Discovery of a novel series of guanidinebenzoates as gut-restricted enteropeptidase and trypsin dual inhibitors for the treatment of metabolic syndrome.

A novel series of guanidinebenzoate enteropeptidase and trypsin dual inhibitors has been discovered and SAR studies were conducted. Optimization was focused on improving properties for gut restriction, including increased aqueous solubility, lower cellular permeability, and reduced oral bioavailability. Lead compounds were identified with efficacy in a mouse fecal protein excretion study.

Enteropeptidase inhibitor SCO-792 effectively prevents kidney function decline and fibrosis in a rat model of chronic kidney disease.

BACKGROUND: Inhibiting enteropeptidase, a gut serine protease regulating protein digestion, suppresses food intake and ameliorates obesity and diabetes in mice. However, the effects of enteropeptidase inhibition on kidney parameters are largely unknown. Here, we evaluated the chronic effects of an enteropeptidase inhibitor, SCO-792, on kidney function, albuminuria and kidney pathology in spontaneously hypercholesterolaemic (SHC) rats, a rat chronic kidney disease (CKD) model. METHODS: SCO-792, an orally available enteropeptidase inhibitor, was administered [0.03% and 0.06% (w/w) in the diet] to 20-week-old SHC rats showing albuminuria and progressive decline in glomerular filtration rate (GFR) for five weeks. The effects of SCO-792 and the contribution of amino acids to these effects were evaluated. RESULTS: SCO-792 increased the faecal protein content, indicating that SCO-792 inhibited enteropeptidase in SHC rats. Chronic treatment with SCO-792 prevented GFR decline and suppressed albuminuria. Moreover, SCO-792 improved glomerulosclerosis and kidney fibrosis. Pair feeding with SCO-792 (0.06%) was less effective in preventing GFR decline, albuminuria and renal histological damage than SCO-792 treatment, indicating the enteropeptidase-inhibition-dependent therapeutic effects of SCO-792. SCO-792 did not affect the renal plasma flow, suggesting that its effect on GFR was mediated by an improvement in filtration fraction. Moreover, SCO-792 increased hydrogen sulphide production capacity, which has a role in tissue protection. Finally, methionine and cysteine supplementation to the diet abrogated SCO-792-induced therapeutic effects on albuminuria. CONCLUSIONS: SCO-792-mediated inhibition of enteropeptidase potently prevented GFR decline, albuminuria and kidney fibrosis; hence, it may have therapeutic potential against CKD.

Enteropeptidase inhibition improves kidney function in a rat model of diabetic kidney disease.

AIM: To examine the effects of an enteropeptidase inhibitor, SCO-792, on kidney function in rats. MATERIALS AND METHODS: The pharmacological effects of SCO-792 were evaluated in Wistar fatty (WF) rats, a rat model of diabetic kidney disease (DKD). RESULTS: Oral administration of SCO-792 increased faecal protein content and improved glycaemic control in WF rats. SCO-792 elicited a rapid decrease in urine albumin-to-creatinine ratio (UACR). SCO-792 also normalized glomerular hyperfiltration and decreased fibrosis, inflammation and tubular injury markers in the kidneys. However, pioglitazone-induced glycaemic improvement had no effect on kidney variables. Dietary supplementation of amino acids (AAs), which bypass the action of enteropeptidase inhibition, mitigated the effect of SCO-792 on UACR reduction, suggesting a pivotal role for enteropeptidase. Furthermore, autophagy activity in the glomerulus, which is impaired in DKD, was elevated in SCO-792-treated rats. Finally, a therapeutically additive effect on UACR reduction was observed with a combination of SCO-792 with irbesartan, an angiotensin II receptor blocker. CONCLUSIONS: This study is the first to demonstrate that enteropeptidase inhibition is effective in improving disease conditions in DKD. SCO-792-induced therapeutic efficacy is likely to be independent of glycaemic control and mediated by the regulation of AAs and autophagy. Taken together with a combination effect of irbesartan, SCO-792 may be a novel therapeutic option for patients with DKD.

Enteropeptidase inhibition improves obesity by modulating gut microbiota composition and enterobacterial metabolites in diet-induced obese mice.

