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This letter describes the further chemical optimization of VU0424238 (auglurant), an mGlu 5 NAM clinical candidate that failed in non-human primate (NHP) 28 day toxicology due to accumulation of a species-specific aldehyde oxidase (AO) metabolite of the pyrimidine head group. Here, we excised the pyrimidine moiety, identified the minimum pharmacophore, and then developed a new series of saturated ether head groups that ablated any AO contribution to metabolism. Putative back-up compounds in this novel series provided increased sp 3 character, uniform CYP 450 -mediated metabolism across species, good functional potency and high CNS penetration. Key to the optimization was a combination of matrix and iterative libraries that allowed rapid surveillance of multiple domains of the allosteric ligand., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This letter describes the on-going SAR efforts to develop PLD1, PLD2 and dual PLD1/2 inhibitors with improved physiochemical and disposition properties as well as securing intellectual property position. Previous PLD inhibitors, based on a triazaspiro[4.5]decanone core proved to be highly selective PLD2 inhibitors, but with low plasma free fraction (rat, human f u  < 0.03), high predicted hepatic clearance (rat CL hep  > 65 mL/min/kg) and very short half-lives in vivo (t 1/2  < 0.15 h). Removal of a nitrogen atom from this core generated a 2,8-diazaspiro[4.5]decanone core, harboring a new chiral center, as well as increased sp 3 character. This new core demonstrated enantioselective inhibition of the individual PLD isoforms, enhanced free fraction (rat, human f u  < 0.13), engendered moderate predicted hepatic clearance (rat CL hep  ∼ 43 mL/min/kg), improved half-lives in vivo (t 1/2  > 3 h), and led to the first issued US patent claiming composition of matter for small molecule PLD inhibitors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Herein, we report the discovery of a novel potent, selective, CNS penetrant, and orally bioavailable mGlu 4 PAM, VU0652957 (VU2957, Valiglurax). VU2957 possessed attractive in vitro and in vivo pharmacological and DMPK properties across species. To advance toward the clinic, a spray-dried dispersion (SDD) formulation of VU2957 was developed to support IND-enabling toxicology studies. Based on its overall profile, VU2957 was evaluated as a preclinical development candidate for the treatment of Parkinson's disease., Competing Interests: The authors declare the following competing financial interest(s): Authors hold patents on mGlu4 PAMs and are actively developing mGlu4 PAMs for PD patients.

Selective activation of the M 1 subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer's disease patients. However, highly potent M 1 ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M 1 , leading to agonist actions in the prefrontal cortex (PFC) that impair cognitive function, induce behavioral convulsions, and result in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M 1 PAM, VU0486846. VU0486846 possesses only weak agonist activity in M 1 -expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427, and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [ 3 H]-NMS binding at the orthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M 1 PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M 1 PAM activity (EC 50 > 100 nM) and pure-PAM activity in native tissues display robust procognitive efficacy without AEs mediated by excessive activation of M 1 . Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development.

Herein, we report the chemical optimization of a new series of M 1 positive allosteric modulators (PAMs) based on a novel pyrrolo[2,3- b ]pyridine core, developed via scaffold hopping and iterative parallel synthesis. The vast majority of analogs in this series proved to display robust cholinergic seizure activity. However, by removal of the secondary hydroxyl group, VU6007477 resulted with good rat M 1 PAM potency (EC 50 = 230 nM, 93% ACh max), minimal M 1 agonist activity (agonist EC 50 > 10 μM), good CNS penetration (rat brain/plasma K p = 0.28, K p,uu = 0.32; mouse K p = 0.16, K p,uu = 0.18), and no cholinergic adverse events (AEs, e.g., seizures). This work demonstrates that within a chemical series prone to robust M 1 ago-PAM activity, SAR can result, which affords pure M 1 PAMs, devoid of cholinergic toxicity/seizure liability., Competing Interests: The authors declare the following competing financial interest(s): Hold IP on M1 PAMs.

