive intervention for brain trauma.

Traumatic brain injury (TBI) is a sudden injury to the brain, accompanied by the production of large amounts of reactive oxygen and nitrogen species (RONS) and acute neuroinflammation responses. Although traditional pharmacotherapy can effectively decrease the immune response of neuron cells via scavenging free radicals, it always involves in short reaction time as well as rigorous clinical trial. Therefore, a noninvasive topical treatment method that effectively eliminates free radicals still needs further investigation. Methods: In this study, a type of catalytic patch based on nanozymes with the excellent multienzyme-like activity is designed for noninvasive treatment of TBI. The enzyme-like activity, free radical scavenging ability and therapeutic efficacy of the designed catalytic patch were assessed in vitro and in vivo. The structural composition was characterized by the X-ray diffraction, X-ray photoelectron spectroscopy and high-resolution transmission electron microscopy technology. Results: Herein, the prepared Cr-doped CeO2 (Cr/CeO2) nanozyme increases the reduced Ce3+ states, resulting in its enzyme-like activity 3-5 times higher than undoped CeO2. Furthermore, Cr/CeO2 nanozyme can improve the survival rate of LPS induced neuron cells via decreasing excessive RONS. The in vivo experiments show the Cr/CeO2 nanozyme can promote wound healing and reduce neuroinflammation of mice following brain trauma. The catalytic patch based on nanozyme provides a noninvasive topical treatment route for TBI as well as other traumas diseases. Conclusions: The catalytic patch based on nanozyme provides a noninvasive topical treatment route for TBI as well as other traumas diseases.

Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF.

In biosynthesis of the pancreatic cancer drug streptozotocin, the tridomain nonheme-iron oxygenase SznF hydroxylates Nδ and Nω' of Nω-methyl-l-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the monoiron cofactor in the enzyme's C-terminal cupin domain, which promotes the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase-like (HO-like) central domain. We leveraged our recent observation that the N-oxygenating µ-peroxodiiron(III/III) intermediate can form in the HO-like domain after the apo protein self-assembles its diiron(II/II) cofactor to solve structures of SznF with both of its iron cofactors bound. These structures of a biochemically validated member of the emerging heme-oxygenase-like diiron oxidase and oxygenase (HDO) superfamily with intact diiron cofactor reveal both the large-scale conformational change required to assemble the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability-a trait shared by the other validated HDOs. During cofactor (dis)assembly, a ligand-harboring core helix dynamically (un)folds. The diiron cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulations. The additional carboxylate ligand is conserved in another N-oxygenating HDO but not in two HDOs that cleave carbon-hydrogen and carbon-carbon bonds to install olefins. Among ∼9,600 sequences identified bioinformatically as members of the emerging HDO superfamily, ∼25% conserve this additional carboxylate residue and are thus tentatively assigned as N-oxygenases.

Detoxication versus Bioactivation Pathways of Lapatinib In Vitro: UGT1A1 Catalyzes the Hepatic Glucuronidation of Debenzylated Lapatinib.

O-Dealkylation of the tyrosine kinase inhibitor lapatinib by cytochrome P450 3A enzymes is implicated in the development of lapatinib-induced hepatotoxicity. Conjugative metabolism of debenzylated lapatinib (M1) via glucuronidation and sulfation is thought to be a major detoxication pathway for lapatinib in preclinical species (rat and dog), limiting formation of the quinoneimine reactive metabolite. Glucuronidation of M1 by human recombinant UDP-glucuronosyltransferases (UGTs) has been reported in vitro; however, the relative UGT enzyme contributions are unknown, and the interspecies differences in the conjugation versus bioactivation pathways of M1 have not been fully elucidated. In the present study, reaction phenotyping experiments using human recombinant UGT enzymes and enzyme-selective chemical inhibitors demonstrated that UGT1A1 was the major hepatic UGT enzyme involved in lapatinib M1 glucuronidation. Formation of the M1-glucuronide by human liver microsomes from UGT1A1-genotyped donors was significantly correlated with UGT1A1 activity as measured by 17ß-estradiol 3-glucuronidation (R 2 = 0.90). Interspecies differences were found in the biotransformation of M1 in human, rat, and dog liver microsomal and 9000g supernatant (S9) fractions via glucuronidation, sulfation, aldehyde oxidase-mediated oxidation, and bioactivation to the quinoneimine trapped as a glutathione (GSH) conjugate. Moreover, we demonstrated the sequential metabolism of lapatinib in primary human hepatocytes to the M1-glucuronide, M1-sulfate, and quinoneimine-GSH conjugate. M1 glucuronidation was highly correlated with the rates of M1 formation, suggesting that O-dealkylation may be the rate-limiting step in lapatinib biotransformation. Interindividual variability in the formation and clearance pathways of lapatinib M1 likely influences the hepatic exposure to reactive metabolites and may affect the risk for hepatotoxicity. SIGNIFICANCE STATEMENT: We used an integrated approach to examine the interindividual and interspecies differences in detoxication versus bioactivation pathways of lapatinib, which is associated with idiosyncratic hepatotoxicity. In addition to cytochrome P450 (P450)-mediated bioactivation, we report that multiple non-P450 pathways are involved in the biotransformation of the primary phenolic metabolite of lapatinib in vitro, including glucuronidation, sulfation, and aldehyde oxidase mediated oxidation. UGT1A1 was identified as the major hepatic enzyme involved in debenzylated lapatinib glucuronidation, which may limit hepatic exposure to the potentially toxic quinoneimine.