Knockdown of Helicoverpa armigera protease genes affects its growth and mortality via RNA interference.

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), the cotton bollworm, is a destructive pest which is famous for its resistance to a variety of insecticides. RNA interference is a posttranscriptional gene silencing mechanism that has become a popular tool to control insect pests, triggered by double-stranded RNAs (dsRNAs). The effect of ingestion and injection delivery methods of dsRNA related to some protease genes including Trypsin (Ha-TRY39 and Ha-TRY96), Chymotrypsin (Ha-CHY), and Cathepsin L (Ha-CAT) on growth and development of H. armigera was investigated in this study. All protease genes encoded full ORFs and were expressed in all H. armigera larvae stages and tissues. In both injection and feeding bioassays, Ha-RNAi CHY's performance outperformed that of other protease genes. CHY enzyme activity in the midgut of larvae was significantly reduced after treatment with ds-HaCHY. Oral administration of ds-CHY also resulted in significant mortality of H. armigera larvae. However, because of the high RNase activity in the midgut lumen of lepidoptera, a large amount of dsRNA was needed to effectively kill instars of H. armigera. To reduce dsRNA degradation, bacterial expression and dsRNA formulation were used. After oral administration, it was toxic to H. armigera larvae. Before oral administration, bacterial cells were sonicated to increase dsRNA release. The RNA interference efficiency of sonicated bacteria was significantly increased, resulting in higher larval mortality when administered orally. All of these findings point to Ha-CHY as a new candidate for developing an effective dsRNA-based pesticide for H. armigera control.

Study of the effects of ultrafine carbon black on the structure and function of trypsin.

Due to the rapid development of industrial society, air pollution is becoming a serious problem which has being a huge threat to human health. Ultrafine particles (UFPs), one of the major air pollutants, are often the culprits of human diseases. At present, most of the toxicological studies of UFPs focus on their biological effects on lung cells and tissues, but there are less researches taking aim at the negative effects on functional proteins within the body. Therefore, we experimentally explored the effects of ultrafine carbon black (UFCB) on the structure and function of trypsin. After a short-term exposure to UFCB, the trypsin aromatic amino acid microenvironment, protein backbone and secondary structure were changed significantly, and the enzyme activity showed a trend that rose at first, then dropped. In addition, UFCB interacts with trypsin in the form of a complex. These studies demonstrated the negative effects of UFCB on trypsin, evidencing potential effects on animals and humans.

A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa.

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor's stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

Leucine regulates α-amylase and trypsin synthesis in dairy calf pancreatic tissue in vitro via the mammalian target of rapamycin signalling pathway.

Starch digestion in the small intestines of the dairy cow is low, to a large extent, due to a shortage of syntheses of α-amylase. One strategy to improve the situation is to enhance the synthesis of α-amylase. The mammalian target of rapamycin (mTOR) signalling pathway, which acts as a central regulator of protein synthesis, can be activated by leucine. Our objectives were to investigate the effects of leucine on the mTOR signalling pathway and to define the associations between these signalling activities and the synthesis of pancreatic enzymes using an in vitro model of cultured Holstein dairy calf pancreatic tissue. The pancreatic tissue was incubated in culture medium containing l-leucine for 3 h, and samples were collected hourly, with the control being included but not containing l-leucine. The leucine supplementation increased α-amylase and trypsin activities and the messenger RNA expression of their coding genes (P <0.05), and it enhanced the mTOR synthesis and the phosphorylation of mTOR, ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1 (P <0.05). In addition, rapamycin inhibited the mTOR signal pathway factors during leucine treatment. In sum, the leucine regulates α-amylase and trypsin synthesis in dairy calves through the regulation of the mTOR signal pathways.

Intriguing role of water in protein-ligand binding studied by neutron crystallography on trypsin complexes.

