Nikolai Zvonok

Covalent Inhibitors of Human Monoacylglycerol Lipase: Ligand-Assisted Characterization of the Catalytic Site by Mass Spectrometry and Mutational Analysis

The active site of recombinant hexa-histidine-tagged human monoacylglycerol lipase (hMGL) is characterized by mass spectrometry using the inhibitors 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701), and N-arachidonylmaleimide (NAM) as probes. Carbamylation of Ser(129) by AM6701 in the putative hMGL catalytic triad demonstrates this residues essential role in catalysis. Partial NAM alkylation of hMGL cysteine residues 215 and/or 249 was sufficient to achieve approximately 80% enzyme inhibition. Although Cys(215) and/or Cys(249) mutations to alanine(s) did not affect hMGL hydrolytic activity as compared with nonmutated hMGL, the C215A displayed heightened NAM sensitivity, whereas the C249A evidenced reduced NAM sensitivity. These data conclusively demonstrate a sulfhydryl-based mechanism for NAM inhibition of hMGL in which Cys(249) is of paramount importance. Identification of amino acids critical to the catalytic activity and pharmacological modulation of hMGL informs the design of selective MGL inhibitors as potential drugs.

Membrane phospholipid bilayer as a determinant of monoacylglycerol lipase kinetic profile and conformational repertoire

The membrane‐associated serine hydrolase, monoacylglycerol lipase (MGL), is a well‐recognized therapeutic target that regulates endocannabinoid signaling. Crystallographic studies, while providing structural information about static MGL states, offer no direct experimental insight into the impact of MGLs membrane association upon its structure–function landscape. We report application of phospholipid bilayer nanodiscs as biomembrane models with which to evaluate the effect of a membrane system on the catalytic properties and conformational dynamics of human MGL (hMGL). Anionic and charge‐neutral phospholipid bilayer nanodiscs enhanced hMGLs kinetic properties [apparent maximum velocity (Vmax) and substrate affinity (Km)]. Hydrogen exchange mass spectrometry (HX MS) was used as a conformational analysis method to profile experimentally the extent of hMGL–nanodisc interaction and its impact upon hMGL structure. We provide evidence that significant regions of hMGL lid‐domain helix α4 and neighboring helix α6 interact with the nanodisc phospholipid bilayer, anchoring hMGL in a more open conformation to facilitate ligand access to the enzymes substrate‐binding channel. Covalent modification of membrane‐associated hMGL by the irreversible carbamate inhibitor, AM6580, shielded the active site region, but did not increase solvent exposure of the lid domain, suggesting that the inactive, carbamylated enzyme remains intact and membrane associated. Molecular dynamics simulations generated conformational models congruent with the open, membrane‐associated topology of active and inhibited, covalently‐modified hMGL. Our data indicate that hMGL interaction with a phospholipid membrane bilayer induces regional changes in the enzymes conformation that favor its recruiting lipophilic substrate/inhibitor from membrane stores to the active site via the lid, resulting in enhanced hMGL catalytic activity and substrate affinity.

Full Mass Spectrometric Characterization of Human Monoacylglycerol Lipase Generated by Large-Scale Expression and Single-Step Purification

The serine hydrolase monoacylglycerol lipase (MGL) modulates endocannabinoid signaling in vivo by inactivating 2-arachidonoylglycerol (2-AG), the main endogenous agonist for central CB1 and peripheral CB2 cannabinoid receptors. To characterize this key endocannabinoid enzyme by mass spectrometry-based proteomics, we first overexpressed recombinant hexa-histidine-tagged human MGL (hMGL) in Escherichia coli and purified it in a single chromatographic step with high yield (approximately 30 mg/L). With 2-AG as substrate, hMGL displayed an apparent V max of 25 micromol/(microg min) and K m of 19.7 microM, an affinity for 2-AG similar to that of native rat-brain MGL (rMGL) (Km=33.6 microM). hMGL also demonstrated a comparable affinity (Km approximately 8-9 microM) for the novel fluorogenic substrate, arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE), in a sensitive, high-throughput fluorometric MGL assay. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) unequivocably demonstrated the mass (34,126 Da) and purity of this hMGL preparation. After in-solution tryptic digestion, hMGL full proteomic characterization was carried out, which showed (1) an absence of intramolecular disulfide bridges in the functional, recombinant enzyme and (2) the post-translational removal of the enzymes N-terminal methionine. Availability of sufficient quantities of pure, well-characterized hMGL will enable further molecular and structural profiling of this key endocannabinoid-system enzyme.

Mass spectrometry-based proteomics of human cannabinoid receptor 2: covalent cysteine 6.47(257)-ligand interaction affording megagonist receptor activation.

The lack of experimental characterization of the structures and ligand-binding motifs of therapeutic G-protein coupled receptors (GPCRs) hampers rational drug discovery. The human cannabinoid receptor 2 (hCB2R) is a class-A GPCR and promising therapeutic target for small-molecule cannabinergic agonists as medicines. Prior mutational and modeling data constitute provisional evidence that AM-841, a high-affinity classical cannabinoid, interacts with cysteine C6.47(257) in hCB2R transmembrane helix 6 (TMH6) to afford improved hCB2R selectivity and unprecedented agonist potency. We now apply bottom-up mass spectrometry (MS)-based proteomics to define directly the hCB2R-AM-841 interaction at the amino-acid level. Recombinant hCB2R, overexpressed as an N-terminal FLAG-tagged/C-terminal 6His-tagged protein (FLAG-hCB2R-6His) with a baculovirus system, was solubilized and purified by immunochromatography as functional receptor. A multiplex multiple reaction monitoring (MRM)-MS method was developed that allowed us to observe unambiguously all seven discrete TMH peptides in the tryptic digest of purified FLAG-hCB2R-6His and demonstrate that AM-841 modifies hCB2R TMH6 exclusively. High-resolution mass spectra of the TMH6 tryptic peptide obtained by Q-TOF MS/MS analysis demonstrated that AM-841 covalently and selectively modifies hCB2R at TMH6 cysteine C6.47(257). These data demonstrate how integration of MS-based proteomics into a ligand-assisted protein structure (LAPS) experimental paradigm can offer guidance to structure-enabled GPCR agonist design.