Previous reports from our laboratory disclosed the structure and activity of a novel 1H-pyrazolo[4,3-b]pyridine-3-amine scaffold (VU8506) which showed excellent potency, selectivity and in vivo efficacy in preclinical rodent models of Parkinson's disease. Unfortunately, this compound suffered from significant CYP1A2 induction as measured through upstream AhR activation (125-fold) and thus was precluded from further advancement in chronic studies. Herein, we report a new scaffold developed recently which was systematically studied in order to mitigate the CYP1A2 liabilities presented in the earlier scaffolds. We have identified a novel structure that maintains the potency and selectivity of other mGlu 4 PAMs, leading to 9i (hmGlu 4 EC 50  = 43 nM; AhR activation = 2.3-fold)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Because of the increase in the number of immunocompromised patients, the incidence of invasive fungal infections is growing, but the treatment efficiency remains unacceptably low. The most potent clinical systemic antifungals (azoles) are the derivatives of two scaffolds: ketoconazole and fluconazole. Being the safest antifungal drugs, they still have shortcomings, mainly because of pharmacokinetics and resistance. Here, we report the successful use of the target fungal enzyme, sterol 14α-demethylase (CYP51), for structure-based design of novel antifungal drug candidates by minor modifications of VNI [( R)- N-(1-(2,4-dichlorophenyl)-2-(1 H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)], an inhibitor of protozoan CYP51 that cures Chagas disease. The synthesis of fungi-oriented VNI derivatives, analysis of their potencies to inhibit CYP51s from two major fungal pathogens ( Aspergillus fumigatus and Candida albicans), microsomal stability, effects in fungal cells, and structural characterization of A. fumigatus CYP51 in complexes with the most potent compound are described, offering a new antifungal drug scaffold and outlining directions for its further optimization.

This letter describes the chemical optimization of a new series of M 1 positive allosteric modulators (PAMs) based on a novel benzomorpholine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0486846 (7), devoid of adverse effect liability. This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0486846 and details all of the challenges faced in allosteric modulator programs (both steep and flat SAR, as well as subtle structural changes affecting CNS penetration and overall physiochemical and DMPK properties)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Based on previous work that established fused heterocycles as viable alternatives for the picolinamide core of our lead series of mGlu 5 negative allosteric modulators (NAMs), we designed a novel series of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide mGlu 5 NAMs. These new quinoline derivatives also contained carbon linkers as replacements for the diaryl ether oxygen atom common to our previously published chemotypes. Compounds were evaluated in a cell-based functional mGlu 5 assay, and an exemplar analog 27 was >60-fold selective versus the other seven mGlu receptors. Selected compounds were also studied in metabolic stability assays in rat and human S9 hepatic fractions and exhibited a mixture of P450- and non-P450-mediated metabolism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aldehyde oxidase (AO) and xanthine oxidase (XO) are molybdo-flavoenzymes that catalyze oxidation of aromatic azaheterocycles. Differences in AO activity have been reported among various species, including rats, humans, and monkeys. Herein we report a species difference in the enzymes responsible for the metabolism of the negative allosteric modulator of metabotropic glutamate receptor subtype 5 (mGlu 5 NAM) VU0424238 (VU238, auglurant). Hepatic S9 incubations with AO and XO specific inhibitors hydralazine and allopurinol indicated that rats and cynomolgus monkeys both oxidized VU238 to the 6-oxopyrimidine metabolite M1 via an AO-mediated pathway, whereas secondary oxidation to the 2,6-dioxopyrimidine metabolite M2 was mediated predominantly by AO in monkeys and XO in rats. Despite differences in enzymatic pathways, intrinsic clearance (CL int ) of M1 was similar between species (cynomolgus and rat CL int = 2.00 ± 0.040 and 2.19 ± 0.201 μ l/min per milligram of protein, respectively). Inhibitor studies in the S9 of multiple species indicated that oxidation of VU238 to M1 was mediated predominantly by AO in humans, cynomolgus and rhesus monkeys, rats, mice, guinea pigs, and minipigs. Oxidation of M1 to M2 was mediated predominantly by XO in rats and mice and by AO in monkeys and guinea pigs, whereas low turnover prevented enzyme phenotyping in humans and minipigs. Additionally, inhibitor experiments indicated that oxidation at the 2-position of the pyrimidine ring of the known AO substrate, BIBX1382, was mediated by AO in all species, although production of this metabolite was comparatively low in rats and mice. These data may suggest low reactivity of rat AO toward 2-oxidation of pyrimidine-containing compounds and highlight the importance of thoroughly characterizing AO-metabolized drug candidates in multiple preclinical species., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Herein, we report the structure-activity relationships within a series of mGlu 7 NAMs based on an N -(2-(1 H -1,2,4-triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamide core with excellent CNS penetration ( K p 1.9-5.8 and K p,uu 0.4-1.4). Analogues in this series displayed steep SAR. Of these, VU6010608 ( 11a ) emerged with robust efficacy in blocking high frequency stimulated long-term potentiation in electrophysiology studies., Competing Interests: The authors declare no competing financial interest.