Hydrogen bonds are key interactions determining protein-ligand binding affinity and therefore fundamental to any biological process. Unfortunately, explicit structural information about hydrogen positions and thus H-bonds in protein-ligand complexes is extremely rare and similarly the important role of water during binding remains poorly understood. Here, we report on neutron structures of trypsin determined at very high resolutions ≤1.5 Å in uncomplexed and inhibited state complemented by X-ray and thermodynamic data and computer simulations. Our structures show the precise geometry of H-bonds between protein and the inhibitors N-amidinopiperidine and benzamidine along with the dynamics of the residual solvation pattern. Prior to binding, the ligand-free binding pocket is occupied by water molecules characterized by a paucity of H-bonds and high mobility resulting in an imperfect hydration of the critical residue Asp189. This phenomenon likely constitutes a key factor fueling ligand binding via water displacement and helps improving our current view on water influencing protein-ligand recognition.

Trypsin-independent porcine epidemic diarrhea virus US strain with altered virus entry mechanism.

BACKGROUND: Porcine Epidemic Diarrhea Virus (PEDV) is a coronavirus that infects the intestinal tract and causes diarrhea and vomiting in older pigs or extreme dehydration and death that could reach 100% mortality in neonatal piglets. In the US, the first PEDV outbreaks occurred in 2013 and since then US PEDV strains have quickly spread throughout the US and worldwide, causing significant economic and public health concerns. Currently two conditionally approved vaccines exist in the US, but there is no live attenuated vaccine, which is considered the best option in controlling PEDV by inducing transferrable mucosal immunity to susceptible neonatal piglets. In this study, we passaged an US PEDV isolate under various conditions to generate three strains and characterized their growth and antigenicity in cell culture using various assays including Western blot analysis, serum neutralization assay, sequencing analysis and confocal microscopy. Finally, these strains were evaluated for pathogenicity in nursing piglets (1-4 days old). RESULTS: One of the PEDV strains generated in this study (designated as PEDV 8aa) is able to replicate in cells without any protease and grows to a high titer of >8 log10 TCID50/ml in cell culture. Interestingly, replication of PEDV 8aa was severely reduced by trypsin and this correlated with the inhibition of virus attachment and entry into the cells. In neonatal nursing piglets, PEDV 8aa (passage number 70 or 105) was found to be fully attenuated with limited virus shedding. CONCLUSIONS: These results suggest that applying selective pressure during viral passages can facilitate attainment of viral attenuation and that PEDV 8aa warrants further investigation as an attenuated vaccine.

Tranexamic acid and the gut barrier: Protection by inhibition of trypsin uptake and activation of downstream intestinal proteases.

OBJECTIVE: Intraluminal pancreatic trypsin and other digestive enzymes injure the gut barrier following trauma-hemorrhagic shock (T/HS). Intestinal proteases (sheddases) exert important effects on normal gut function but may cause barrier disruption due to exaggerated production following T/HS. We hypothesized that the protective mechanism of TXA on the gut barrier following T/HS includes inhibition of these "downstream" proteases. This was studied in vitro. METHODS: Trypsin, matrix metalloproteinase (MMP-9) and ADAM-17 activity were measured in intestinal epithelial cells (IEC) exposed to HR + trypsin. TXA was added to IEC subsets. Pulmonary microvascular endothelial cells (HMVEC) were exposed to IEC supernatants and syndecan release and ICAM-1 expression determined. RESULTS: Trypsin activity and the activity of the "downstream" sheddases ADAM-17, MMP was increased in IEC lysates following exposure to HR + trypsin. Syndecan and ICAM-1 were increased in HMVEC exposed to IEC supernatants. TXA administration 'early' abrogated these effects. CONCLUSIONS: TXA administration early after shock protects the gut barrier by inhibiting trypsin uptake and activity and the subsequent downstream protease cascade. To be effective, TXA should be administered early in all "at risk" patients.

Efficacy of protease inhibitor from marine Streptomyces sp. VITBVK2 against Leishmania donovani - An in vitro study.

