Discovery of highly potent small molecule kallikrein inhibitors

Uncontrolled kallikrein activation is involved in diseases such as hereditary angioedema, bacterial septic shock and procedures such as cardiopulmonary bypass. Here we report a series of small molecule compounds that potently inhibit kallikrein activity in vitro. Kinetic studies indicate that some of these compounds are slow binding inhibitors of kallikrein with Ki final less than a nanomolar. The ability of these compounds to inhibit the activity of kallikrein was further confirmed in a plasma model by quantitating the release of bradykinin, an endogenous cleavage product of plasma kallikrein. To understand the inhibitory mechanism of the selected compounds toward kallikrein, the interactions between the selected compounds and kallikrein was explored using molecular modeling based on the information of crystal structures of TF/FVIIa and kallikrein. The information presented in the current study provides an initial approach to develop more selective and therapeutically useful small molecule inhibitors.     

Role of the EGF-like domains in mammalian tolloid (mTLD) secretion and procollagen C-proteinase activity

Introduction Bone morphogenetic protein (BMP)‐1 and its larger splice variant mammalian tolloid (mTLD) belong to the tolloid group of astacin‐like metalloproteinases that are fundamental to tissue patterning and extracellular matrix assembly. BMP‐1 and mTLD exhibit similar substrate specificity in vitro; however, BMP‐1 is a much better procollagen C‐proteinase than mTLD. mTLD consists of a prodomain (which is cleaved by a furin‐like enzyme) ( Leighton & Kadler 2003 ), a zinc metalloproteinase domain and a C‐terminal part comprising five CUB domains thought to be important for protein–protein interactions ( Hartigan et al. 2003 ), and two EGF‐like domains, which in other proteins are involved in calcium ion binding. BMP‐1 lacks the most C‐terminal two CUB domains and one EGF‐like domain. mTLD activity is known to be calcium ion dependent, as demonstrated for the chick homologue ( Hojima et al. 1985 ). In our current work, we are studying the role of the EGF‐like domains in the secretion and procollagen C‐proteinase activity of mTLD, and the contribution that these domains made to calcium ion dependency. Materials and methods We designed proteins lacking EGF1, EGF2 or both. NotI sites were introduced by PCR at the borders of the EGF domain of a cDNA clone encoding a V5‐His mTLD. Restriction enzyme digestion was used to delete individual domains. The mutant constructs in pCEP4 were stably transfected into 293‐EBNA cells. Expression was analysed by Western blot. The wild‐type and the mutant enzymes were purified on a nickel ion column, and their activity was determined by cleavage of type‐I procollagen in the presence or absence of 5 mm CaCl₂. Results We showed that (1) the mTLD proteins lacking EGF1, EGF2 or EGF1 + EGF2 were poorly secreted into the culture medium compared to mTLD and (2) the EGF deletion mutants remained calcium ion dependent, but some differences were seen. Most notably, the ΔEGF2 and ΔEGF1 + ΔEGF2 mutants were found to be better C‐proteinases than the wild‐type enzyme in the presence of calcium ions. Conclusion From these preliminary data, we concluded that (1) the EGF domains are necessary for efficient secretion (2) both EGF1 and EGF2 domains contribute to the calcium ion dependency of mTLD and (3) the EGF2 domain might be a Ca²⁺‐activated hinge that ‘swings’ the CUB‐4 and CUB‐5 domains away from the active site. The ?EGF2 mTLD might be expected to have an open conformation, thereby making it a better C‐proteinase than the wild‐type enzyme, and (?4) Ca²⁺ ions are bound by other domains in mTLD and not only by the EGF‐like domains.     

Discovery and characterization of potent small molecule inhibitors of the high affinity proline transporter

The mammalian proline transporter (PROT) is a high affinity Na⁺/Cl⁻-dependent transporter expressed in specific regions of the brain. It is homologous to other neurotransmitter transporters such as glycine, norepinephrine, serotonin, and dopamine transporters. PROT is enriched in glutamatergic synaptic terminals and may play an important role in the regulation of excitatory neurotransmission. No non-peptide small molecule inhibitors have been described for this transporter. To study its physiological role in the central nervous system and evaluate its potential as a therapeutic target, we established cell lines that stably express recombinant hPROT and characterized its kinetic properties for proline uptake. We then screened for inhibitors and identified a series of compounds that inhibit hPROT-mediated proline uptake. A known compound, benztropine, was found to inhibit hPROT with an IC₅₀ of 0.75 μM. A series of novel compounds were also found, one of which, LP-403812, showed an IC₅₀ of approximately 0.1 μM on both recombinant human and mouse PROT without significant inhibition of glycine and dopamine transporters at concentrations up to 10 μM. This compound also inhibited proline transporter activity of mouse brain synaptosomes with the same potency. These inhibitors provide important tools for the understanding of PROT functions in the brain and may lead to the development of therapeutic agents for certain neurological disorders.     

