Synthesis of proline analogues as potent and selective cathepsin S inhibitors

Cathepsin S is a potential target of autoimmune disease. A series of proline derived compounds were synthesized and evaluated as cathepsin S inhibitors. We discovered potent cathepsin S inhibitors through structure–activity relationship studies of proline analogues. In particular, compound 19-(S) showed promising in vitro/vivo pharmacological activities and properties as a selective cathepsin S inhibitor.     

Bile duct hyperplasia in mice infected with Schistosoma mansoni

Schistosoma mansoni releases large amounts of proline into the hepatoenteric circulation. Because proline release has been linked to bile duct hyperplasia in fascioliasis, the current investigation tested the possibility that such hyperplasia might occur in schistosomiasis. The lumenal perimeter and wall thickness in bile ducts was compared between infected and uninfected mice. In those harboring 5 week old S. mansoni infections there was a 180% increase in the lumenal perimeter of the duct (P<0.001) and a 580% increase in the thickness of the duct wall (P<0.001). These results tend to support data linking proline to bile duct and liver fibroblast proliferation.     

Discovery and SAR studies of methionine–proline anilides as dengue virus NS2B-NS3 protease inhibitors

A series of methionine–proline dipeptide derivatives and their analogues were designed, synthesized and assayed against the serotype 2 dengue virus NS2B-NS3 protease, and methionine–proline anilides 1 and 2 were found to be the most active DENV 2 NS2B-NS3 competitive inhibitors with K_i values of 4.9 and 10.5 μM. The structure and activity relationship and the molecular docking revealed that l-proline, l-methionine and p-nitroaniline in 1 and 2 are the important characters in blocking the active site of NS2B-NS3 protease. Our current results suggest that the title dipeptidic scaffold represents a promising structural core to discover a new class of active NS2B-NS3 competitive inhibitors.     

Hormonal regulation of proline synthesis and glucose release in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata

In the adult males of the Colorado potato beetle, Leptinotarsa decemlineata, corpora cardiaca extract injected in vivo gives rise to an increase in glucose and a decrease in alanine concentration of the haemolymph. The regulation of proline synthesis and glucose release in the fat body of the Colorado potato beetle was investigated further in vitro. Proline regulates its own synthesis by a feedback inhibition. Moreover, a factor present in extracts from the corpora cardiaca stimulates synthesis in the fat body in vitro. This effect was demonstrated with corpora cardiaca extracts from beetles, locusts and cokroaches. Also, synthetic adipokinetic hormone stimulates proline synthesis in the fat body of the Colorado beetle. In addition, a release of glucose from the fat body of the beetle could be evoked by the addition of locust and beetle corpora cardiaca extracts or synthetic adipokinetic hormone. The physiological significance of both effects is discussed.     

Hormone stimulated lipolysis and proline synthesis in the fat body of the adult tsetse fly, Glossina morsitans

G. morsitans fat cells incubated in vitro with l-[U-¹⁴C]-leucine incorporated the radiolabel, mainly into triglycerides. Aqueous extracts of corpora cardiaca, midbrain, or thoracic ganglion stimulated the release of radiolabelled material from prelabelled fat cells in vitro. Corpora cardiaca extracts were the most active, approx. 1 × 10^?3 gland pairs/μl elicited the maximal response. At concentrations above 1 × 10^?3 gland pairs/μl the activity of corpora cardiaca extracts was inhibited by a substance which could be removed by gel filtration. The stimulatory factor in nervous-tissue extracts was destroyed by proteolytic enzymes and was recoverable in a single peak by Sephadex G15 gel filtration. Results suggest that it is a peptide hormone produced mainly by the median neurosecretory cells of the midbrain with the corpora cardiaca being the site of storage and release. No hormone was detectable in fresh haemolymph, but it was found at high concentration in boiled haemolymph, implying the presence of a heat labile inhibitor.Under the in vitro conditions used the hormone stimulated the synthesis of proline from alanine and the hydrolysis of triglycerides to free fatty acids. The probable functions of the hormone are to stimulate proline synthesis in response to demand for flight and/or to mobilise lipid for larval nutrition. The relative importance of these apparent functions in vivo could not be determined.     

Fasciola hepatica: Azetidine inhibition of bile duct hyperplasia in the infected rat

In controlled experiments the luminal bile duct perimeters were compared between rats infused with l-azetidine-2-carboxylic acid (AZC) (120 μmole/rat/day) and rats infused with saline after both groups had been implanted with adult Fasciola. The average bile duct lumen in the group receiving AZC did not enlarge relative to controls, and was about one-half the average perimeter of ducts from rats infused with saline that had worm implants. These results indicate that azetidine can inhibit bile duct hyperplasia induced by Fasciola which inhabit the duct. The results also support the hypothesis that the hyperplasia of fascioliasis is mediated through the release of free proline from the worms where it is present in high concentrations.     