In the present study the leishmanicidal effect of potential protease inhibitor producing marine actinobacterial isolate has been investigated against Leishmania donovani, the causative agent of visceral leishmaniasis. Among 89 marine actinobacteria isolated from a salt pan in Kanyakumari, only one isolate (BVK2) showed 97% of protease inhibition activity against trypsin. Moderate to high protease inhibitor activity was shown by isolate BVK2 on proteinase (30%) and chymotrypsin (85%). In optimization study for protease inhibitor production glucose as carbon source and casein as nitrogen source showed the best activity. In the in-vitro Fluorescence-activated cell sorting (FACS) assay, 100 µg/ml of BVK2 extract was active against amastigotes in infected J774A.1 macrophages and showed 87% of parasitic inhibition. The isolate BVK2 showed significant anti-parasitic activity with an IC50 of 27.1 µg/ml after double doses were administered. The potential isolate was identified by molecular 16S rRNA gene sequencing as Streptomyces sp. VITBVK2. The results obtained suggest that the marine actinobacterial extract which have novel metabolites can be considered as a potential source for the development of drugs.

Inhibition of Amyloid-like Fibril Formation of Trypsin by Red Wines.

The aim of the present study was to examine the potential role and applicability of dietary supplements in reducing the risk of development of amyloid diseases associated with the gastrointestinal tract, such as type II diabetes. Trypsin, a well-known serine protease was used as a model protein in our experiments. The effect of various red wines on the formation of amyloid-like fibrils of trypsin was studied in vitro, in aqueous ethanol, at pH 7.0. Turbidity measurements, aggregation kinetics experiments, Congo red binding assays and electronic circular dichroism spectroscopic measurements were used to follow the aggregation process in the presence or absence of various red wines. The results suggest that red wines effectively inhibit the formation of amyloid-like fibrils of trypsin and the inhibitory effect is dose-dependent. The extent of inhibition was found to be proportional to the total concentration of phenolic compounds.

A potent bidirectional promoter from the monocot cereal Eleusine coracana.

Ragi bifunctional α-amylase-trypsin inhibitor (RBI) of Eleusine coracana (L.) Gaertn. (finger millet) simultaneously inhibits α-amylase and trypsin. In continuation of previous work on the cloning, expression and characterization of RBI, a bidirectional promoter from finger millet was explored on the basis of experimental observations. Two trypsin inhibitors were identified while purifying RBI from a trypsin-Sepharose column eluent. Using an FPLC gel filtration column, these three inhibitors were purified to homogeneity and subjected to MALDI-TOF-TOF-MS/MS analysis and N-terminal sequencing. Both ragi trypsin inhibitors (RTIs) showed the same N-terminal sequence and considerable sequence similarity to RBI, indicating the presence of a multigene protease inhibitor family in finger millet. To gain insight into the evolution of these genes, the upstream region of RBI was explored by Genome Walking. Interestingly, on sequencing, a genome walking product of ∼1 Kb showed presence of an N-terminal RBI specific primer sequence twice but in opposite directions and leaving an intervening region of ∼0.9 Kb. The intervening region was presumed to represent an E. coracana bidirectional promoter (EcBDP), intuitively having a divergent RBI-RTI gene pair at two sides. For assaying the bidirectionality of promoter activity, a dual reporter GUS-GFP vector construct was made for plant expression containing the reporter genes at two ends of EcBDP, which was used to transform Agrobacterium tumefaciens LBA 4404. Transient plant transformation by recombinant Agrobacterium cells was carried out in onion scale epidermal cells and finger millet seedling leaves. Simultaneous expression of GUS and GFP under EcBDP established it as a potent natural bidirectional promoter from monocot origin, thereby potentially having vast application in cereal gene manipulations. In addition, inducibility of the EcBDP by either abscisic acid or cold treatment, as determined by transient transformation in onion, would substantiate more precise control of gene expression to mitigate the effects of adverse environmental conditions.

Marizomib irreversibly inhibits proteasome to overcome compensatory hyperactivation in multiple myeloma and solid tumour patients.

