Perspectives in the design of small molecule enzyme inhibitors as useful drugs

An irreversible inhibitor is often assessed as a potential drug on the basis of its specificity towards a target enzyme in vitro. This is inadequate because in vivo the enzyme may be re-synthesized and the inhibitor may react with other major body constituents, e.g. glutathione.Guidelines are given for selecting the class of inhibitor to be studied for use in a particular clinical area.     

A quantitative model of liver regeneration in the rat

The ability of various theories to generate the kinetics of rat-liver regeneration is considered. Wound hormone and functional demand theories are shown to be either inadequate or overly complex. A simple model based on a liver-produced mitotic inhibitor is, however, able to match the experimental results on the mitotic rate and thymidine uptake of parenchymal cells in the liver as a function of time following various degrees of partial hepatectomy. The inhibitor itself would belong to the class of molecules known as chalones and differential equations describing the mechanism of its action are derived and solved numerically. The only arbitrary parameters required for the solution are those giving the halflife and dose-response curve of the inhibitor. Optimal matching of theory to data is obtained when the half-life is about 3 h and the dose-response curve is given by a negative exponential function. Experimental procedures for measuring these parameters are discussed and an explanation of the uneven distribution of mitoses in regenerating liver is given.     

The influence of drugs on the kinin-forming system in relation to pregnancy and parturition in the rat.

The duration of normal gestation and parturition in the rat can be changed by treatment with drugs which alter the equilibrium of the kallikrein-kinin system. The kallikrein inhibitor, aprotinin, when given from Days 19-22 of pregnancy prolongs gestation. Treatment with aprotinin from Days 20-22 of pregnancy prolongs the parturient process, as does a single dose given on the morning of Day 22. Kallikrein, when administered from Days 19-22 of pregnancy, results in a prolongation of gestation and abolishes the pre-parturient behaviour ('labour'). Parturition is prolonged and many fetuses are stillborn. Soya bean trypsin inhibitor when given from Days 19-22 of pregnancy delays and prolongs parturition; maternal haemorrhage occurs during birth and many fetuses are born dead or are abandoned at birth. It is suggested that the kallikrein-kinin system plays a functional role in the normal process of parturition in the rat.     

The effect of a nitrification inhibitor on the concentration of nitrate in grass during growth

Summary In an experiment on clay-loam soil ammonium sulphate with or without the nitrification inhibitor 2-chloro-6-(trichloromethyl) pyridine (at 2% of the weight of N in fertilizer) or sodium nitrate were applied at 100 or 200 lb N/acre to the seedbed before sowing S22 Italian ryegrass and again after the first cut. Eighty-four days after the first dressing, all the grass given 100 lb N/acre contained similar amounts of nitrate-N; with 200 lb N/acre, grass given ammonium sulphate alone contained most nitrate-N and grass given ammonium sulphate plus inhibitor least. Forty-two days after the second dressing, all the grass given 100 lb N/acre again contained similar amounts of nitrate-N and with 200 lb N/acre, grass given sodium nitrate contained most nitrate-N and that given ammonium sulphate plus inhibitor least.     

Pepstatin- and leupeptin-loaded liposomes: a tool in protein breakdown studies

The preparation of proteinase inhibitor loaded "solid" positively charged liposomes is described. Some properties of the vesicle preparations are given and the findings are tentatively summarized in a scheme of liposomophagy.     

Inhibition of the soluble adenosine triphosphatase from mitochondria by adenylyl imidodiphosphate

1. Adenylyl imidodiphosphate is an inhibitor with high affinity for the soluble ATPase (adenosine triphosphatase) from mitochondria. 2. The reaction of the inhibitor with the ATPase is slow and estimates for the association and dissociation reaction rate constants are given. 3. The number of binding sites for the inhibitor appears to be doubled in the presence of 2,4-dinitrophenol. 4. Adenylyl imidodiphosphate is less effective as an inhibitor of the ATPase activity of this enzyme than of the inosine triphosphatase activity. It is also less effective on the ATPase of frozen-thawed or intact mitochondria and did not inhibit ADP-stimulated respiration by intact mitochondria.     