Thioproline-induced accumulation of proline in barley leaf sections

Abstract Treatment of barley leaf sections with 0.1 mol m^?3 thioproline (L-thiazolidine-4-carboxylic acid) was found to induce a marked increase in proline together with some decrease in glutamate, whereas the levels of other andno acids were not influenced. This result is discussed in relation to the significance of the increase in proline in tissues treated with abscisic acid or subjected to water stress.     

In crystallo screening for proline analog inhibitors of the proline cycle enzyme PYCR1

Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the biosynthetic half-reaction of the proline cycle by reducing Δ¹-pyrroline-5-carboxylate (P5C) to proline through the oxidation of NAD(P)H. Many cancers alter their proline metabolism by up-regulating the proline cycle and proline biosynthesis, and knockdowns of PYCR1 lead to decreased cell proliferation. Thus, evidence is growing for PYCR1 as a potential cancer therapy target. Inhibitors of cancer targets are useful as chemical probes for studying cancer mechanisms and starting compounds for drug discovery; however, there is a notable lack of validated inhibitors for PYCR1. To fill this gap, we performed a small-scale focused screen of proline analogs using X-ray crystallography. Five inhibitors of human PYCR1 were discovered: l-tetrahydro-2-furoic acid, cyclopentanecarboxylate, l-thiazolidine-4-carboxylate, l-thiazolidine-2-carboxylate, and N-formyl l-proline (NFLP). The most potent inhibitor was NFLP, which had a competitive (with P5C) inhibition constant of 100 μm. The structure of PYCR1 complexed with NFLP shows that inhibitor binding is accompanied by conformational changes in the active site, including the translation of an α-helix by 1 Å. These changes are unique to NFLP and enable additional hydrogen bonds with the enzyme. NFLP was also shown to phenocopy the PYCR1 knockdown in MCF10A H-RAS^V12 breast cancer cells by inhibiting de novo proline biosynthesis and impairing spheroidal growth. In summary, we generated the first validated chemical probe of PYCR1 and demonstrated proof-of-concept for screening proline analogs to discover inhibitors of the proline cycle.     

Characterization of sheep antibodies involved in precipitate formation with surface antigens of Fasciola hepatica in vitro

Sandeman R. M. and Howell M. J. 1982. Characterization of sheep antibodies involved in precipitate formation with surface antigens of Fasciola hepatica in vitro. International Journal for Parasitology12: 467–471. The role of sheep antibodies which precipitate with surface antigens of Fasciola hepatica is unclear. In an attempt to clarify their function these antibodies were characterized as to their immunoglobulin class and ability to affect the survival of fluke in rats. The ability of fluke antigens complexed with sheep antibody to vaccinate rats against infection was also tested. IgM antibodies were involved in precipitate formation on the teguments of fluke 3 weeks after infection but IgG₁ predominated at later stages of infection. The decreased survival of fluke in rats after culture with increasing levels of sheep antibodies suggests that the antibodies exert some deleterious effect on the fluke in vitro. The fluke antigen-sheep antibody complex failed to immunize rats against infection. Since sheep antibodies to F. hepatica can impair the ability of fluke to resist further attack in rats but not sheep, it is suggested that some effector mechanism other than antibody is defective in the latter.     

Optimization of spirocyclic proline tryptophan hydroxylase-1 inhibitors

As a follow-up to the discovery of our spirocyclic proline-based TPH1 inhibitor lead, we describe the optimization of this scaffold. Through a combination of X-ray co-crystal structure guided design and an in vivo screen, new substitutions in the lipophilic region of the inhibitors were identified. This effort led to new TPH1 inhibitors with in vivo efficacy when dosed as their corresponding ethyl ester prodrugs. In particular, 15b (KAR5585), the prodrug of the potent TPH1 inhibitor 15a (KAR5417), showed robust reduction of intestinal serotonin (5-HT) levels in mice. Furthermore, oral administration of 15b generated high and sustained systemic exposure of the active parent 15a in rats and dogs. KAR5585 was selected for further pharmacological evaluation in disease models associated with a dysfunctional peripheral 5-HT system.     

Fasciola hepatica: Energy sources and metabolism

Gas chromatographic determination of biliary glucose in the rat showed only low levels were present. Although flukes took up glucose from the bile in vivo, biliary glucose could not be a major energy source as occurs in Hédon Fleig solution in vitro. Alanine, arginine, glutamate, histidine, phenylalanine, and serine were not metabolised in vitro to volatile fatty acids, and mixtures of peptides or amino acids failed to spare glycogen breakdown in the absence of glucose. Although glycerol was metabolised in vitro, the main energy source of the fluke in vivo has not been identified. Labelling studies confirmed that glucose taken up in vitro is degraded via glycolysis rather than the pentose phosphate pathway.     