Biochemical and mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase inhibition.

The mechanism of inactivation of human enzyme N-acylethanolamine-hydrolyzing acid amidase (hNAAA), with selected inhibitors identified in a novel fluorescent based assay developed for characterization of both reversible and irreversible inhibitors, was investigated kinetically and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 1-Isothiocyanatopentadecane (AM9023) was found to be a potent, selective and reversible hNAAA inhibitor, while two others, 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-Benzyloxycarbonyl-L-serine β-lactone (N-Cbz-serine β-lactone), inhibited hNAAA in a covalent and irreversible manner. MS analysis of the hNAAA/covalent inhibitor complexes identified modification only of the N-terminal cysteine (Cys126) of the β-subunit, confirming a suggested mechanism of hNAAA inactivation by the β-lactone containing inhibitors. These experiments provide direct evidence of the key role of Cys126 in hNAAA inactivation by different classes of covalent inhibitors, confirming the essential role of cysteine for catalysis and inhibition in this cysteine N-terminal nucleophile hydrolase enzyme. They also provide a methodology for the rapid screening and characterization of large libraries of compounds as potential inhibitors of NAAA, and subsequent characterization or their mechanism through MALDI-TOF MS based bottom up-proteomics.

Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s)

Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs.

Mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase.

N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme that primarily degrades palmitoylethanolamine (PEA), a lipid amide that inhibits inflammatory responses. We developed a HEK293 cell line stably expressing the NAAA pro-enzyme (zymogen) and a single step chromatographic purification of the protein from the media. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry MALDI-TOF MS analysis of the zymogen (47.7 kDa) treated with peptide-N-glycosidase F (PNGase F) identified 4 glycosylation sites, and acid cleavage of the zymogen into α- and β-subunits (14.6 and 33.3 kDa) activated the enzyme. Size exclusion chromatography estimated the mass of the active enzyme as 45 ± 3 kDa, suggesting formation of an α/β heterodimer. MALDI-TOF MS fingerprinting covered more than 80% of the amino acid sequence, including the N-terminal peptides, and evidence for the lack of a disulfide bond between subunits. The significance of the cysteine residues was established by their selective alkylation resulting in almost complete loss of activity. The purified enzyme was kinetically characterized with PEA and a novel fluorogenic substrate, N-(4-methyl coumarin) palmitamide (PAMCA). The production of sufficient quantities of NAAA and a high throughput assay could be useful in discovering novel inhibitors and determining the structure and function of this enzyme.

Functional selectivity at G-protein coupled receptors: Advancing cannabinoid receptors as drug targets

ABSTRACT The phenomenon of functional selectivity, whereby a ligand preferentially directs the information output of a G‐protein coupled receptor (GPCR) along (a) particular effector pathway(s) and away from others, has redefined traditional GPCR signaling paradigms to provide a new approach to structure‐based drug design. The two principal cannabinoid receptors (CBRs) 1 and 2 belong to the class‐A GPCR subfamily and are considered tenable therapeutic targets for several indications. Yet conventional orthosteric ligands (agonists, antagonists/inverse agonists) for these receptors have had very limited clinical utility due to their propensity to incite on‐target adverse events. Chemically distinct classes of cannabinergic ligands exhibit signaling bias at CBRs towards individual subsets of signal transduction pathways. In this review, we discuss the known signaling pathways regulated by CBRs and examine the current evidence for functional selectivity at CBRs in response to endogenous and exogenous cannabinergic ligands as biased agonists. We further discuss the receptor and ligand structural features allowing for selective activation of CBR‐dependent functional responses. The design and development of biased ligands may offer a pathway to therapeutic success for novel CBR‐targeted drugs.

Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase

Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH, we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs.

Active-site inhibitors modulate the dynamic properties of human monoacylglycerol lipase: a hydrogen exchange mass spectrometry study.

Human monoacylglycerol lipase (hMGL) regulates endocannabinoid signaling primarily by deactivating the lipid messenger 2-arachidonoylglycerol. Agents that carbamylate hMGLs catalytic Ser(122) constitute a leading class of therapeutically promising hMGL inhibitors. We have applied peptide-level hydrogen/deuterium exchange mass spectrometry to characterize hMGLs conformational responses to two potent carbamylating inhibitors, AM6580 (irreversible) and AM6701 (slowly reversible). A dynamic, solvent-exposed lid domain is characteristic of hMGLs solution conformation. Both hMGL inhibitors restricted backbone enzyme motility in the active-site region and increased substrate binding-pocket solvent exposure. Covalent reaction of AM6580 with hMGL generates a bulkier carbamylated Ser(122) residue as compared to the more discrete Ser(122) modification by AM6701, a difference reflected in AM6580s more pronounced effect upon hMGL conformation. We demonstrate that structurally distinct carbamylating hMGL inhibitors generate particular conformational ensembles characterized by region-specific hMGL dynamics. By demonstrating the distinctive influences of two hMGL inhibitors on enzyme conformation, this study furthers our understanding at the molecular level of the dynamic features of hMGL interaction with small-molecule ligands.