A survey of mathematical models of competition with an inhibitor

Mathematical models of the effect of inhibitors on microbial competition are surveyed. The term inhibitor is used in a broad sense and includes toxins, contaminants, allelopathic agents, etc. This includes both detoxification where the inhibitor is viewed as a pollutant and control where the inhibitor is viewed as an aid to controlling a bioreactor. The inhibitor may be supplied externally or may be created as an anti-competitor toxin. This includes plasmid-bearing, plasmid-free competition. The literature is spread across journals in different disciplines and with different notation. The survey attempts to present the mathematical models and the results of the corresponding analysis within a common framework and notation. Detailed mathematical proofs are not given but the methods of proof are indicated, references cited, and the results presented in tables. Open problems are indicated where there is a gap in the theory.     

IDENTIFICATION AND QUANTIFICATION OF 3-METHOXY -4-HYDROXYPHENYLLACTIC ACID (VLA) IN CEREBROSPINAL FLUID AND 3-METHOXY-4-HYDROXY PHENYLPYRUVIC ACID (VPA) IN THE URINE OF PARKINSONIAN PATIENTS TREATED WITH L-DOPA

The identification and quantification of 3-methoxy-4-hydroxyphenyllactic acid in cerebrospinal fluid, and 3-methoxy-4-hydroxyphenylpyruvic acid in the urine of Parkinsonian patients on medication with l -3,4-dihydroxyphenylalanine with or without a peripheral decarboxylase inhibitor are described. Data are also given for other catecholamine- and catecholamine precursor metabolites in urine and cerebrospinal fluid. Analyses were performed by means of gas chromatography with flame ionization or mass spectrometric detection. We have concluded that following the administration of l -3.4-dihydroxyphenylalanine + peripheral decarboxylase inhibitor. 3-methoxy-4-hydroxyphenyllactic acid must be considered as a quantitatively important metabolite of L-3.4-dihydroxyphenylalanine in cerebrospinal fluid. In addition, some comment is given on the presence of 3-methoxy-4-hydroxy-phenylpyruvic acid in the urine of these patients.     

Ipodate and 8-anilino-1-naphthalene sulfonic acid block receptor binding of T3 in rat liver

We have determined that 8-anilino-1-naphthalene sulfonic acid (ANS) and ipodate are effective inhibitor in vitro of 125I-T3 binding to rat hepatic nuclei receptors. Both of these agents are estimated to have a Kd for the T3 receptor of about 1--2 x 10(-4) M. Indirect preliminary studies suggest that ANS is a non-competitive inhibitor and ipodate is a competitive inhibitor of T3 binding. Compounds such as tyropanoate and diatrizoate and iodide had no effect on T3 receptor binding. Further in vivo studies with ipodate suggested that T3 receptor binding inhibition also occurred when ipodate was given intravenously to rats.     

An Inhibitor of Salt Absorption in the Root Tissues of Red Beet

The absorption of manganese ions by discs of beet-root tissueoccurs in two phases. Evidence suggests that the first is physicalin nature, the second physiological. The interval between thetwo phases becomes longer as the thickness of the discs is increasedand reasons are given for attributing this to the presence ofan inhibitor of the physiological absorption process. Aqueous extracts of beet-root contain an inhibitor of ion absorptionwhich also delays the germination of mustard and cress seeds.The extract does not affect the respiration rate of beet-roottissue.     

Haemophilia B: database of point mutations and short additions and deletions, 7th edition.

The seventh edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1535 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.     

