(γ)-glutamyl carboxylase (GGCX) is an integral membrane
protein responsible for the post-translational catalytic conversion
of select glutamic acid (Glu) residues to γ-carboxy glutamic
acid (Gla) in vitamin K-dependent (VKD) proteins. Understanding the
mechanism of carboxylation and the role of GGCX in the vitamin K cycle
is of biological interest in the development of therapeutics for blood
coagulation disorders. Historically, biophysical investigations and
structural characterizations of GGCX have been limited due to complexities
involving the availability of an appropriate model membrane system.
In previous work, a hydrogen exchange mass spectrometry (HX MS) platform
was developed to study the structural configuration of GGCX in a near-native
nanodisc phospholipid environment. Here we have applied the nanodisc–HX
MS approach to characterize specific domains of GGCX that exhibit
structural rearrangements upon binding the high-affinity consensus
propeptide (pCon; AVFLSREQANQVLQRRRR). pCon binding
was shown to be specific for monomeric GGCX-nanodiscs and promoted
enhanced structural stability to the nanodisc-integrated complex while
maintaining catalytic activity in the presence of carboxylation co-substrates.
Noteworthy modifications in HX of GGCX were prominently observed in
GGCX peptides 491–507 and 395–401 upon pCon association,
consistent with regions previously identified as sites for propeptide
and glutamate binding. Several additional protein regions exhibited
minor gains in solvent protection upon propeptide incorporation, providing
evidence for a structural reorientation of the GGCX complex in association
with VKD carboxylation. The results herein demonstrate that nanodisc–HX
MS can be utilized to study molecular interactions of membrane-bound
enzymes in the absence of a complete three-dimensional structure and
to map dynamic rearrangements induced upon ligand binding.
We report the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggest that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. These first-in-class covalent K-Ras inhibitors demonstrate that irreversible targeting of the K-Ras guanine-nucleotide binding site is potentially a viable therapeutic strategy for inhibition of Ras signaling.
Cancer; Covalent inhibitor; Drug design; K-Ras
The higher order structure of protein therapeutics can be interrogated with hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS is now a widely used tool in the structural characterization of protein therapeutics. In this article, HDX-MS based workflows designed for both protein therapeutic discovery and development processes are presented, focusing on the specific applications of epitope mapping for protein/drug interactions and biopharmaceutical comparability studies. Future trends in the application of HDX-MS to protein therapeutics characterization are also described.
Peptide drugs have traditionally suffered from poor pharmacokinetic properties due to their conformational flexibility and the interaction of proteases with backbone amide bonds. “Stapled Peptides” are cyclized using an all-hydrocarbon cross-linking strategy to reinforce their α-helical conformation, yielding improved protease resistance and drug-like properties. Here we demonstrate that Hydrogen Exchange-Mass Spectrometry (HX-MS) effectively probes the conformational dynamics of Stapled Peptides derived from the survivin-borealin protein-protein interface and predicts their susceptibility to proteolytic degradation. In Stapled Peptides, amide exchange was reduced by over five orders-of-magnitude versus the native peptide sequence depending on staple placement. Furthermore, deuteration kinetics correlated directly with rates of proteolysis to reveal the optimal staple placement for improved drug properties.
helix; stapled-peptide; protein folding; deuterium
Noisy and overlapped mass spectrometry data hinders the sequence coverage that can be obtained from Hydrogen Deuterium exchange analysis, and places a limit on the complexity of the samples that can be studied by this technique. Advances in instrumentation have addressed these limits, but as the complexity of the biological samples under investigation increases, these problems are reencountered. Here we describe the use of binomial distribution fitting with asymmetric linear squares regression for calculating the accurate deuterium content for mass envelopes of low signal or that contain significant overlap. The approach is demonstrated with a test data set of HIV Env gp140 wherein inclusion of the new analysis regime resulted in obtaining exchange data for 42 additional peptides, improving the sequence coverage by 11%. At the same time, the precision of deuterium uptake measurements was improved for nearly every peptide examined. The improved processing algorithms also provide an efficient method for deconvolution of bimodal mass envelopes and EX1 kinetic signatures. All these functions and visualization tools have been implemented in the new version of the freely available software, HX-Express v2.