Proteasome inhibitors (PIs) are highly active in multiple myeloma (MM) but resistance is commonly observed. All clinical stage PIs effectively inhibit chymotrypsin-like (CT-L) activity; one possible mechanism of resistance is compensatory hyperactivation of caspase-like (C-L) and trypsin-like (T-L) subunits, in response to CT-L blockade. Marizomib (MRZ), an irreversible PI that potently inhibits all three 20S proteasome subunits with a specificity distinct from other PIs, is currently in development for treatment of MM and malignant glioma. The pan-proteasome pharmacodynamic activity in packed whole blood and peripheral blood mononuclear cells was measured in two studies in patients with advanced solid tumours and haematological malignancies. Functional inhibition of all proteasome subunits was achieved with once- or twice-weekly MRZ dosing; 100% inhibition of CT-L was frequently achieved within one cycle at therapeutic doses. Concomitantly, C-L and T-L activities were either unaffected or increased, suggesting compensatory hyperactivation of these subunits. Importantly, this response was overcome by continued administration of MRZ, with robust inhibition of T-L and C-L (up to 80% and 50%, respectively) by the end of Cycle 2 and maintained thereafter. This enhanced proteasome inhibition was independent of tumour type and may underlie the clinical activity of MRZ in patients resistant to other PIs.

Composition and biological activities of the essential oil of Piper corcovadensis (Miq.) C. DC (Piperaceae).

Essential oil from fresh leaves of the shrub Piper corcovadensis (Miq.) C. DC was obtained in 0.21% (w/w) yield by hydrodistillation in a Clevenger type apparatus. Thirty-one components, accounting for 96.61% of the leaf oil, were identified by gas chromatography-mass spectrometry. The major constituents of the oil were 1-butyl-3,4-methylenedioxybenzene (30.62%), terpinolene (17.44%), trans -caryophyllene (6.27%), α-pinene (5.92%), δ-cadinene (4.92%), and Limonene (4.46%). Bioassays against larvae of the Dengue mosquito (Aedes aegypti) revealed that leaf oil (LC50 = 30.52 ppm), terpinolene (LC50 = 31.16 ppm), and pure 1-butyl-3,4-methylenedioxybenzene (LC50 = 22.1 ppm) possessed larvicidal activities and are able to interfere with the activity of proteases from L4 gut enzymes. Additionally, the essential oil exhibited a strong oviposition deterrent activity at 50 and 5 ppm. This paper constitutes the first report of biological activities associated with the essential oil of leaves of P. corcovadensis.

Effects of α,ß-unsaturated lactones on larval survival and gut trypsin as well as oviposition response of Aedes aegypti.

Lactones are organic cyclic esters that have been described as larvicides against Aedes aegypti and as components of oviposition pheromone of Culex quinquefasciatus. This work describes the effect of six α,ß-unsaturated lactones (5a-5f) on survival of A. aegypti fourth instar larvae (L4). It is also reported the effects of the lactones on L4 gut trypsin activity and oviposition behavior of A. aegypti females. Five lactones were able to kill L4 being the lactones 5a (LC50 of 39.05 ppm), 5e (LC50 of 36.30 ppm) and 5f (LC50 of 40.46 ppm) the most promising larvicides. Only the lactone 5a inhibited L4 gut trypsin activity, with an IC50 of 115.15 µg/mL. Lactones 5a, 5c, 5d and 5e did not exert deterrent or stimulatory effects on oviposition, whereas lactone 5b exhibited a strong deterrent oviposition activity. In conclusion, this work introduces new α,ß-unsaturated lactones as promising alternatives to control A. aegypti dissemination. The larvicidal mechanism of the lactone 5a can involve the disruption of proteolysis at larval gut.

Inhibition of Human Prolyl Oligopeptidase Activity by the Cyclotide Psysol 2 Isolated from Psychotria solitudinum.

