Generation of recombinant antibodies against human tissue kallikrein 7 to treat skin diseases.

Human tissue kallikreins (KLKs) constitute a family of 15 serine proteases that are distributed in various tissues and implicated in several pathological disorders. KLK7 is an unusual serine protease that presents both trypsin-like and chymotrypsin-like specificity and appears to be upregulated in pathologies that are related to skin desquamation processes, such as atopic dermatitis, psoriasis and Netherton syndrome. In recent years, various groups have worked to develop specific inhibitors for this enzyme, as KLK7 represents a potential target for new therapeutic procedures for diseases related to skin desquamation processes. In this work, we selected nine different single-chain variable fragment antibodies (scFv) from a human naïve phage display library and characterized their inhibitory activities against KLK7. The scFv with the lowest IC50 against KLK7 was affinity maturated, which resulted in the generation of four new scFv-specific antibodies for the target protease. These new antibodies were expressed in the scFv-Fc format in HEK293-6E cells, and the characterization of their inhibitory activities against KLK7 showed that three of them presented IC50 values lower than that of the original antibody. The cytotoxicity analysis of these recombinant antibodies demonstrated that they can be safely used in a cellular model. In conclusion, our research showed that in our case, a phage-display methodology in combination with enzymology assays can be a very suitable tool for the development of inhibitors for KLKs, suggesting a new strategy to identify therapeutic protease inhibitors for diseases related to uncontrolled kallikrein activity.

Detection of Active Granzyme A in NK92 Cells with Fluorescent Activity-Based Probe.

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.

Identification of Zika Virus NS2B-NS3 Protease Inhibitors by Structure-Based Virtual Screening and Drug Repurposing Approaches.

The NS2B-NS3 protease has been identified as an attractive target for drug development against Zika virus (ZIKV) and combined drug repurposing and structure-based virtual screening has improved the development of antiviral drugs. In this study, we performed a structure-based virtual screening of 1861 Food and Administration (FDA) approved drugs available in DrugBank by the selection and docking validation of crystal structure of ZIKV NS2B-NS3 protease (PDB ID 5H4I ) using Glide and DOCK 6 software. The antihistaminic chlorcyclizine (Grid score -24.8 kcal/mol) exhibited the most promising interaction with NS2B-NS3 protease in comparison to crystallography ligand (Grid score -15.6 kcal/mol) by interaction to Tyr161 by hydrophobic interactions in the binding site of NS2B-NS3 which is recognized as an important amino acid in substrate molecular recognition. Cytotoxicity and global antiviral activity assay in Vero cells by MTT method showed that chlorcyclizine reduced the ZIKV induced cytopathic effect (EC50 of 69.0 ± 7.3 µM and SI = 1.9), and explicit molecular dynamics simulations implemented on a NAMD program indicated great stability of chlorcyclizine in protease binding site, suggesting the repurposing of chlorcyclizine as a promising finding in anti-ZIKV drug development.

Venoms of Iranian Scorpions (Arachnida, Scorpiones) and Their Potential for Drug Discovery.

Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including Androctonus crassicauda, Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus, in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.

Novel peptidyl α-aminoalkylphosphonates as inhibitors of hepatitis C virus NS3/4A protease.

Herein, we describe the synthesis and application of novel phosphonic inhibitors designed to target the NS3/4A protease, which is crucial for the life cycle of hepatitis C virus. We examined the inhibitory potency of our synthesized compounds against two genotypes (1a and 1b) of NS3/4A protease and four mutant strains of HCV. The most potent inhibitors displayed k2/KI values of 79 850 M-1s-1 and 60 850 M-1s-1 against genotype 1a and 1b protease, respectively. Further in vitro evaluation of the most potent inhibitors revealed that vastly reduced HCV replication. Cellular toxicity, plasma stability, reactivity with selected human proteases as well the stability of inhibitor-protease complex and its intracellular availability are also discussed.

The prolyl oligopeptidase inhibitor IPR19 ameliorates cognitive deficits in mouse models of schizophrenia.