To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers was compared to their simplest aggregated form, i.e., dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry (H/DX-MS) and other biophysical measurements was used to make the comparison. The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen deuterium exchange compared to its dimer form. In this case, differences observed were located in specific functional regions of the CH2 domain and the hinge region between CH1 and CH2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.
antibody dimerization; aggregation; size-exclusion chromatography (SEC); hydrogen-deuterium exchange mass spectrometry (H/DX-MS); differential scanning calorimetry (DSC); N-linked glycosylation; small-angle x-ray solution scattering (SAXS); domain swapping
Many proteins are post-translationally modified by acylation targetting them to lipid membranes. While methods such as X-ray crystallography and NMR are available to determine the structure of folded proteins in solution, the precise position of folded domains relative to a membrane remains largely unknown. We used neutron and X-ray reflection methods to measure the displacement of the core domain of HIV Nef from lipid membranes upon insertion of the N-terminal myristate group. Nef is one of several HIV-1 accessory proteins and an essential factor in AIDS progression. Upon insertion of the myristate and residues from the N-terminal arm, Nef transitions from a closed to open conformation that positions the core domain 70 Å from the lipid headgroups. This work rules out speculation that the Nef core remains closely associated with the membrane to optimize interactions with the cytoplasmic domain of MHC-1.
Notch receptors are single-pass transmembrane proteins that regulate development and tissue homeostasis in all metazoan organisms. Prior to ligand-induced signaling, Notch receptors adopt a proteolytic-resistant conformation maintained by a critical interdomain interface within a negative regulatory region (NRR), which sits immediately external to the plasma membrane. Signaling is initiated when ligand binding induces exposure of the proteolytic cleavage site, termed S2, within the NRR. Here, we use hydrogen exchange in conjunction with mass spectrometry (HX-MS) to study the dynamics of the human Notch3 NRR in four distinct biochemical states: in its unmodified quiescent form, in a proteolytically “on” state induced by EDTA, and in complex with either agonist or inhibitory antibodies. Induction of the “on” state by either EDTA or the agonist monoclonal antibody leads to accelerated deuteration in the region of the S2 cleavage site, reflecting an increase in S2 dynamics. In contrast, complexation of the Notch3 NRR with an inhibitory antibody retards deteuration not only across its discontinuous binding epitope, but also around the S2 site, stabilizing the NRR in its “off” state. Together with previous work investigating the dynamics of the Notch1 NRR, these studies show that key features of autoinhibition and activation are shared among different Notch receptors, and provide additional insights into mechanisms of Notch activation and inhibition by modulatory antibodies.
Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.
Human monoacylglycerol lipase (hMGL) regulates endocannabinoid signaling primarily by deactivating the lipid messenger 2-arachidonoylglycerol. Agents that carbamylate hMGLs catalytic Ser122 constitute a leading class of therapeutically promising hMGL inhibitors. We have applied peptide-level hydrogen/deuterium exchange mass spectrometry to characterize hMGL’s conformational responses to two potent carbamylating inhibitors, AM6580 (irreversible) and AM6701 (slowly reversible). A dynamic, solvent-exposed lid domain is characteristic of hMGL’s 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 Ser122 residue as compared to the more discrete Ser122 modification by AM6701, a difference reflected in AM6580’s more pronounced effect upon hMGL conformation. We demonstrate that structurally distinct carbamylating hMGL inhibitors generate particular conformational ensembles characterized by regionspecific 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.
Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, exist and in many cases are important for protein function. The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days. In this review we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples. Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of inter- and intra-molecular interactions in the global control of kinase function. Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signaling proteins. HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both classification of and greater understanding of the prevalence of protein motion.
Src-family kinase; Hck; Lck; SH3 domain; SH2 domain; Abl; deuterium; HDX; protein dynamics, flexibility
Understanding the conformation of antibodies, especially those of therapeutic value, is of great interest. Many of the current analytical methods used to probe protein conformation face issues in the analysis of antibodies, either due to the nature of the antibody itself or the limitations of the method. One method that has recently been utilized for conformational analysis of antibodies is hydrogen/deuterium exchange mass spectrometry (H/DX MS). H/DX MS can be used to probe the conformation and dynamics of proteins in solution, requires small sample quantities, is compatible with many buffer systems, and provides peptide-level resolution. The application of H/DX MS to immunoglobulin gamma 1 (IgG1) recombinant monoclonal antibodies can provide information about IgG1 conformation, dynamics, and changes to conformation as a result of protein modification(s), changes in storage conditions, purification procedures, formulation, and many other parameters. In this article we provide a comprehensive HD/X MS protocol for the analysis of an antibody.
immunoglobin; deuterium; conformation; protein structure; antibody; hydrogen exchange
Itk and Btk are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of six of the 619 amino acid residues of Itk with those of Btk was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the observed differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties depending on the cellular context. We suggest that the weaker catalytic activities observed for T cell–specific kinases is one mechanism to regulate cellular activation and prevent aberrant immune responses.