Based on a hypothesis that an intramolecular hydrogen bond was present in our lead series of picolinamide mGlu 5 NAMs, we reasoned that an inactive nicotinamide series could be modified through introduction of a fused heterocyclic core to generate potent mGlu 5 NAMs. In this Letter, we describe the synthesis and evaluation of compounds that demonstrate the viability of that approach. Selected analogs were profiled in a variety of in vitro assays, and two compounds were evaluated in rat pharmacokinetic studies and a mouse model of obsessive-compulsive disorder. Ancillary pharmacology screening revealed that members of this series exhibited moderate inhibition of the dopamine transporter (DAT), and SAR was developed that expanded the selectivity for mGlu 5 versus DAT., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Herein, we report the structure-activity relationships within a series of mGlu 7 PAMs based on a pyrazolo[1,5- a ]pyrimidine core with excellent CNS penetration ( K p s > 1 and K p,uu s > 1). Analogues in this series proved to display a range of Group III mGlu receptor selectivity, but VU6005649 emerged as the first dual mGlu 7/8 PAM, filling a void in the Group III mGlu receptor PAM toolbox and demonstrating in vivo efficacy in a mouse contextual fear conditioning model.

Herein, we detail the optimization of the mGlu 3 NAM, VU0650786, via a reductionist approach to afford a novel, simplified mGlu 3 NAM scaffold that engenders potent and selective mGlu 3 inhibition (mGlu 3 IC 50 = 245 nM, mGlu 2 IC 50 > 30 μM) with excellent central nervous system penetration (rat brain/plasma K p = 1.2, K p,uu = 0.40). Moreover, this new chemotype, exemplified by VU6010572, requires only four synthetic steps and displays improved physiochemical properties and in vivo efficacy in a mouse tail suspension test (MED = 3 mg/kg i.p.).

Herein, we detail the optimization of the mGlu 2 negative allosteric modulator (NAM), VU6001192, by a reductionist approach to afford a novel, simplified mGlu 2 NAM scaffold. This new chemotype not only affords potent and selective mGlu 2 inhibition, as exemplified by VU6001966 (mGlu 2 IC 50 = 78 nM, mGlu 3 IC 50 > 30 μM), but also excellent central nervous system (CNS) penetration ( K p = 1.9, K p,uu = 0.78), a feature devoid in all previously disclosed mGlu 2 NAMs ( K p s ≈ 0.3, K p,uu s ≈ 0.1). Moreover, this series, based on overall properties, represents an exciting lead series for potential mGlu 2 PET tracer development.

Both historical clinical and recent preclinical data suggest that the M 1 muscarinic acetylcholine receptor is an exciting target for the treatment of Alzheimer's disease and the cognitive and negative symptom clusters in schizophrenia; however, early drug discovery efforts targeting the orthosteric binding site have failed to afford selective M 1 activation. Efforts then shifted to focus on selective activation of M 1 via either allosteric agonists or positive allosteric modulators (PAMs). While M 1 PAMs have robust efficacy in rodent models, some chemotypes can induce cholinergic adverse effects (AEs) that could limit their clinical utility. Here, we report studies aimed at understanding the subtle structural and pharmacological nuances that differentiate efficacy from adverse effect liability within an indole-based series of M 1 ago-PAMs. Our data demonstrate that closely related M 1 PAMs can display striking differences in their in vivo activities, especially their propensities to induce adverse effects. We report the discovery of a novel PAM in this series that is devoid of observable adverse effect liability. Interestingly, the molecular pharmacology profile of this novel PAM is similar to that of a representative M 1 PAM that induces severe AEs. For instance, both compounds are potent ago-PAMs that demonstrate significant interaction with the orthosteric site (either bitopic or negative cooperativity). However, there are subtle differences in efficacies of the compounds at potentiating M 1 responses, agonist potencies, and abilities to induce receptor internalization. While these differences may contribute to the differential in vivo profiles of these compounds, the in vitro differences are relatively subtle and highlight the complexities of allosteric modulators and the need to focus on in vivo phenotypic screening to identify safe and effective M 1 PAMs.