Cyclotides are head-to-tail cyclized peptides comprising a stabilizing cystine-knot motif. To date, they are well known for their diverse bioactivities such as anti-HIV and immunosuppressive properties. Yet little is known about specific molecular mechanisms, in particular the interaction of cyclotides with cellular protein targets. Native and synthetic cyclotide-like peptides from Momordica plants are potent and selective inhibitors of different serine-type proteinases such as trypsin, chymotrypsin, matriptase, and tryptase-beta. This study describes the bioactivity-guided isolation of a cyclotide from Psychotria solitudinum as an inhibitor of another serine-type protease, namely, the human prolyl oligopeptidase (POP). Analysis of the inhibitory potency of Psychotria extracts and subsequent fractionation by liquid chromatography yielded the isolated peptide psysol 2 (1), which exhibited an IC50 of 25 µM. In addition the prototypical cyclotide kalata B1 inhibited POP activity with an IC50 of 5.6 µM. The inhibitory activity appeared to be selective for POP, since neither psysol 2 nor kalata B1 were able to inhibit the proteolytic activity of trypsin or chymotrypsin. The enzyme POP is well known for its role in memory and learning processes, and it is currently being considered as a promising therapeutic target for the cognitive deficits associated with several psychiatric and neurodegenerative diseases, such as schizophrenia and Parkinson's disease. In the context of discovery and development of POP inhibitors with beneficial ADME properties, cyclotides may be suitable starting points considering their stability in biological fluids and possible oral bioavailability.

Digestive enzyme inhibitors from grains as potential components of nutraceuticals.

Inhibitor activity against digestive enzymes, such as α-amylase from human saliva and porcine pancreas and trypsin from bovine pancreas, of three cereal grains species were studied as potential components of nutraceuticals strengthening diabetes and obesity treatment. Significant differences were demonstrated: the highest antitryptic activity was found in the grain of the rye varieties studied, whereas the grain of the wheat varieties had significantly higher ability to inhibit α-amylases from human saliva and porcine pancreas. Additionally, seeds of Puma, one of the studied wheat varieties, demonstrated especially low antitryptic activity. Such a beneficial arrangement of inhibitors, i.e. high level of inhibitors of α-amylase from human saliva and porcine pancreas and simultaneously low level of trypsin inhibitors from bovine pancreas, indicate the possibility of the application of seeds with such properties to the preparation of nutraceuticals for people with obesity or suffering from diabetes.

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin.

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine ß-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3-18) and its analog [Phe(8)]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.

Disruption of the mucosal barrier during gut ischemia allows entry of digestive enzymes into the intestinal wall.

Intestinal ischemia is associated with high morbidity and mortality, but the underlying mechanisms are uncertain. We hypothesize that during ischemia the intestinal mucosal barrier becomes disrupted, allowing digestive enzymes access into the intestinal wall initiating autodigestion. We used a rat model of splanchnic ischemia by occlusion of the superior mesenteric and celiac arteries up to 30 min with and without luminal injection of tranexamic acid as a trypsin inhibitor. We determined the location and activity of digestive proteases on intestinal sections with in situ zymography, and we examined the disruption of two components of the mucosal barrier: mucin isoforms and the extracellular and intracellular domains of E cadherin with immunohistochemistry and Western blot techniques. The results indicate that nonischemic intestine has low levels of protease activity in its wall. After 15-min ischemia, protease activity was visible at the tip of the villi, and after 30 min, enhanced activity was seen across the full thickness of the intestinal wall. This activity was accompanied by disruption of the mucin layer and loss of both intracellular and extracellular domains of E cadherin. Digestive protease inhibition in the intestinal lumen with tranexamic acid reduced morphological damage and entry of digestive enzymes into the intestinal wall. This study demonstrates that disruption of the mucosal epithelial barrier within minutes of intestinal ischemia allows entry of fully activated pancreatic digestive proteases across the intestinal barrier triggering autodigestion.

Identification and transcription profiling of trypsin in Aedes taeniorhynchus (Diptera: Culicidae): developmental regulation, blood feeding, and permethrin exposure.