The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from >30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5-6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data.
Mass spectrometry; aspergillopepsin; factor XIII; hydrogen exchange; rice field eel; Monopterus albus Zuiew; online digestion
Although the use of hydrogen exchange (HX) mass spectrometry (MS) to study proteins and protein conformation is now over 20 years old, the perception lingers that it still has “issues”. Is this method, in fact, still in the quicksand with many remaining obstacles to overcome? We do not think so. This critical insight addresses the “issues” and explores several broad questions including: have the limitations of HX MS been surmounted and has HX MS achieved “indispensable” status in the pantheon of protein structural analysis tools.
A major component of a hydrogen exchange mass spectrometry experiment is the analysis of protein and peptide mass spectra to yield information about deuterium incorporation. The processing of data that are produced includes the identification of each peptic peptide to create a master table/array of peptide sequence, retention time and retention time range, mass range and undeuterated mass. The amount of deuterium incorporated into each of the peptides in this array must then be determined. Various software platforms have been developed in order to perform this specific type of data analysis. We describe the fundamental parameters to be considered at each step along the way and how data processing, either by an individual or by software, must approach the analysis.
Deuterium; software; algorithm; protein dynamics; isotope
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.
HIV-1 has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host-cell enzymes. The HIV-1 virion infectivity factor, one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and down-regulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution including the APOBEC3G/F binding site and HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.
Accessory protein; APOBEC3F/G; E3 Ligase; hydrogen exchange; mass spectrometry; deuterium; Vif
Although small molecule actin modulators have been widely used as research tools, only one cell permeable small molecule inhibitor of actin depolymerization (jasplakinolide) is commercially available. We report that the natural product cucurbitacin E inhibits actin depolymerization and show that its mechanism of action is different from jasplakinolide. In assays using pure fluorescently labeled actin, cucurbitacin E specifically affected depolymerization without affecting polymerization. It inhibited actin depolymerization at sub-stoichiometric concentrations up to 1:6 cucurbitacin:actin E. Cucurbitacin E specifically binds to filamentous actin (F-actin) forming a covalent bond at residue Cys257, but not to monomeric actin (G-actin). Based on its compatibility with phalloidin staining, we show that cucurbitacin E occupies a different binding site on actin filaments. Using loss of fluorescence after localized photoactivation, we found that cucurbitacin E inhibited actin depolymerization in live cells. Cucurbitacin E is a widely available plant-derived natural product, making it a useful tool to study actin dynamics in cells and actin-based processes such as cytokinesis.
The function, efficacy, and safety of protein biopharmaceuticals are tied to their three-dimensional structure. The analysis and verification of this higher-order structure are critical in demonstrating manufacturing consistency and in establishing the absence of structural changes in response to changes in production. It is, therefore, essential to have reliable, high-resolution and high sensitivity biophysical tools capable of interrogating protein structure and conformation. Here, we demonstrate the use of hydrogen/deuterium exchange mass spectrometry (H/DX-MS) in biopharmaceutical comparability studies. H/DX-MS measurements can be conducted with good precision, consume only picomoles of protein, interrogate nearly the entire molecule with peptide level resolution, and can be completed in a few days. Structural comparability or lack of comparability was monitored for different preparations of interferon-β-1a. We present specific graphical formats for the display of H/DX-MS data that aid in rapidly making both the qualitative (visual) and quantitative assessment of comparability. H/DX-MS is capable of making significant contributions in biopharmaceutical characterization by providing more informative and confident comparability assessments of protein higher order structure than are currently available within the biopharmaceutical industry.
biotherapeutic; interferon; protein conformation; biosimilar; protein drug; structure
Pepsin was immobilized on ethyl-bridged hybrid (BEH) particles and digestion performance was evaluated in a completely online format, with the specific intent of using the particles for hydrogen deuterium exchange mass spectrometry (HDX MS) experiments. Because the BEH particles are mechanically strong, they could withstand prolonged, continuous high-pressure at 10,000 psi. Online digestion was performed under isobaric conditions with continuous solvent flow, in contrast to other approaches where the pressure or flow is cycled. As expected, digestion efficiency at 10,000 psi was increased and reproducibly produced more peptic peptides versus digestion at 1,000 psi. Prototype columns made with the BEH pepsin particles exhibited robust performance and deuterium back-exchange was similar to that of other immobilized pepsin particles. These particles can be easily incorporated in existing HDX MS workflows to provide more peptide coverage in experiments where fast, efficient, and reproducible online pepsin digestion is desired.