Human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain inhibition, mainly because of its restricted expression in nociceptors within the peripheral nervous system. However, constrained by species differences across Mrgprs , drug candidates that activate MRGPRX1 do not activate rodent receptors, leaving no responsive animal model to test the effect on pain in vivo. Here, we generated a transgenic mouse line in which we replaced mouse Mrgprs with human MrgprX1 This humanized mouse allowed us to characterize an agonist [bovine adrenal medulla 8-22 (BAM8-22)] and a positive allosteric modulator (PAM), ML382, of MRGPRX1. Cellular studies suggested that ML382 enhances the ability of BAM8-22 to inhibit high-voltage-activated Ca 2+ channels and attenuate spinal nociceptive transmission. Importantly, both BAM8-22 and ML382 effectively attenuated evoked, persistent, and spontaneous pain without causing obvious side effects. Notably, ML382 by itself attenuated both evoked pain hypersensitivity and spontaneous pain in MrgprX1 mice after nerve injury without acquiring coadministration of an exogenous agonist. Our findings suggest that humanized MrgprX1 mice provide a promising preclinical model and that activating MRGPRX1 is an effective way to treat persistent pain.

Abnormalities in the signaling of the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) within cortical and limbic brain regions are thought to underlie many of the complex cognitive and affective symptoms observed in individuals with schizophrenia. The M 1 muscarinic acetylcholine receptor (mAChR) subtype is a closely coupled signaling partner of the NMDAR. Accumulating evidence suggests that development of selective positive allosteric modulators (PAMs) of the M 1 receptor represent an important treatment strategy for the potential normalization of disruptions in NMDAR signaling in patients with schizophrenia. In the present studies, we evaluated the effects of the novel and highly potent M 1 PAM, VU6004256, in ameliorating selective prefrontal cortical (PFC)-mediated physiologic and cognitive abnormalities in a genetic mouse model of global reduction in the NR1 subunit of the NMDAR (NR1 knockdown [KD]). Using slice-based extracellular field potential recordings, deficits in muscarinic agonist-induced long-term depression (LTD) in layer V of the PFC in the NR1 KD mice were normalized with bath application of VU6004256. Systemic administration of VU6004256 also reduced excessive pyramidal neuron firing in layer V PFC neurons in awake, freely moving NR1 KD mice. Moreover, selective potentiation of M 1 by VU6004256 reversed the performance impairments of NR1 KD mice observed in two preclinical models of PFC-mediated learning, specifically the novel object recognition and cue-mediated fear conditioning tasks. VU6004256 also produced a robust, dose-dependent reduction in the hyperlocomotor activity of NR1 KD mice. Taken together, the current findings provide further support for M 1 PAMs as a novel therapeutic approach for the PFC-mediated impairments in schizophrenia., Competing Interests: The authors declare the following competing financial interest(s): Over the past year, C.W.L. consulted for Abbott. M.B., T.M.B., C.W.L., P.J.C., and C.K.J. received research/salary support from AstraZeneca and/or Bristol Myers Squibb. C.M.N., C.W.L., and P.J.C. are inventors on multiple composition of matter patents protecting allosteric modulators of GPCRs. The remaining authors declare no competing financial interests.

Herein, we report the structure-activity relationships within a series of potent, selective, and orally bioavailable muscarinic acetylcholine receptor 4 (M 4 ) positive allosteric modulators (PAMs). Compound 6c (VU0467485) possesses robust in vitro M 4 PAM potency across species and in vivo efficacy in preclinical models of schizophrenia. Coupled with an attractive DMPK profile and suitable predicted human PK, 6c (VU0467485) was evaluated as a preclinical development candidate., Competing Interests: The authors declare the following competing financial interest(s): The authors are developing M4 PAMs for the treatment of schizophrenia and hold patents on the same.