Discovery of substituted isoxazolecarbaldehydes as potent spermicides, acrosin inhibitors and mild anti-fungal agents

BACKGROUND: The continued endeavour to design novel, non-detergent molecules that can be useful as topical, prophylactic contraceptives has led to the discovery of substituted isoxazolecarbaldehydes as a new class of compounds exhibiting both spermicidal and acrosin inhibitory activities simultaneously. METHODS: Normal human semen samples were used to detect the spermicidal and acrosin inhibitory activities of the new compounds. Lactobacillus, HeLa and Candida cultures were used to determine the safety of compounds towards normal vaginal flora, their cytotoxicity and anti-fungal activity. Supravital staining and the hypo-osmotic swelling test (HOST) were used to detect the effect on sperm membrane integrity. Nonoxynol-9 (N-9) was used as a reference standard. RESULTS: The 5- and 3-substituted isoxazolecarbaldehydes showed significant spermicidal [minimum effective concentration (MEC)=0.005-2.5%] and acrosin inhibitory (IC50=3.9-58 x 10(-4) mol/l) activities in several molecules along with weak fungicidal activity against Candida albicans. Lineweaver-Burk and Dixon plot analysis of a representative structure showed non-competitive inhibition of human acrosin enzyme, and the most potent acrosin inhibitors also considerably diminished the induction of the acrosome reaction by Ca2+ ionophore. Some compounds were found to be significantly safer than N-9 towards Lactobacillus acidophilus in vitro at their respective spermicidal MECs. In the cytotoxicity assay, the IC50 of these compounds towards the HeLa cell line was of the same order as N-9 (0.9-0.1 mmol/l); however, in contrast, the compounds exhibited only a moderate effect on sperm membrane integrity. CONCLUSIONS: This study indicates that 5- and 3-substituted isoxazolecarbaldehydes are 'first generation' multifunctional, spermicidal molecules that hold promise for development as topical contraceptives with useful associated activities that can add considerably to their effectiveness, safety and prophylaxis.     

Esomeprazole: potent acid suppression in the treatment of acid-related disorders

Esomeprazole (S-omeprazole), an enantiomer of the racemate omeprazole, is the first proton pump inhibitor to be developed as an isomer. This confers improved pharmacokinetics and pharmacodynamics compared with the racemate R/S-omeprazole. The difference in the pharmacokinetics of esomeprazole compared with omeprazole and the R-isomer is due to reductions in total body clearance and first-pass metabolism in the liver. Pharmacodynamic studies showed that esomeprazole 40 mg provides greater intragastric acid control than respective doses of all the other proton pump inhibitors on the market. Several well-designed clinical trials, employing both endoscopic and symptomatic response criteria, have compared the efficacy of esomeprazole with that of other proton pump inhibitors in the management of gastroesophageal reflux disease patients, and in the eradication of Helicobacter pylori. In addition, the efficacy of esomeprazole for the healing and prevention of nonsteroidal anti-inflammatory drug-associated dyspeptic symptoms and ulcers has been established. The aim of this review is to provide an overview of the pharmacokinetics, pharmacodynamics and consequent clinical importance of esomeprazole in the treatment of acid-related disorders.     

3-(1-imidazolylmethyl) indoles: potent and selective inhibitors of human blood platelet thromboxane synthetase

Several 3-(1-imidazolylmethyl) indoles were tested for inhibition of the microsomal enzymes which catalyse the biosynthesis of thromboxane A₂, prostaglandin I₂, and prostaglandin endoperoxides. These products were measured by bioassay to assess levels of enzyme activity. The highest activity against human blood platelet thromboxane A₂-synthetase was obtained with 2-cyclopropyl-3(1-imidazolylmethyl) indole (IC₅₀ 1×10^–10 M). This compound also exhibited the highest activity against pig aorta prostaglandin I₂-synthetase (IC₅₀ 8.4×10^–7 M). Of much more potential therapeutic interest, 2-isopropyl-3-(1-imidazolylmethyl) indole and 3-(1-imidazolylmethyl) indole showed almost complete selectivity against thromboxane A₂-synthetase. Both compounds exhibited IC₅₀'s of 2×10^–8 M against the latter enzyme but showed only weak effects (IC₅₀'s>10^–4 M) against prostaglandin I₂-synthetase and ram seminal vesicle PGH₂-synthetase.     