Pressure; online proteolysis; enzyme; bioreactor; ultra performance liquid chromatography; UPLC; LC; ethyl-bridged hybrid; BEH; acid protease
Discovery of EX1 kinetics in hydrogen exchange (HX) mass spectrometry (MS) experiments is rare. Proteins follow the EX1 kinetic regime when cooperative unfolding events simultaneously expose multiple residues to solvent such that they all become deuterated together before the region is able to refold. A number of factors can contribute to what we call “false EX1” in which it appears as though EX1 occurs in a protein when it probably does not. One of the contributors to false EX1 is peptide carryover between chromatographic runs. In this work, we explore the origins of peptide carryover in HX MS, describe how carryover causes mass spectra to indicate false EX1 kinetics and then describe an optimized washing protocol that can be used to eliminate peptide carryover. A series of solvent injections was developed and found to efficiently eliminate carryover signatures such that analysis of deuterium incorporation could be reliably followed for two proteins prone to high carryover.
Peptide carryover; EX1 kinetics; hydrogen exchange; mass spectrometry; deuterium
The HIV-1 accessory protein Nef is N-terminally myristoylated and this posttranslational modification is essential for Nef function in AIDS progression. Transfer of a myristate group from myristoyl coenzyme A to Nef occurs cotranslationally and is catalyzed by human N-myristoyl transferase-1 (NMT). To investigate the conformational effects of myristoylation on Nef structure as well as to probe the nature of the Nef: NMT complex, we investigated various forms of Nef with hydrogen exchange mass spectrometry. Conformational changes in Nef were not detected as a result of myristoylation and NMT had no effect on deuterium uptake by Nef in a myrNef:NMT complex. However, myrNef binding did have an effect on NMT deuterium uptake. Major HX differences in NMT were primarily located around the active site, with more subtle differences, at the longer timepoints, across the structure. At the shortest timepoint, significant differences between the two states were observed in two regions which interact strongly with the phosphate groups of coenzyme A. Based on our results, we propose a model of the Nef:NMT complex in which only the myristoyl moiety holds the two proteins together in complex and speculate that perhaps NMT chaperones Nef to the membrane and thereby protect the myristic acid group from the cytosol rather than Nef operating through a myristic acid switch mechanism.
Hydrogen exchange; mass spectrometry; protein dynamics; myristoyl coenzyme A; myristoylation
Biologics such as monoclonal antibodies are much more complex than small-molecule drugs, which raises challenging questions for the development and regulatory evaluation of follow-on versions of such biopharmaceutical products (also known as biosimilars) and their clinical use once patent protection for the pioneering biologic has expired. With the recent introduction of regulatory pathways for follow-on versions of complex biologics, the role of analytical technologies in comparing biosimilars with the corresponding reference product is attracting substantial interest in establishing the development requirements for biosimilars. Here, we discuss the current state of the art in analytical technologies to assess three characteristics of protein biopharmaceuticals that regulatory authorities have identified as being important in development strategies for biosimilars: post-translational modifications, three-dimensional structures and protein aggregation.
In the mammalian central nervous system, monoacylglycerol
(MGL) is principally responsible for inactivating the endocannabinoid
signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1
receptor (CB1R) desensitization and signal intensity. MGL is also
a drug target for diseases in which CB1R stimulation may be therapeutic.
To inform the design of human MGL (hMGL) inhibitors, we have engineered
a Leu(Leu169;Leu176)-to-Ser(Ser169;Ser176) double hMGL mutant (sol-hMGL) which exhibited
enhanced solubility properties, and we further mutated this variant
by substituting its catalytic-triad Ser122 with Cys (sol-S-hMGL).
The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter
substrate with comparable affinities. Our results suggest that the
hMGL cysteine mutant maintains the same overall architecture as wild-type
hMGL. The results also underscore the superior nucleophilic nature
of the reactive catalytic Ser122 residue as compared to
that of Cys122 in the sol-S-hMGL mutant and suggest that
the nucleophilic character of the Cys122 residue is not
commensurately enhanced within the three dimensional architecture
of hMGL. The interaction of the sol-hMGL variants with the irreversible
inhibitors AM6580 and N-arachidonylmaleimide (NAM)
and the reversible inhibitor AM10212 was profiled. LC/MS analysis
of tryptic digests from sol-S-hMGL directly demonstrate covalent modification
of this variant by NAM and AM6580, consistent with enzyme thiol alkylation
and carbamoylation, respectively. These data provide insight into
hMGL catalysis, the key role of the nucleophilic character of Ser122, and the mechanisms underlying hMGL inhibition by different
classes of small molecules.
Active site; catalytic mechanism; drug design; enzyme inhibition; serine hydrolase