This letter describes the further deconstruction of the known PAR4 inhibitor chemotypes (MWs 490-525 and with high plasma protein binding) to identify a minimum PAR4 pharmacophore devoid of metabolic liabilities and improved properties. This exercise identified a greatly simplified 2-methoxy-6-arylimidazo[2,1-b][1,3,4]thiadiazole scaffold that afforded nanomolar inhibition of both activating peptide and γ-thrombin mediated PAR4 stimulation, while reducing both molecular weight and the number of hydrogen bond donors/acceptors by ∼50%. This minimum PAR4 pharmacophore, with competitive inhibition, versus non-competitive of the larger chemotypes, allows an ideal starting point to incorporate desired functional groups to engender optimal DMPK properties towards a preclinical candidate., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This Letter describes the further lead optimization of the CHT inhibitor probe, ML352 (VU0476201), and the development of VU6001221, an improved in vivo tool. A multi-dimensional optimization effort encountered steep SAR, and ultimately, subtle tuning of the electronics of the central phenyl core provided VU6001221, a CHT inhibitor with comparable potency for choline uptake inhibition as ML352, yet improved PK and CNS penetration. Moreover, VU6001221 enabled evaluation, for the first time, of a CHT inhibitor in a standard preclinical rodent cognition model, novel object recognition (NOR). We observed VU6001221 to elicit a dose-responsive increase in NOR, raising the possibility of agonism of synaptic α7 nicotinic ACh receptors by elevated extracellular choline, that if confirmed would represent a novel molecular strategy to enhance cognition., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cyclooxygenase enzymes (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin G2. The inhibitory activity of rapid, reversible COX inhibitors (ibuprofen, naproxen, mefenamic acid, and lumiracoxib) demonstrated a significant increase in potency and time dependence of inhibition against double tryptophan murine COX-2 mutants at the 89/90 and 89/119 positions. In contrast, the slow, time-dependent COX inhibitors (diclofenac, indomethacin, and flurbiprofen) were unaffected by those mutations. Further mutagenesis studies suggested that mutation at position 89 was principally responsible for the changes in inhibitory potency of rapid, reversible inhibitors, whereas mutation at position 90 may exert some effect on the potency of COX-2-selective diarylheterocycle inhibitors; no effect was observed with mutation at position 119. Several crystal structures with or without NSAIDs indicated that placement of a bulky residue at position 89 caused a closure of a gap at the lobby, and alteration of histidine to tryptophan at position 90 changed the electrostatic profile of the side pocket of COX-2. Thus, these two residues, especially Val-89 at the lobby region, are crucial for the entrance and exit of some NSAIDs from the COX active site., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

A series of novel indomethacin analogues with carbaboranes as three-dimensional substitutes for the chlorophenyl ring have been prepared. Their cyclooxygenase (COX) inhibition and enzyme selectivity has been tested and compared to the corresponding adamantyl analogues. Surprisingly, only the ortho-carbaborane derivatives were active compounds. Preliminary biological studies gave an interesting insight into the validity of employing carbaboranes as pharmacophores., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their pharmacological activities by inhibiting cyclooxygenase (COX)-1 and COX-2. Previous studies have shown that esters and amides of non-selective inhibitors such as indomethacin are selective against COX-2, which is the therapeutically relevant isoform. Structure-activity analysis indicates that substituted phenyl rings are tolerated as ester components. In the present study, the introduction of inorganic ortho- and meta-carbaborane moieties was explored with the aim to create COX-2 inhibitors and more importantly to investigate the validity of using these boron clusters as drug entities. Interestingly, only the ortho-carbaborane ester was active whereas the meta isomer was not. A similar lack of inhibitory potency was observed when an adamantyl substituent or alkylene spacers at the carbaborane were introduced in the ester functionality., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Herein we report the discovery, synthesis, and evaluation of a series of N-(4-acetamido)-phenylpicolinamides as positive allosteric modulators of mGlu(4). Compounds from the series show submicromolar potency at both human and rat mGlu(4). In addition, pharmacokinetic studies utilizing subcutaneous dosing demonstrated good brain exposure in rats.

Lumiracoxib is the first example of a marketed COX-2 inhibitor of the arylacetic acid class, and it is reported to be the most selective COXIB in vivo. However, the molecular basis of its COX-2 inhibition has not been completely defined. Using standard assays, lumiracoxib was found to be a poor inhibitor of purified ovine COX-1 and a relatively weak inhibitor of purified human COX-2. The extent of COX-2 inhibition plateaued at around 50% and suggested that the inhibitor may be reversibly bound to the enzyme. Kinetic studies with lumiracoxib demonstrated that it was a time-dependent and slowly reversible inhibitor of human COX-2 that exhibited at least two binding steps during inhibition. Derivatives of lumiracoxib were synthesized with or without the methyl group on the phenylacetic acid ring and with various substitutions on the lower aniline ring. Inhibition studies demonstrated that the methyl group on the phenylacetic acid ring is required for COX-2 selectivity. The chemical identity and position of the substituents on the lower aniline ring were important in determining the potency and extent of COX inhibition as well as COX-2 selectivity. Mutation of Ser-530 to Ala or Val-349 to Ala or Leu abolished the potent inhibition observed with wild-type human COX-2 and key lumiracoxib analogs. Interestingly, a Val-349 to Ile mutant was inhibited with equal potency to human COX-2 with 2,6-dichloro-, 2,6-dimethyl-, or 2-chloro-6-methyl-substituted inhibitors and, in the case of lumiracoxib, actually showed an increase in potency. Taken together with a recent crystal structure of a lumiracoxib-COX-2 complex, the kinetic analyses presented herein of the inhibition of mutant COX-2s by lumiracoxib allows the definition of the molecular basis of COX-2 inhibition.