Organ crosstalk: the potent roles of inflammation and fibrotic changes in the course of organ interactions

Inflammation Research - Organ crosstalk can be defined as the complex and mutual biological communication between distant organs mediated by signaling factors. Normally, crosstalk helps to...     

Mechanical stress triggers selective release of fibrotic mediators from bronchial epithelium

Transforming growth factor-beta (TGF-beta) and endothelin (ET) are found in elevated amounts in the airways of individuals with asthma. The cellular source of these peptides and their role in mediating the airway fibrosis of chronic asthma are unknown. In response to mechanical stresses similar to those occurring in vivo during airway constriction, bronchial epithelial cells increase the steady-state level of mRNA for both ET-1 and ET-2, followed by increased release of ET protein. Mechanical stress also enhances release of TGF-beta2 from a preformed cell-associated pool. TGF-beta2 and ET act individually and, more importantly, synergistically to promote fibrotic protein synthesis in reporter fibroblasts. To confirm the role of these intermediates in stress-induced fibrosis, conditioned medium from mechanically stressed bronchial epithelial cells was shown to elicit fibrotic protein synthesis in reporter fibroblasts; this effect was significantly inhibited by combined treatment with ET receptor antagonists and a neutralizing antibody to TGF-beta2. These data are consistent with a primary pathogenic role for mechanical stress-induced release of both TGF-beta2 and ET in the subepithelial fibrosis that characterizes chronic asthma.     

Structure of alveolar epithelial cells in patients with fibrotic lung disorders

Ultrastructural studies were made of the types of alveolar epithelial cells in fibrotic lungs from 34 patients, including 20 with idiopathic pulmonary fibrosis, five with collagen-vascular diseases, six with sarcoidosis, one with lymphangioleiomyomatosis, one with histiocytosis X, and one with chronic eosinophilic pneumonia. In 28 patients, proliferation of type II alveolar epithelial cells was recognized on the basis of lamellar bodies in the cytoplasm, microvilli in the luminal surface, focal microfoldings of the basal plasma membrane, close interaction with underlying mesenchymal cells, and unilayered arrangement. Two types of cuboidal epithelial cells were recognized and were considered to be derived from bronchiolar basal cells (type A cuboidal cells) and from cuboidal cells in respiratory bronchioles (type B cuboidal cells). Type A cuboidal cells frequently contained large numbers of cytoskeletal filaments, and their basal plasma membranes possessed hemidesmosomes in close association with anchoring fibrils. Type B cells lacked hemidesmosomes and anchoring fibrils, Proliferation of either or both types of cuboidal cells was found in 30 patients. In 10 patients (average degree of fibrosis = 3.5 on a scale of 0 to +4), the proliferation involved type A cells; in 10 other patients (average degree of fibrosis = 2.5), type B cells in nine patients (average degree of fibrosis = 3.4), both type A and type B cells; in one patient cuboidal cells were identified only by light microscopy. In 17 patients, proliferating cuboidal cells formed foci of epithelial pseudostratification. Type II alveolar epithelial cells did not participate in the process of multilayering. Thus, type II alveolar epithelial cells and two types of cuboidal epithelial cells are sources of epithelial renewal in damaged alveoli in fibrotic lungs. Type II cells proliferate mainly in areas of less severe degrees of fibrosis; cuboidal cells become the main source of epithelial renewal in areas of very severe lung damage.     