Characterization of a lactate dehydrogenase inhibitor protein from rabbit skeletal muscle mitochondrial fraction

A lactate dehydrogenase inhibitor protein is isolated from rabbit skeletal muscle crude mitochondrial fraction. The molecular weight of the inhibitor is approximately 20,000 as determined by size exclusion HPLC. The inhibitor isoelectricpH is 5.3 as determined by agarose or polyacrylamide gel isoelectric focusing. The amino acid composition of the inhibitor is given. The presence of the inhibitor gives an acidic characteristic to the alkaline M₄ lactate dehydrogenase isozyme and the lactate dehydrogenase-inhibitor complex is more stable than the enzyme alone.     

The pharmacology of recombinant hirudin

A review about comprehensive studies on pharmacological properties of hirudin produced by genetic engineering is given. Anticoagulant effects, influence on platelet functions, studies on toxicity and side effects as well as the pharmacokinetic behaviour and antithrombotic actions of this highly effective thrombin inhibitor are described.     

Acute and conditioned hypoxic tolerance augmented by endothelial nitric oxide synthase inhibition in mice.

To identify a possible role for nitric oxide (NO) in acute hypoxic tolerance (HT) we measured hypoxic survival time (HST), effect of hypoxic conditioning (HC), and survival following hypoxic conditioning while blocking or mimicking the action of nitric oxide synthase (NOS). To inhibit NOS, CD-1 mice were given supplemental endogenous NOS inhibitor asymmetrical dimethylarginine (ADMA) or a synthetic NOS inhibitor N(omega)-nitro-L-arginine (L-NNA), both of which nonselectively inhibit three of the isoforms of NOS [inducible (iNOS), neuronal (nNOS), and endothelial NOS (eNOS)]. ADMA (10 mg/kg i.p.) or saline vehicle was given 5 min before HST testing. L-NNA was given orally at 1 g/l in drinking water with tap water as the control for 48 h before testing. Both ADMA and L-NNA significantly increased HST and augmented the HC effect on HST. Neither the nNOS selective inhibitor 7-nitroindazole (7-NI) nor the iNOS selective inhibitor N-{[3-(aminomethyl)phenyl]methyl}-enthanimidamide (1400W) had a statistically significant effect on HST or HT. The NO donor, 3-morpholinosydnoeimine, when given alone did not significantly decrease HT, but it did mitigate the increased HT effect of L-NNA. These data confirm that acute hypoxic conditioning increases HT and that NOS inhibition by endogenous (ADMA) and a synthetic NOS inhibitor (L-NNA) further increases HT, whereas iNOS and nNOS inhibition does not, suggesting that it is the inhibition of eNOS that mediates enhancement of HT.     

CRYSTALLINE SOYBEAN TRYPSIN INHIBITOR : II. GENERAL PROPERTIES

A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mµ.     

An inhibitor of acylCoA: cholesterol acyltransferase increases expression of ATP-binding cassette transporter A1 and thereby enhances the ApoA-I-mediated release of cholesterol from macrophages

The effect of inhibition of acylCoA: cholesterol acyltransferase (ACAT) was studied on high density lipoprotein (HDL) metabolism. An inhibitor of ACAT, MCC-147, was given mouse peritoneal macrophages and expression of ATP-binding cassette transporter A1 (ABCA1) was examined. ABCA1 was increased both at the mRNA and protein levels, only when the cells are cholesterol-loaded and thereby the inhibitor decreased esterified cholesterol and increased unesterified cholesterol. In this condition, the ACAT inhibitor increased reversible binding of apoA-I to the cells and enhanced apoA-I-mediated release of cellular cholesterol and phospholipid, but did not influence nonspecific cellular cholesterol efflux to lipid microemulsion. It was therefore concluded that the ACAT inhibitor increased the release of cholesterol from the cholesterol-loaded macrophages by increasing the expression of ABCA1, putatively through shifting cholesterol distribution from the esterified to the free compartments.     