tert-Butyl acetylene (tBA) is a mechanism-based inactivator of cytochromes P450 2E1 and 2E1 T303A; however, the inactivation of the T303A mutant could be reversed by overnight dialysis. The inactivation of P450 2E1 T303A, but not the wild-type 2E1 enzyme, by tBA resulted in the formation of a novel reversible acetylene-iron spectral intermediate with an absorption maximum at 485 nm. The formation of this intermediate required oxygen and could be monitored spectrally with time. Although the alternate oxidants tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP) supported the inactivation of wild-type P450 2E1 by tBA in a reductase- and NADPH-free system, only tBHP supported the inactivation of the 2E1 T303A mutant. The losses in enzymatic activity occurred concomitantly with losses in the native P450 heme, which were accompanied by the formation of tBA-adducted heme products. The inactivations supported by tBHP and CHP were completely irreversible with overnight dialysis. Spectral binding constants (K(s)) for the binding of tBA to the 2E1 P450s together with models of the enzymes with the acetylenic inactivator bound in the active site suggest that the T303A mutation results in increased hydrophobic interactions between tBA and nearby P450 residues, leading to a higher binding affinity for the acetylene compound in the mutant enzyme. Together, these data support a role for the highly conserved T303 residue in proton delivery to the active site of P450 2E1 and in the inactivation of the 2E1 P450s by small acetylenic compounds.

This report investigates and characterizes the mechanism for the novel reversible inactivation of a T303A mutant of rabbit cytochrome P450 (P450) 2E1 by tert-butyl acetylene (tBA). P450 2E1 T303A was inactivated in a time-, concentration-, and NADPH-dependent manner through the formation of two tBA adducts to the P450 heme. Interestingly, losses in enzymatic activity and in the reduced CO spectrum of the tBA-inactivated T303A mutant could be restored to the samples after an overnight incubation at 4 degrees C. Removal of free tBA and NADPH from the tBA-inactivated T303A samples by spin column gel filtration demonstrated that the observed reversibility was time-dependent and was not significantly affected by the presence or absence of NADPH or tBA. Furthermore, the recovery of native heme was dependent on the native P450 enzyme structure. Electrospray ionization liquid chromatography-tandem mass spectrometry analysis under nondenaturing conditions of a preacidified tBA-inactivated T303A sample yielded two tBA adducts (m/z of 661 Da) with ion fragmentation patterns characteristic of a tBA adduct to the P450 heme. These adducts were absent in nonacidified samples subjected to the same conditions. In contrast, tandem mass spectrometry analysis of both non- and preacidified tBA-inactivated wild-type 2E1 samples yielded two tBA adducts (m/z of 661 Da) with ion fragmentation patterns similar to the preacidified T303A mutant adducts. These results lend insight into the reversible inactivation mechanism of the tBA-inactivated T303A mutant and suggest a role for the highly conserved threonine 303 residue in proton donation to the P450 2E1 active site and the stabilization of a reactive intermediate during substrate metabolism by P450.

tert-Butyl 1-methyl-2-propynyl ether (tBMP) was analyzed for its ability to act as a mechanism-based inactivator of p450 2B4. tBMP inactivated p450 2B4 in a time-, concentration-, and NADPH-dependent manner. Losses in activity occurred with concurrent losses in the reduced CO spectrum and native p450 heme; however, there was a greater loss in activity than could be accounted for by reduced CO spectra or native heme loss. LC/MS analysis demonstrated that the losses in native heme were accompanied by the appearance of two modified hemes with m/z values of 705Da, consistent with tBMP adducted hemes. Both adducts had identical fragmentation patterns when analyzed by LC/MS/MS. The spectra were consistent with a tBMP molecule and an oxygen atom attached to iron-depleted heme. Proton NMR studies suggest that the two modified hemes in p450 2B1 are N-alkylated on pyrrole rings A and D.