E-series prostaglandins are potent growth inhibitors for some B lymphomas

The ability of prostaglandins (PG) to inhibit the growth of B cell lymphomas was investigated. Macrophage-secreted PGE2 was previously shown to promote unresponsiveness to antigen in normal B lymphocytes. This observation suggested that B lymphomas might also be regulated by prostanoids. Five non-PG-secreting Ly-1+ B lymphomas (CH12, CH31, CH33, NBL and WEHI-231) were incubated for 24-72 h with PGE2, PGE1 or PGF2 alpha. The level of lymphoma growth at the end of culture was determined using a colorimetric assay which detects only viable cells. A marked heterogeneity was observed with respect to the sensitivity of these lymphomas to PGE2 and PGE1. CH31 was very sensitive, being growth inhibited by as little as 10(-8) M PGE. In contrast, CH12, a more mature lymphoma, was highly resistant, whereas CH33, NBL and WEHI-231 were of intermediate resistance. All five lymphomas demonstrated little or no growth inhibition when cultured with PGF2 alpha. Moreover, unlike PGE2, PGF2 alpha failed to elevate intracellular cAMP levels. It was previously shown that CH31, CH33 and WEHI-231 could be growth inhibited by anti-immunoglobulin antibodies which cross-link surface immunoglobulin. Interestingly, these three lymphomas were rendered more sensitive to this treatment if PGE2 was present. For example, 10(-8) M PGE2 alone had little effect on CH33, but significantly augmented growth inhibition induced by suboptimal quantities of anti-immunoglobulin antibody. Cholera toxin, another agent which was found to rapidly elevate intracellular cAMP levels, also synergized with suboptimal doses of anti-immunoglobulin to induce growth inhibition. Overall these data suggest that, in vivo, macrophage-secreted prostanoids may slow the growth of some lymphomas and that anti-immunoglobulin or anti-idiotype treatment may be more effective in the presence of agents which elevate cAMP such as E-series PG.     

Ultrastructure of pulmonary mast cells in patients with fibrotic lung disorders.

The topographic distribution, population density, and ultrastructural features of metachromatic cells (mast cells and basophilic leukocytes) were studied in lung biopsies from five control patients and 17 patients with fibrotic lung disorders. The great majority of metachromatic cells were mast cells. The average number of metachromatic cells per square millimeter of tissue section was much larger in patients with fibrotic lung disorders (45.8 +/- 6.5) than in control patients (2.6 +/- 1.6). In control patients, mast cells were most frequently seen in subpleural and perivascular connective tissue. In contrast, the vast majority of mast cells in patients with fibrotic lung disorders was present in thickened, fibrous alveolar septa; mast cells also were found within the alveolar epithelial layer and alveolar lumina. The quantitative distribution of different types of mast cell granules differed in the two groups of patients: granules composed of scrolls were more frequent in control patients, and granules of the combined type (containing mixtures of different components within the same granule) were more frequent in patients with fibrotic lung disorders. Mast cells in the latter patients appeared to migrate through defects in the basement membrane into the epithelial layer and alveolar lumina; mast cells in these areas often showed reduced numbers of granules and disorganized granule content. These changes suggest that pulmonary parenchymal mast cells in fibrotic lung disorders undergo a chronic process of partial degranulation which differs from that found in anaphylaxis; this chronic release of mast cell products may contribute to the continuing alveolar injury and the ventilation-perfusion inequalities observed in the fibrotic lung disorders.     

The pharmacogenetics of the selective serotonin reuptake inhibitors

Summary Citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline are selective serotonin reuptake inhibitors (SSRIs), which are thought to act as antidepressants through their ability to inhibit presynaptic serotonin reuptake in the brain. The elimination of the SSRIs proceeds predominantly via oxidation catalyzed by cytochrome P450 in the liver. Paroxetine and fluoxetine are potent inhibitors of cytochrome P4502D6 and hence may cause serious interactions with drugs metabolized by this isozyme, notably tricyclic antidepressants, some neuroleptics, and some antiarrhythmics. Citalopram, fluvoxamine and sertraline do not share this property. Fluvoxamine is the only SSRI that is a potent inhibitor of cytochrome P4501A2 and hence causes serious pharmacokinetic interactions with amitriptyline, clomipramine, imipramine, theophylline, and presumably caffeine and other drugs which are metabolized by the isozyme. Citalopram and fluoxetine are administered as racemates, but practically nothing is known about the stereoselective metabolism of the two drugs. Citalopram is partially metabolized via the mephenytoin oxidation polymorphism, and paroxetine is partially metabolized via the sparteine/debrisoquine oxidation polymorphism. The pharmacogenetic differences in the oxidation of the SSRIs themselves are probably of no clinical relevance.Abbreviations CYP cytochrome P450 - EM extensive metabolizer - PM poor metabolizer - SSRI selective serotonin reuptake inhibitor     

The future of selective serotonin reuptake inhibitors (SSRIs) in psychiatric treatment

Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed and widely regarded as a first-line treatment for depression. Yet, a growing body of evidence indicates that these agents are only moderately more effective than placebo in treating major depressive disorder. In recent years, it has been debated whether SSRIs offer any clinically meaningful advantage over placebos. As part of this debate, it has been argued that these agents are first-line treatments for some forms of depression but not necessarily for others. The present paper examines two hypotheses that are central to these issues. The first hypothesis is that SSRIs are more effective than placebo for some types of depression but not for others. The second is that SSRIs are more effective than psychotherapies for some types of depression than others. A review of the empirical literature reveals three main classifications of depression that are relevant to the first hypothesis: (a) more vs. less severe depression, (b) melancholic vs. non-melancholic depression, and (c) depression defined according to associated genetic factors (particularly the long vs. short allele of the serotonin transporter gene promoter). There is no strong or consistent support for (a) or (b). There is, however, emerging and consistent evidence for (c), and so the first hypothesis is tentatively supported, but only for (c). Most of the empirical evidence does not support the second hypothesis. Psychotherapies (cognitive-behavioral and interpersonal therapies) and SSRIs generally have equivalent efficacy, regardless of the severity of depression. The research literature also suggests a third hypothesis that remains to be evaluated: that SSRIs are more effective for treating anxiety disorders (and possibly other disorders) than they are for treating depression. If that hypothesis is supported by subsequent research, then the future of SSRIs may lie largely in the treatment of anxiety disorders, and in the management of particular subtypes of depression.     

Chlorpheniramine, selective serotonin-reuptake inhibitors (SSRIs) and over-the-counter (OTC) treatment

Some old antihistamines were selective serotonin-reuptake inhibitors (SSRIs) and the SSRI effect was discovered by Nobel Laureate Professor Arvid Carlsson as early as 1969. Chlorpheniramine was the most active of the tested drugs, and it compares favourably with amitriptyline and imipramine with respect to actions on both serotonergic and noradrenergic neurons. Chlorpheniramine can be called a SSRI, since the blocking of 5HT is stronger than the effect on noradrenaline neurons; however it might also be called a selective serotonin and noradrenaline reuptake inhibitor (SSNRI) and be compared with new drugs, such as venlafaxine. Carlsson suggested the potential value of clinical studies of the antidepressant properties of this and related antihistamine drugs. But, in the event, no such trials were ever performed at the time. However, later clinical observations of the benefits of dex-chlorpheniramine treatment in panic disorder have been published. Clinical experience suggests that patients using chlorpheniramine, and having also a concomitant depression or panic disorder, may experience a return of symptoms when their old drug is changed to a new antihistamine lacking SSRI effects. Yet this phenomenon is not known to many doctors, and even less known to the large number of patients buying chlorpheniramine under various trade names over-the-counter (OTC) at a low price for self-treatment of hay fewer or as a cold remedy. Chlorpheniramine was introduced in USA under the name Chlor-Trimeton as long ago as July 1950, and is still on the market. Therefore, this SSRI is now over 50 years old. If chlorpheniramine had been tested in depression in the nineteen seventies, it is probable that a safe, inexpensive SSRI drug could have been used some 15 years earlier than fluoxetine - which became available in 1987. Chlorpheniramine might have been the first safe, non-cardiotoxic and well-tolerated antidepressant. Billions of dollars in the development and marketing costs would have been saved, and the suffering of millions of patients alleviated.     

Guidance for the diagnosis and treatment of hypolipidemia disorders

The Abetalipoproteinemia and Related Disorders Foundation was established in 2019 to provide guidance and support for the life-long management of inherited hypocholesterolemia disorders. Our mission is “to improve the lives of individuals and families affected by abetalipoproteinemia and related disorders”. This review explains the molecular mechanisms behind the monogenic hypobetalipoproteinemia disorders and details their specific pathophysiology, clinical presentation and management throughout the lifespan. In this review, we focus on abetalipoproteinemia, homozygous hypobetalipoproteinemia and chylomicron retention disease; rare genetic conditions that manifest early in life and cause severe complications without appropriate treatment. Absent to low plasma lipid levels, in particular cholesterol and triglyceride, along with malabsorption of fat and fat-soluble vitamins are characteristic features of these diseases. We summarize the genetic basis of these disorders, provide guidance in their diagnosis and suggest treatment regimens including high dose fat-soluble vitamins as therapeutics. A section on preconception counseling and other special considerations pertaining to pregnancy is included. This information may be useful for patients, caregivers, physicians and insurance agencies involved in the management and support of affected individuals.