Enteropeptidase is a transmembrane serine protease localized in the lumen of the duodenum that acts as a key enzyme for protein digestion. SCO-792 is an orally available enteropeptidase inhibitor that has been reported to have therapeutic effects on obesity and diabetes in mice. However, the mechanism underlying the therapeutic effect of SCO-792 has not yet been fully elucidated. In this study, we evaluated the role of gut microbiota on SCO-792-induced body weight (BW) reduction in high-fat diet-induced obese (DIO) mice. Chronic administration of SCO-792 substantially decreased BW and food intake in DIO mice. While the pair-fed study uncovered food intake-independent mechanisms of BW reduction by SCO-792. Interestingly, antibiotics-induced microbiota elimination in the gut canceled SCO-792-induced BW reduction by nearly half without affecting the anorectic effect, indicating the involvement of gut microbiota in the anti-obesity mechanism that is independent of food intake reduction. Microbiome analysis revealed that SCO-792 altered the gut microbiota composition in DIO mice. Notably, it was found that the abundance of Firmicutes decreased while that of Verrucomicrobia increased at the phylum level. Increased abundance of Akkermansia muciniphila, a bacterium known to be useful for host metabolism, was observed in SCO-792-treated mice. Fecal metabolome analysis revealed increased amino acid levels, indicating gut enteropeptidase inhibition. In addition, SCO-792 was found to increase the level of short-chain fatty acids, including propionate, and bile acids in the feces, which all help maintain gut health and improve metabolism. Furthermore, it was found that SCO-792 induced the elevation of colonic immunoglobulin A (IgA) concentration, which may maintain the microbiota condition, in DIO mice. In conclusion, this study demonstrates the contribution of microbiota to SCO-792-induced BW reduction. Enteropeptidase-mediated regulation of microbiota, enterobacterial metabolites, and IgA in the gut may coordinately drive the therapeutic effects of SCO-792 in obesity.

Targeting Enteropeptidase with Reversible Covalent Inhibitors To Achieve Metabolic Benefits.

Inhibition of the serine protease enteropeptidase (EP) opens a new avenue to the discovery of chemotherapeutics for the treatment of metabolic diseases. Camostat has been used clinically for treating chronic pancreatitis in Japan; however, the mechanistic basis of the observed clinical efficacy has not been fully elucidated. We demonstrate that camostat is a potent reversible covalent inhibitor of EP, with an inhibition potency (k inact/KI) of 1.5 × 104 M-1s-1 High-resolution liquid chromatography-mass spectrometry (LC-MS) showed addition of 161.6 Da to EP after the reaction with camostat, consistent with insertion of the carboxyphenylguanidine moiety of camostat. Covalent inhibition of EP by camostat is reversible, with an enzyme reactivation half-life of 14.3 hours. Formation of a covalent adduct was further supported by a crystal structure resolved to 2.19 Å, showing modification of the catalytic serine of EP by a close analog of camostat, leading to formation of the carboxyphenylguanidine acyl enzyme identical to that expected for the reaction with camostat. Of particular note, minor structural modifications of camostat led to changes in the mechanism of inhibition. We observed from other studies that sustained inhibition of EP is required to effect a reduction in cumulative food intake and body weight, with concomitant improved blood glucose levels in obese and diabetic leptin-deficient mice. Thus, the structure-activity relationship needs to be driven by not only the inhibition potency but also the mechanistic and kinetic characterization. Our findings support EP as a target for the treatment of metabolic diseases and demonstrate that reversible covalent EP inhibitors show clinically relevant efficacy. SIGNIFICANCE STATEMENT: Interest in targeted covalent drugs has expanded in recent years, particularly so for kinase targets, but also more broadly. This study demonstrates that reversible covalent inhibition of the serine protease enteropeptidase is a therapeutically viable approach to the treatment of metabolic diseases and that mechanistic details of inhibition are relevant to clinical efficacy. Our mechanistic and kinetic studies outline a framework for detailed inhibitor characterization that is proving essential in guiding discovery efforts in this area.

Discovery and characterization of a small-molecule enteropeptidase inhibitor, SCO-792.

Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO-792, a novel inhibitor of enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of enteropeptidase inhibitors, and compound optimization resulted in identification of SCO-792, which inhibited enteropeptidase activity in vitro, with IC 50 values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of enteropeptidase by SCO-792 was potentiated by increased incubation time, and the calculated Kinact/KI was 82 000/mol/L s. An in vitro dissociation assay showed that SCO-792 had a dissociation half-life of almost 14 hour, with a calculated koff rate of 0.047/hour, which suggested that SCO-792 is a reversible enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO-792 effectively inhibited plasma elevation of branched-chain amino acids in an oral protein challenge test, which indicated that SCO-792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO-792 as a potent and reversible inhibitor against enteropeptidase. SCO-792 slowly dissociated from enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO-792 could reveal the effects of inhibiting enteropeptidase on biological actions.

SCO-792, an enteropeptidase inhibitor, improves disease status of diabetes and obesity in mice.

AIMS: Enteropeptidase is a serine protease localized on the duodenal brush border that catalyzes the conversion of inactive trypsinogen into active trypsin, thereby regulating protein breakdown in the gut. We evaluated the effects of SCO-792, a novel enteropeptidase inhibitor, in mice. MATERIALS AND METHODS: In vivo inhibition of enteropeptidase was evaluated via an oral protein challenge. Pharmacological effects were evaluated in normal mice, in diet-induced obese (DIO) mice and in obese and diabetic ob/ob mice. RESULTS: A single oral administration of SCO-792 inhibited plasma branched-chain amino acids (BCAAs) in an oral protein challenge test in mice, indicating in vivo inhibition of enteropeptidase. Repeated treatment with SCO-792 induced reduction in food intake and decrease in body weight in DIO and ob/ob mice. Plasma FGF21 levels were increased in SCO-792-treated DIO mice, an observation that was probably independent of reduction in food intake. Hyperglycaemia was markedly improved in SCO-792-treated ob/ob mice. A hyperinsulinaemic-euglycaemic clamp study revealed improved muscle insulin sensitivity in SCO-792-treated ob/ob mice. SCO-792 also improved plasma and liver lipid profiles and decreased plasma alanine transaminase, suggesting a potential treatment for liver diseases. Dietary supplementation with essential amino acids attenuated the effect of SCO-792 on reduction in food intake and decrease in body weight in normal mice, suggesting a pivotal role for enteropeptidase in these biological phenomena. CONCLUSIONS: SCO-792 inhibited enteropeptidase in vivo, reduced food intake, decreased body weight, increased insulin sensitivity, improved glucose and lipid control, and ameliorated liver parameters in mouse models with obesity and/or diabetes. SCO-792 may exhibit similar effects in patients.

Inhibition effect of enteropeptidase on RANKL-RANK signalling by cleavage of RANK.

Enteropeptidase can cleave trypsinogen on the sequence of Asp-Asp-Asp-Asp-Lys and plays an important role in food digestion. The RANKL-RANK signalling pathway plays a pivotal role in bone remodelling. In this study, we reported that enteropeptidase can inhibit the RANKL-RANK signalling pathway through the cleavage of RANK. A surrogate peptide blocking assay indicated that enteropeptidase could specifically cleave RANK on the sequence NEEDK. Osteoclast differentiation assay and NF-κB activity assay confirmed that enteropeptidase could inhibit osteoclastogenesis in vitro through the cleavage of RANK. This is the first study to prove that the RANKL-RANK signalling pathway can be inhibited by cleavage of RANK instead of targeting RANKL.

Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

The serine protease inhibitor protein C inhibitor (PCI) is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP) is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4) M(-1) s(-1). Low molecular weight (LMWH) and unfractionated heparin (UFH) slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition) value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml). By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

Biochemical characterization of human enteropeptidase light chain.

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.

Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide.

Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)4-Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 A resolution in complex with the inhibitor Val-(Asp)4-Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the Nzeta atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)4-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp)4-Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol(-1) of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen.

Autolysis of bovine enteropeptidase heavy chain: evidence of fragment 118-465 involvement in trypsinogen activation.

Variations in bovine enteropeptidase (EP) activity were shown to result from autolysis caused by the loss of calcium ions; the cleavage sites were determined. The native enzyme preferred its natural substrate, trypsinogen (KM=2.4 microM), to the peptide and fusion protein substrates (KM=200 and 125 microM, respectively). On the other hand, the truncated enzyme composed of the C-terminal fragment 466-800 of EP heavy chain and intact light chain did not distinguish these substrates. The results suggest that the N-terminal fragment 118-465 of the enteropeptidase heavy chain contains a secondary substrate-binding site that interacts directly with trypsinogen.