Cognitive deficits are considered a key feature of schizophrenia, and they usually precede the onset of the illness and continue after psychotic symptoms appear. Current antipsychotic drugs have little or no effect on the cognitive deficits of this disorder. Prolyl oligopeptidase (POP) is an 81-kDa monomeric serine protease that is expressed in brain and other tissues. POP inhibitors have shown neuroprotective, anti-amnesic and cognition-enhancing properties. Here we studied the potential of IPR19, a new POP inhibitor, for the treatment of the cognitive symptoms related to schizophrenia. The efficacy of the inhibitor was evaluated in mouse models based on subchronic phencyclidine and acute dizocilpine administration, and in adult offspring from mothers with immune reaction induced by polyinosinic:polycytidylic acid administration during pregnancy. Acute IPR19 administration (5mg/kg, i.p.) reversed the cognitive performance deficits of the three mouse models in the novel object recognition test, T-maze, and eight-arm radial maze. The compound also ameliorates deficits of the prepulse inhibition response. The in vitro inhibitory efficacy and selectivity, brain penetration and exposure time after injection of IPR19 were also addressed. Our results indicate that the inhibition of POP using IPR19 may offer a promising strategy to develop drugs to ameliorate the cognitive deficits of schizophrenia.

The potential effect of the angiotensin II receptor blocker telmisartan in regulating OVA-induced airway remodeling in experimental rats.

BACKGROUND: Bronchial asthma is a true ascending clinical problem. Angiotensin II is now accused to be potentially implicated in its pathogenesis, being a potent pro-inflammatory mediator with remodeling effects. OBJECTIVE: This study aims to evaluate the possible protective effect of telmisartan, an angiotensin II receptor blocker, on experimentally-induced bronchial asthma. METHODS: Animals were divided into 5 groups; a normal control group, an asthma control group, a reference treatment group, receiving dexamethasone, and two treatment groups, receiving telmisartan in two dose levels. Bronchial asthma was induced by intraperitoneal sensitization followed by intranasal challenge with ovalbumin (OVA). Test agents were administered prior to each intranasal OVA challenge. Lung function tests, namely tidal volume (TV) and peak expiratory flow rate (PEF) were assessed 1h after the last challenge. One day after the last challenge, absolute eosinophil counts (AEC) in blood and bronchoalveolar lavage fluids (BALF) were assessed. Serum immunoglobulin E (IgE) as well as BALF total nitrate/nitrite (NOx) were assessed. Oxidative and inflammatory biomarkers, namely lung tissue superoxide dismutase (SOD), glutathione reduced (GSH), tumor necrosis factor-alpha (TNF-α) and interleukin-5 (IL-5), were also assessed, in addition to histopathological study. RESULTS: Telmisartan administration in both doses significantly improved TV, PEF, AEC, IgE, NOx, GSH, SOD, TNF-α and IL-5 values compared to asthma control values. Histopathological study strongly supported the results of biochemical estimations, particularly regarding airway remodeling. CONCLUSION: These results suggest that telmisartan may have potential protecting effects against experimental bronchial asthma, probably due to its bronchodilator, antioxidant and anti-inflammatory effects.

Pharmacological properties and pathophysiological significance of a Kunitz-type protease inhibitor (Rusvikunin-II) and its protein complex (Rusvikunin complex) purified from Daboia russelii russelii venom.

A 7.1 kDa basic peptide (Rusvikunin-II) was purified from a previously described protein complex (Rusvikunin complex, consists of Rusvikunin and Rusvikunin-II) of Daboia russelii russelii venom. The N-terminal sequence of Rusvikunin-II was found to be blocked, but peptide mass fingerprinting analysis indicated its identity as Kunitz-type basic protease inhibitor 2, previously reported from Russell's Viper venom. A tryptic peptide sequence of Rusvikunin-II containing the N-terminal sequence HDRPTFCNLFPESGR demonstrated significant sequence homology to venom basic protease inhibitors, Kunitz-type protease inhibitors and trypsin inhibitors. The secondary structure of Rusvikunin-II was dominated by ß-sheets (60.4%), followed by random coil (38.2%), whereas α-helix (1.4%) contributes the least to its secondary structure. Both Rusvikunin-II and the Rusvikunin complex demonstrated dose-dependent anticoagulant activity; however, the anticoagulant potency of latter was found to be higher. Both inhibited the amidolytic activity of trypsin > plasmin >> FXa, fibrinogen clotting activity of thrombin, and, to a lesser extent, the prothrombin activation property of FXa; however, the inhibitory effect of the Rusvikunin complex was more pronounced. Neither Rusvikunin-II nor Rusvikunin complex inhibited the amidolytic activity of chymotrypsin and thrombin. Rusvikunin-II at 10 µg/ml was not cytotoxic to Colo-205, MCF-7 or 3T3 cancer cells; conversely, Rusvikunin complex showed ∼30% reduction of MCF-7 cells under identical experimental conditions. Rusvikunin-II (5.0 mg/kg body weight, i.p. injection) was not lethal to mice or House Geckos; nevertheless, it showed in vivo anticoagulant action in mice. However, the Rusvikunin complex (at 5.0 mg/kg) was toxic to NSA mice, but not to House Geckos, suggesting it has prey-specific toxicity. Rusvikunin complex-treated mice exhibited dyspnea and hind-limb paresis prior to death. The present study indicates that the Kunitz-type protein complex Rusvikunin from Russell's Viper venom significantly contributes to venom toxicity, and an important biological role in venoms appears to be facilitation of prey subjugation.