Fluorescent lipoprotein probe

The fluorescent cholesterol analog cholesta-5,7,9(11)triene-3-β-ol was used to label high-density and low-density lipoproteins in vivo (rabbit) and in vitro (human). Rabbit feeding experiments demonstrated that in vivo both the esterified and nonesterified forms of the fluorophore were incorporated by these particles. Using in vitro labeling techniques, it was possible to selectively incorporate either the free form of the fluorophore or both the free and the esterified forms depending upon the presence or absence of serum esterases during incubation. Subsequent to labeling, the thermotropic behavior of the low- and high-density lipoproteins was evaluated using temperature-dependent fluorescence intensity measurements. Purified low-density lipoprotein samples (human and rabbit) containing both forms of the fluorophore were observed to undergo a thermotropic transition between 27 and 32°C. However, this transition was not observed in low-density lipoprotein samples containing only the nonesterified form of the probe nor was it observed in any of the high-density lipoprotein samples, even those containing both forms of the fluorophore. These results provide further evidence that the previously reported thermotropic transition in low-density lipoprotein is due to a reordering of the low-density lipoprotein cholesterol ester core (Deckelbaum, R.J., Shipley, G. G., Small, D. M., Lees, R. S., and George, P. K. (1975) Science190, 392–394; Sears, B., Deckelbaum, R. J., Janiak, M. J., Shipley, G. G., and Small, D. M. (1976) Biochemistry15, 4151–4157; Chana, G. S., Sheppard, R. J., Mills, G. L., and Grant, E. H. (1980) Phys. Med. Biol.25, 427–432).     

Pyrroline-5-carboxylic acid reductase from soybean leaves

Pyrroline-5-carboxylic acid reductase was purified 40-fold from soybean leaves (Glycine max L. var Corsoy). The enzyme was fairly unstable, had a broad pH optimum, and was inactivated by heat and acid; NADH and NADPH both served as cofactors. It had a higher activity with NADH (about 4 ×) compared to NADPH, but a lower K_m for NADPH. NADP⁺ inhibited both the NADH- and NADPH-dependent activity. Sulfhydryl group blocking agents reduced the activity as did the carbonyl blocking agent, NH₂OH. Thiazolidine-4-carboxylic acid and phosphate inhibited the enzyme and proline inhibited only at high concentrations. ATP, GTP, and CTP were all effective inhibitors of both the NADH- and NADPH-dependent activity. Phosphorylated nucleotide inhibition was reversed by Mg²⁺ ions.     

Synthetic thiosemicarbazones as a new class of Mycobacterium tuberculosis protein tyrosine phosphatase A inhibitors

Mycobacterium tuberculosis secretes two protein tyrosine phosphatases as virulence factors, PtpA and PtpB. Inhibition studies of these enzymes have shown significant attenuation of the M. tuberculosis growth in vivo. As PtpA mediates many effects on the regulation of host signaling ensuring the intracellular survival of the bacterium we report, for the first time, thiosemicarbazones as potential novel class of PtpA inhibitors. Several compounds were synthesized and biologically evaluated, revealing interesting results. Enzyme kinetic assays showed that compounds 5, 9 and 18 are non-competitive inhibitors of PtpA, with K_i values ranging from 1.2 to 5.6?µM. Modeling studies clarified the structure-activity relationships observed in vitro and indicated a possible allosteric binding site in PtpA structure. To the best of our knowledge, this is the first disclosure of potent non-competitive inhibitors of PtpA with great potential for future studies and development of analogues.     

In vitro development of the strobilar stage of Mesocestoides corti

Thompson R. C. A., Jue Sue L. P. and Buckley S. J. 1982. In vitro development of the strobilar stage of Mesocestoides corti. International Journal for Parasitology12: 303–314. Sexually mature strobilated adults of Mesocestoides corti were grown consistently from undifferentiated tetrathyridia in vitro using a conventional diphasic culture system. Development (growth, strobilisation and maturation) was compared in vitro and in vivo. Although growth and strobilisation were comparable in vitro and in vivo, during the first 18 days, total length and numbers of proglottids decreased in vivo but continued to increase in vitro after day 18. Both male and female reproductive systems appeared to develop normally in vitro and self copulation was frequently observed in cultured worms. However, fully developed oncospheres were not produced in vitro.     