Inhibitor fingerprinting of metalloproteases using microplate and microarray platforms: an enabling technology in Catalomics

One of the most fundamental properties of an enzyme is its selectivity, a property that has proved highly challenging to understand. Recent developments offer methodologies to rapidly establish activity-dependent profiles of enzymes. In this protocol, we describe methods to elucidate inhibitor fingerprints of enzymes. By taking advantage of well-defined small-molecule inhibitor libraries and the screening throughput offered from microplate and microarray platforms, we provide step-by-step application of the methodology toward the global characterization of metalloproteases, an important class of enzymes involved in numerous diseases and cellular processes. The same strategy is nonetheless applicable to virtually any given enzyme class, provided suitable experimental design and chemical inhibitor libraries are carefully implemented. We are able to routinely fingerprint as many as 2,000 independent enzyme interactions on the microplate platform within a span of approximately 7 h; however, the same throughput is attained within 5 h on the microarray platform.     

Substrate and dilution effects on the inhibition of acetylcholinesterase by carbamates

1. The kinetics of acetylcholinesterase (EC 3.1.1.7) activity and its inhibition by eserine or by Sevin (1-naphthyl N-methylcarbamate) have been studied over the substrate concentration range 5x10(-8) to 2.5x10(-2)m. 2. Equations are given for inhibition as a function of time, substrate and inhibitor concentrations, and the relevant parameters determined at 25 degrees and 37 degrees . 3. The observed and calculated effects of time, dilution, substrate addition and enzyme concentration were in good agreement and consistent with a steady-state carbamylation by eserine or by Sevin in the presence of excess of inhibitor. 4. The quantitative destruction of either inhibitor at high enzyme concentrations implied by the carbamylation hypothesis has been confirmed experimentally. 5. The importance and possibility of allowing quantitatively for dilution and substrate effects when estimating carbamate inhibition are demonstrated.     

Acute antihypertensive synergism of angiotensin-converting enzyme inhibitors and diuretics

In spontaneously hypertensive rats (SHR), after 1 day of dosing with an angiotensin-converting enzyme (ACE) inhibitor (captopril or enalapril) plus a diuretic (hydrochlorothiazide), a synergistic antihypertensive effect was observed when a second dose of the combination or ACE inhibitor alone but not the diuretic alone was given the next day. Bilateral ureteral ligation did not prevent the synergism, which indicates that diuresis per se was not the mechanism. Vascular responses to various agonists did not differ in SHR given ACE inhibitor or ACE inhibitor plus diuretic. SHR given combination treatment had higher and more prolonged increases in plasma renin activity. Aprotinin or indomethacin did not alter the synergism, which suggests that endogenous kinins and prostaglandins did not play a role. These data suggest that the mechanism for the synergistic antihypertensive effect resulted from the combination treatment's shifting the blood pressure regulation system to be renin dependent and responding more to drugs affecting the renin-angiotensin system (RAS). Evidence was presented that the RAS can be shifted rapidly to assume a greater role in blood pressure regulation in SHR as well as in normotensive and two-kidney, one-clip Goldblatt renal hypertensive dogs by restricting sodium intake. The data may partly explain the various degrees of antihypertensive responsiveness of essential hypertensive patients to ACE inhibitors.     

Structural basis for the activity of drugs that inhibit phosphodiesterases

Phosphodiesterases (PDEs) comprise a large family of enzymes that catalyze the hydrolysis of cAMP or cGMP and are implicated in various diseases. We describe the high-resolution crystal structures of the catalytic domains of PDE4B, PDE4D, and PDE5A with ten different inhibitors, including the drug candidates cilomilast and roflumilast, for respiratory diseases. These cocrystal structures reveal a common scheme of inhibitor binding to the PDEs: (i) a hydrophobic clamp formed by highly conserved hydrophobic residues that sandwich the inhibitor in the active site; (ii) hydrogen bonding to an invariant glutamine that controls the orientation of inhibitor binding. A scaffold can be readily identified for any given inhibitor based on the formation of these two types of conserved interactions. These structural insights will enable the design of isoform-selective inhibitors with improved binding affinity and should facilitate the discovery of more potent and selective PDE inhibitors for the treatment of a variety of diseases.