Discovery of novel 2-hydroxydiarylamide derivatives as TMPRSS4 inhibitors.

TMPRSS4 is a novel type II transmembrane serine protease that has been implicated in the invasion and metastasis of colon cancer cells. In this study, a novel series of 2-hydroxydiarylamide derivatives were synthesized and evaluated for inhibiting TMPRSS4 serine protease activity and suppressing cancer cell invasion. These derivatives demonstrated good inhibitory activity against TMPRSS4 serine protease, which correlated with the promising anti-invasive activity of colon cancer cells overexpressing TMPRSS4.

PIVL, a new serine protease inhibitor from Macrovipera lebetina transmediterranea venom, impairs motility of human glioblastoma cells.

A novel Kunitz-type serine proteinase inhibitor, termed PIVL, was purified to homogeneity from the venom of the Tunisian snake Macrovipera lebetina transmediterranea. It is a monomeric polypeptide chain cross-linked by three disulfide linkages with an isotope-averaged molecular mass of 7691.7 Da. The 67-residue full-length PIVL sequence was deduced from a venom gland cDNA clone. Structurally, PIVL is built by a single Kunitz/BPTI-like domain. Functionally, it is able to specifically inhibit trypsin activity. Interestingly, PIVL exhibits an anti-tumor effect and displays integrin inhibitory activity without being cytotoxic. Here we show that PIVL is able to dose-dependently inhibit the adhesion, migration and invasion of human glioblastoma U87 cells. Our results also show that PIVL impairs the function of αvß3 and to a lesser extent, the activity of αvß6, αvß5, α1ß1 and α5ß1 integrins. Interestingly, we demonstrate that the (41)RGN(43) motif of PIVL is likely responsible for its anti-cancer effect. By using time lapse videomicroscopy, we found that PIVL significantly reduced U87 cells motility and affected cell directionality persistence by 68%. These findings reveal novel pharmacological effects for a Kunitz-type serine proteinase inhibitor.

Pro-soft Val-boroPro: a strategy for enhancing in vivo performance of boronic acid inhibitors of serine proteases.

Val-boroPro, 1, is a potent, but relatively nonspecific inhibitor of the prolyl peptidases. It has antihyperglycemic activity from inhibition of DPPIV but also striking anticancer activity and a toxicity for which the mechanisms are unknown. 1 cyclizes at physiological pH, which attenuates its inhibitory potency >100-fold, which is a "soft drug" effect. Here we show that this phenomenon can be exploited to create prodrugs with unique properties and potential for selective in vivo targeting. Enzyme-mediated release delivers 1 to the target in the active form at physiological pH; cyclization attenuates systemic pharmacological effects from subsequent diffusion. This "pro-soft" design is demonstrated with a construct activated by and targeted to DPPIV, including in vivo results showing improved antihyperglycemic activity and reduced toxicity relative to 1. Pro-soft derivatives of 1 can help to illuminate the mechanisms underlying the three biological activities, or to help localize 1 at a tumor and thereby lead to improved anticancer agents with reduced toxicity. The design concept can also be applied to a variety of other boronic acid inhibitors.

Effects of diverse psychopharmacological substances on the activity of brain prolyl oligopeptidase.