The cDNA of a trypsin gene from Aedes (Ochlerotatus) taeniorhynchus (Weidemann) was cloned and sequenced. The full-length mRNA sequence (890 bp) for trypsin from Ae. taeniorhynchus (AetTryp1) was obtained, which encodes an open reading frame of 765 bp (i.e., 255 amino acids). To detect whether AetTryp is developmentally regulated, a quantitative real-time polymerase chain reaction was used to examine AetTrypl mRNA expression levels in different developmental stages of Ae. taeniorhynchus. AetTryp1 was expressed at low levels in egg, larval, and pupal stages, but was differentially expressed in adult Ae. taeniorhynchus, with highest levels found in 5-d-old female adults when compared with teneral adults. In addition, AetTryp1 mRNA expression differed between sexes, with expression levels much lower in males. However, in both males and females, there was a significant increase in AetTryp1 transcription levels as age increased and peaked in 5-d-old adults. AetTrypl expressed in 5-d-old female Ae. taeniorhynchus significantly increased after 30 min postblood feeding compared with the control. The AetTryp1 mRNA expression in 5-d-old female Ae. taeniorhynchus was affected by different concentrations of permethrin.

Interdicting protease-activated receptor-2-driven inflammation with cell-penetrating pepducins.

Protease-activated receptor-2 (PAR2), a cell surface receptor for trypsin-like proteases, plays a key role in a number of acute and chronic inflammatory diseases of the joints, lungs, brain, gastrointestinal tract, and vascular systems. Despite considerable effort by the pharmaceutical industry, PAR2 has proven recalcitrant to targeting by small molecule inhibitors, which have been unable to effectively prevent the interaction of the protease-generated tethered ligand with the body of the receptor. Here, we report the development of first-in-class cell-penetrating lipopeptide "pepducin" antagonists of PAR2. The design of the third intracellular (i3) loop pepducins were based on a structural model of a PAR2 dimer and by mutating key pharmacophores in the receptor intracellular loops and analogous pepducins. Individual pharmacophores were identified, which controlled constitutive, agonist, and antagonist activities. This approach culminated in the identification of the P2pal-18S pepducin which completely suppressed trypsin and mast cell tryptase signaling through PAR2 in neutrophils and colon cancer cells. The PAR2 pepducin was highly efficacious in blocking PAR2-dependent inflammatory responses in mouse models. These effects were lost in PAR2-deficient and mast-cell-deficient mice, thereby validating the specificity of the pepducin in vivo. These data provide proof of concept that PAR2 pepducin antagonists may afford effective treatments of potentially debilitating inflammatory diseases and serve as a blueprint for developing highly potent and specific i3-loop-based pepducins for other G protein-coupled receptors (GPCRs).

Quantitative structure-activity relationships for organophosphates binding to trypsin and chymotrypsin.

Organophosphate (OP) nerve agents such as sarin, soman, tabun, and O-ethyl S-[2-(diisopropylamino) ethyl] methylphosphonothioate (VX) do not react solely with acetylcholinesterase (AChE). Evidence suggests that cholinergic-independent pathways over a wide range are also targeted, including serine proteases. These proteases comprise nearly one-third of all known proteases and play major roles in synaptic plasticity, learning, memory, neuroprotection, wound healing, cell signaling, inflammation, blood coagulation, and protein processing. Inhibition of these proteases by OP was found to exert a wide range of noncholinergic effects depending on the type of OP, the dose, and the duration of exposure. Consequently, in order to understand these differences, in silico biologically based dose-response and quantitative structure-activity relationship (QSAR) methodologies need to be integrated. Here, QSAR were used to predict OP bimolecular rate constants for trypsin and α-chymotrypsin. A heuristic regression of over 500 topological/constitutional, geometric, thermodynamic, electrostatic, and quantum mechanical descriptors, using the software Ampac 8.0 and Codessa 2.51 (SemiChem, Inc., Shawnee, KS), was developed to obtain statistically verified equations for the models. General models, using all data subsets, resulted in R(2) values of .94 and .92 and leave-one-out Q(2) values of 0.9 and 0.87 for trypsin and α-chymotrypsin. To validate the general model, training sets were split into independent subsets for test set evaluation. A y-randomization procedure, used to estimate chance correlation, was performed 10,000 times, resulting in mean R(2) values of .24 and .3 for trypsin and α-chymotrypsin. The results show that these models are highly predictive and capable of delineating the complex mechanism of action between OP and serine proteases, and ultimately, by applying this approach to other OP enzyme reactions such as AChE, facilitate the development of biologically based dose-response models.