In vivo and in vitro protective role of proline

Accumulation of proline in response to environmental stresses seems tobe widespread among plants. To elucidate the role of proline in plantresponses,in vivo and in vitro, we studied theeffect of proline on catalase (CAT; EC 1.11.1.6), peroxidase (POD; EC 1.11.1.7)and polyphenol oxidase (PPO; EC 1.14.18.1). In vivo, thesethree enzymes were activated by proline, while CAT and POD were activated andPPO was inactivated by NaCl. In vitro, CAT and POD wereactivated and PPO was inactivated by proline. Proline appeared to protect thesethree enzyme activities. The significance of these findings with regard toenvironmental stress-induced proline accumulation in vivois discussed. The ability of proline to activate the enzymes may suggest alimited conformational change. These results are important for characterisationof metabolic responses to environmental stresses and can be used as a stressindicator.     

The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts.

Collagen secretion by chick embryo fibroblasts was measured by incorporating [¹⁴C]proline into proteins and then analyzing the amount of collagen in the cell and medium separately by using purified bacterial collagenase. In order to produce varying levels of hydroxylation, cells were incubated with varying concentrations of ascorbate or with varying concentrations of α,α′-dipyridyl in the presence of saturating ascorbate. Ascorbate stimulated both the hydroxylation of proline in collagen and the secretion of collagen; the concentration of ascorbate required for half-maximal stimulation of both proesses was approximately 4.5 × 10^?7, m. Since the cells could concentrate ascorbate 10-fold, this K_M for proline hydroxylation is 100-fold lower than values reported for purified prolyl hydroxylase (Abbot, M. T., and Udenfriend, S. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.), p. 173, Academic Press New York; Kivirikko K. I., et al. (1968) Biochim. Biophys. Acta, 151, 558–567). Conversely, α,ga′-dipyridyl inhibited both proline hydroxylation and collagen secretion; half-maximal inhibition of both processes was observed at 7 × 10^?5, m. The results of the two types of experiments show that the secretion of collagen becomes directly proportional to proline hydroxylation when approximately 30% of the proline residues in collagen have been hydroxylated compared to maximal hydroxylation of 50%. Since the stability of triple-helical collagen at 37 °C has been shown to be dependent on the hydroxyproline content of the molecule (Rosenbloom, J., et al. (1973) Arch. Biochem. Biophys., 158, 478–484), we suggest that the observed proportionality between secretion and hydroxylation is a reflection of the increased amount of stable triple helical collagen at 37 °C. When the cells were incubated with a concentration of ascorbate that was saturating for secretion and hydroxylation, there was no significant activation of prolyl hydroxylase as measured in a cell-free extract. These experiments suggest that ascorbate effects collagen secretion by acting at the site of proline hydroxylation but not by increasing the activity of prolyl hydroxylase.     

Fungicidal Activity of Two Spermidine Analogues

The potential use of polyamine analogues as inhibitors of polyamine biosynthesis to control plant pathogenic fungi is well established. However, all of this information relates to the use of putrescine analogues and no data exist for spermidine analogues. In the present work, two spermidine analogues. N¹- and N⁸-acetylspermidine were evaluated against powdery mildew on barley. Post-inoculation treatments reduced infection by 69.7% and 51.5%. respectively. Since the barley powdery mildew fungus cannot be grown axenically. mode of action studies were undertaken using the oat leaf-stripe pathogen Pyrenophora avenae. Neither of the analogues had any effect on polyamine biosynthesis in P. avenae grown in vitro. Although the mechanism(s) by which inhibitors affect in vivo fungal growth and in vitro growth may differ, it is unlikely that the antifungal properties of the analogues are the result of a perturbation in polyamine biosynthesis.     

Synthesis and anti-tumor activity of [1,4] dioxino [2,3-f] quinazoline derivatives as dual inhibitors of c-Met and VEGFR-2

Both c-Met and VEGFR-2 were important targets for cancer therapies. In order to develop reversible and non-covalent c-Met and VEGFR-2 dual inhibitors, a series of [1,4]dioxino[2,3-f]quinazoline derivatives were designed and synthesized. The enzyme assay demonstrated that most target compounds had inhibition potency on both c-Met and VEGFR-2 with IC₅₀ values in nanomolar range especially compounds 7m and 7k. Based on further cell proliferation assay in vitro, compound 7k showed significantly anti-tumor activity in vivo on a hepatocellular carcinoma (MHCC97H cells) xenograft mouse model. We docked the compound 7m with c-Met and VEGFR-2 kinases, and interpreted the SAR of these analogues. All results indicated that the target compounds were dual inhibitors of c-Met and VEGFR-2 kinases that held promising potential in cancer therapy.