Prolyl oligopeptidase (POP) has been connected to learning, memory and mood. Changes in serum or plasma POP activity have been linked to psychiatric disorders. POP has been thought to interfere in these conditions by cleaving neuroactive peptides or via the phosphatidylinositol second messenger system. However, little is known about the possible POP inhibition of commonly used psychoactive drugs. In this study, we measured the effects of various psychotropic drugs, including antidepressants, antipsychotics, mood stabilisers and anxiolytics, on the activity of the rat brain homogenate POP. Of the 38 compounds tested, 18 inhibited POP by at least 20% at 10 µM (buspirone, chlorpromazine, citalopram, clozapine, desipramine, duloxetine, escitalopram, flupenthixol, imipramine, ketanserin, lamotrigine, levomepromazine, prazosin, prochlorperazine, promazine, risperidone ritanserin and thioridazine). Thioridazine and valproate (VPA) acted at therapeutic plasma levels. Kinetically, VPA was a competitive inhibitor, thioridazine a non-competitive inhibitor and ketanserin a mixed type inhibitor. Being lipophilic, many of the psychoactive compounds are present in the brain at several-times higher concentrations than in plasma. At concentrations reported to be reached in the brain, chlorpromazine, clozapine, desipramine, imipramine, prochlorperazine and promazine inhibited POP by 30-50% suggesting that they could inhibit POP in vivo. However, when studied ex vivo, a single dose of 10 mg/kg thioridazine caused a deep sedation in the mice but did not inhibit the activity of POP. In conclusion, compared with conventional POP inhibitors, all psychopharmacological compounds tested are very weak inhibitors in vitro, and we doubt that their POP inhibition would be therapeutically meaningful.

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis.

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops.

Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus.

The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coli expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 microM and 3.5-8 microM, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.

Aflatoxin B1 misregulates the activity of serine proteases: possible implications in the toxicity of some mycotoxin.

Aflatoxins are highly hazardous contaminants of common food and feed. Aflatoxin B1 in particular, the most predominant among aflatoxins, was thoroughly demonstrated to be highly toxic, mutagenic, teratogenic and carcinogenic in many animal species. Besides its established targets and effects, this work investigates on the possible direct interaction between aflatoxin B1 and three major serine proteases, namely elastase, thrombin and trypsin. These proteases belongs to a class of structurally and functionally related proteins pivotal in both direct and indirect regulation of a number of cellular events. Additionally, several pathological processes, including cancer, inflammatory processes and thrombosis, rely upon the subtle equilibrium between these enzymes and their potential modulators: in fact, their misregulation, caused by foreign molecules, could facilitate (or be the cause for) the occurrence of these pathologies. Our results provide the evidence for a reversible binding between AFB1 and these enzymes, likely to have profound implications in the manifestation of aflatoxicosis. Precisely, the toxin behaved as a moderate competitive inhibitor toward the enzymatic activity of the serine proteases in the low micromolar range.

Characteristics of gabexate mesilate-induced cell injury in porcine aorta endothelial cells.

Gabexate mesilate (GM), a serine protease inhibitor, often causes severe vascular injury, when injected in high concentration. In the present study, we investigated the mechanisms for the cytotoxicity of GM on porcine aorta endothelial cells (PAECs). GM (0.5 - 5.0 mM) decreased cell viability in a dose-dependent manner and caused cell injury, whilst nafamostat mesilate (NM), another serine protease inhibitor, or mesilate itself had no effect on cell viability. zVAD-fmk, a pancaspase inhibitor, or zDEVD-fmk, a caspase-3 inhibitor, did not affect the GM (1.5 mM)-induced decrease of cell viability. Apoptotic cells or DNA fragmentation were also not observed after GM treatment. Moreover, Ca(2+) chelators, a nitric oxide (NO) synthase inhibitor, antioxidants, and radical scavengers had no effect on the GM-induced cell injury. On the other hand, cellular ATP content was decreased in the GM (2.0 mM)-treated cells. Surprisingly, GM (2.0 mM) immediately increased cellular uptake of propidium iodine. These findings suggest that GM induces necrotic cell death via injury of the cell membrane.

Inhibition of urokinase plasminogen activator with a novel enzyme inhibitor, WXC-340, ameliorates endotoxin and surgery-accelerated growth of murine metastases.