[An inhibitor of enteropeptidases and trypsin from the bovine duodenum]. / Ingibitor énteropeptidazy i tripsina iz dvenadtsatiperstnoi kishki byka.

Enteropeptidase inhibitor (DI) was isolated from bovine duodenum during purification of this enzyme. DI was purified by affinity chromatography on immobilised trypsin. DI preparations contain two main components: DI-9 (9 kD) and DI-20 (20 kD). The N-terminal amino acid sequence 1-19 of DI-9 is highly homologous to the Kunitz inhibitor (BPI). Molecular weights of DI-9 and BPI are the same (gel electrophoresis data). Fragment 1-19 of DI-9 differs from the corresponding region of BPI only at the position 17: DI-9 contains Ala-17 instead of Arg in BPI. The homology of N-terminal amino acid sequence 1-25 of DI-20 with the corresponding regions of some phospholipases A2 suggests that this protein is a new intestinal phospholipase A2. Inhibitor DI-9 and phospholipase DI-20 are probably isolated in a common lipoprotein complex. The only earlier known in vitro inhibitor of enteropeptidase, BPI, was localised in vivo in different tissues with this enzyme. In our opinion the Kunitz-type inhibitor DI-9 is, a physiological inhibitor of enteropeptidase.

Small intestinal mucosal hyperplasia caused by an enterokinase inhibitor from red kidney bean in rats.

A specific enterokinase inhibitor (EKI) was purified from red kidney bean (RKB). Male weanling rats fed a diet containing this purified EKI (0.06%) for 6 d showed increases in mucosal weights, protein, DNA and lactic dehydrogenase contents in their small intestines compared to age-matched control rats fed a standard diet. Total mucosal EK and disaccharidase activities were, however, decreased in EKI-fed rats. Thus, oral consumption of EKI from RKB led to small intestinal mucosal hyperplasia in rats. The mucosal hyperplasia observed in EKI-fed rats is not likely due to decreased turnover of mucosal proteins as a result of reduced luminal proteases since luminal contents of trypsin, chymotrypsin and elastase in EKI-fed rats were similar to those of control rats. Enterokinase inhibitor may have a direct hyperplastic effect on the small intestine of rats.

A rational approach to the specific chemotherapy of pancreatitis.

Oedematous pancreatitis is pancreatic acinar cell damage with leakage into the peritoneal cavity and circulation of the inactive zymogens of digestive enzymes and active amylase and lipase. Pancreatic oedema and intra-abdominal fat necrosis occur. Necrotising pancreatitis is pancreatic acinar cell damage accompanied by the specific conversion of trypsinogens to trypsins, at a rate, and on a scale, sufficient to overwhelm local defences. Rapid release of the whole spectrum of activated pancreatic enzymes leads to necrosis of parts of the pancreas and blood vessels, and the disseminated enzyme-mediated damage which characterises the molecular pathology of the established severe disease. Chronic pancreatitis, although less well understood, is also associated with trypsinogen activation within the gland. Two mechanisms have emerged as initiators of trypsinogen activation, lysosomal cathepsins and bile-borne enterokinase. Chemotherapeutic strategies against disease initiation include preparation of synthetic enterokinase and Cathepsin B inhibitors. Chemotherapeutic strategies against second-stage mediation of multi-organ damage in the disease, include oligopeptide or organic functionalities with novel catalytic site-directed moieties (such as fluoromethyl ketones) suitable for in vivo use and the specific inhibition of the relevant range of enzymes in complex with alpha 2-macroglobulin. Interference with pancreatic enzyme biosynthesis using proteolysis-resistant constructs mimicking receptor-binding domains of inhibitor peptide hormones as well as inhibitors of pancreatic signal peptidase are promising additional chemotherapeutic approaches worthy of active investigation.

Effect of lectins from leguminous seeds on rat duodenal enterokinase activity.

Enterokinase activity from rat duodenal brush borders was assayed in vitro in the presence of purified lectins from 3 leguminous seeds. Noncompetitive inhibition of the enzyme was observed in each case. Phaseolus hemagglutinin was the most potent inhibitor among the 3 lectins tested.

Isolation and characterization of a specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris).

A specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris) was purified to homogeneity. It showed a single protein band on sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis in the presence of mercaptoethanol, and the Mr was 31000. Aspartic acid was identified as the N-terminus of the inhibitor. The Mr by gel chromatography on Sephadex G-200 was found to be 60000, indicating the dimeric nature of the inhibitor. The inhibitor was found to be a glycoprotein. The monosaccharide moieties were glucose, mannose, glucuronic acid and glucosamine in the proportions 3.15%, 5.0%, 0.85% and 1.3% respectively. The inhibitor was most active on pig enterokinase, followed by bovine and human enterokinases. Maximal inhibitory activity was elicited by preincubation of the inhibitor with the enzyme for 15 min. Digestion with pepsin resulted in loss of inhibitory activity. The inhibitor was stable to exposure to a wide range of pH values (2-10), and exposure to pH above 10 resulted in loss of inhibitory activity. Modification of arginine residues by cyclohexane 1,2-dione and ninhydrin led to complete loss of enterokinase-inhibitory activity.

Identification of a defence mechanism in vivo against the leakage of enterokinase into the blood.

1. The serum proteinase inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin, inter-alpha-trypsin inhibitor and C1-esterase inhibitor were found not to affect the catalytic activity of human enterokinase, whereas bovine trypsin activity was modified essentially as expected. Enterokinase was also not inhibited by Trasylol (trypsin inhibitor from bovine lung) or bovine pancreatic trypsin inhibitor. No other component in human or mouse serum complexing with enterokinase was identified. 2. Human enterokinase administered intravenously into mice was rapidly cleared from the circulation with a half-life of 2.5 min. This removal was not the result of the difference in species, since partially purified mouse enterokinase was cleared at the same rate as the human enzyme. Clearance was mediated by recognition of the carbohydrate portion of enterokinase and not through specific recognition of its catalytic site. Immunofluorescent staining showed that the enzyme accumulated in the liver. Attempts to block the clearance by the simultaneous infusion of competing glycoproteins suggested that enterokinase was taken up by hepatocytes. Of the glycoproteins tested only two, human lactoferrin (terminal fucosyl alpha 1 leads to 3 N-acetylglucosamine) and bovine asialo-fetuin (terminal galactosyl beta 1 leads to 4 N-acetylglucosamine) were weakly competitive. Two inhibitors of endocytosis, Intralipid and Triton WR1339, failed to delay the removal of enterokinase. It is proposed that enterokinase is cleared from the circulation by an as yet uncharacterized hepatocyte receptor.

[The influence of orally applicable antibiotics on the activities of human enterokinase and disaccharidases (author's transl)]. / Beeinflussung der Aktivitäten von Enterokinase und Disaccharidasen beim Menschen durch oral applizierbare Antibiotika

In this study the influence of 14 antibiotics, 12 of them orally applicable, on human enterokinase was investigated. The effects of these substances on the activities of human disaccharidases were also examined. The enterokinase activity is more sensitive to the studied antibiotics than is human lactase, saccharase or isomaltase. Unphysiologically high concentrations of penicillins, cephalexin and chloramphenicol (10(-2) Mol/l) inhibited enterokinase, tetracycline (doxycycline) in a dose of 10(-3) m reduced the activity of this enzyme by 50%, neomycinsulphate and the sulphates of polymyxin B and E have no effect on the disaccharidases. On the contrary, these substances are the best inhibitors of enterokinase among the tested antibiotics. Neomycin or polymyxin (10(-4) Mol/l) causes a 50% inhibition of a physiological quantity of this enzyme. Therapeutic doses of both antibiotics may reduce the enterokinase activity by 70% to 90%, while the activity of trypsin is not affected unless a concentration greater than 10(-2) m is used. The inhibition is not only caused by the anion (SO4) of these antibiotics, since sulphates reduce the enterokinase only in concentrations higher than 10(-3) Mol/l in man. The mechanism of inhibition is not effected by binding cholic acids under test conditions. Both polymyxin and neomycin inhibit the enterokinase activity with and without glycodeoxycholic acid. Further studies showed that the type of inhibition is competitive in both cases. The inhibition constant K2 of neomycin-B-sulphate is 8.7X10(-5) Mol/l, of polymyxin-E-sulphate 8.6X10(-5) Mol/l. The inhibition type of penicillins, cephalosporins and doxycycline is noncompetitive, thus contrasting that of neomycin and polymyxin.