The urokinase plasminogen activator (u-PA) is intimately associated with tumour invasion and metastases. Surgery facilitates accelerated metastatic tumour growth in murine models, a phenomenon related to elevated perioperative bacterial lipopolysaccaride (LPS) and inflammatory cytokine levels. The objectives of the study were to examine the role of u-PA in cytokine-enhanced tumour cell invasion in vitro and surgery-induced accelerated metastatic tumour growth in vivo and to assess the potential benefit of a novel selective u-PA inhibitor WXC-340 in this setting. CT-26 murine colorectal carcinoma cells were stimulated with LPS, tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Cell supernatant u-PA expression and activity were determined using a colorimetric assay and Western blot analysis, respectively. Baseline and cytokine-stimulated in vitro invasion were assessed using ECmatrix invasion chambers. Two established murine models of accelerated metastatic tumour growth were used to investigate the consequences of u-PA inhibition on postoperative metastatic tumour burden. The effect of u-PA inhibition in vitro and in vivo was examined using the novel selective u-PA inhibitor, WXC-340. Proinflammatory cytokine stimulation significantly enhanced in vitro u-PA expression, activity and extracellular matrix invasion by approximately 50% compared to controls (P<0.05). This was abrogated by WXC-340. In vivo WXC-340 almost completely ameliorated both LPS- and surgery-induced, metastatic tumour growth compared to controls (P>0.05). In conclusion, u-PA cascade is actively involved in cytokine-mediated enhanced tumour cell invasion and LPS and surgery-induced metastatic tumour growth. Perioperative u-PA inhibition with WXC-340 may represent a novel therapeutic paradigm.

Inhibitory effects of Actinidia polygama extract and cyclosporine A on OVA-induced eosinophilia and bronchial hyperresponsiveness in a murine model of asthma.

Actinidia polygama is one of the well known herb used in oriental medicine for treatment of anti-inflammatory and many allergic diseases. Anti-asthmatic effects of A. polygama in the development of OVA-induced eosinophilia and hyperresponsiveness in murine model of asthma have not been fully investigated in vivo. Cyclosporine A (CsA) has been shown to inhibit single allergen-induced allergic inflammation such as eosinophilic and lymphocytic infiltration and mRNA expression for interleukin (IL)-4 and IL-5. Asthma is a chronic inflammatory disease of the mucosa and is associated with excess production of Th2 cytokines and eosinophil influx in lung. To clarify the anti-inflammatory and anti-asthmatic effects of A. polygama and CsA, we examined the influence of A. polygama fructus extract (APF) and CsA on the development of pulmonary eosinophilic inflammation in murine model of asthma. Our results have shown that APF and CsA have profound inhibitory effects on the accumulation of eosinophills into airways, with the reduction of eosinophil and total lung leukocyte number by reducing IL-4, IL-5, IL-13 and IgE levels in the BALF. Moreover, APF decreased eosinophil CCR3 expression and CD11b expression in lung cells. These results indicate that APF has a deep inhibitory effect on airway inflammation and hyperresponsiveness in murine model of asthma and play a crucial role as an immunomodulator which possess anti-inflammatory and anti-asthmatic property by modulating the relationship between Th1/Th2 cytokine imbalance.

Serine hydrolase targets of organophosphorus toxicants.

Acetylcholinesterase (AChE) is one of several hundred serine hydrolases in people potentially exposed to about 80 organophosphorus (OP) compounds important as insecticides or chemical warfare agents. The toxicology of OPs was interpreted until recently almost solely on the basis of AChE inhibition. It is assumed that each serine hydrolase has a specific function and proposed that every OP compound has a unique inhibitory profile. This review considers the progress in sifting the expanding list of potential serine hydrolase toxicological targets. About 50 serine hydrolase targets have been recognized but only a few studied thoroughly. The toxicological relevance of known secondary OP targets is established mainly from observations with humans (butyrylcholinesterase and neuropathy target esterase-lysophospholipase) and studies with mice (cannabinoid CB1 receptor, carboxylesterase, lysophospholipase and platelet activating factor acetylhydrolase) and hen eggs (arylformamidase or kynurenine formamidase). Pesticides most commonly shown to inhibit these targets in experimental vertebrates are chlorpyrifos and tribufos. Generally the levels of environmental and occupational OP pesticide exposure are well below those causing in vivo inhibition of secondary serine hydrolase targets. Although exposure to OP insecticides is decreasing from stricter regulations and the development of resistant pest strains, it will continue to some degree for decades in the future. Only two OPs are used as pharmaceuticals, i.e. echothiophate as an ophthalmic for treatment of glaucoma and metrifonate as an anthelmintic for Schistosoma (and formerly as a candidate drug for improved cognitive function in Alzheimer's disease). In safety evaluations, knowledge on known OP targets must be balanced against major gaps in current understanding since more than 75% of the serine hydrolases are essentially unknown as to OP targeting and relevance, i.e. it is not clear if they play a role in OP toxicology.