Synthesis of a series of stromelysin-selective thiadiazole urea matrix metalloproteinase inhibitors.

The synthesis and enzyme inhibition data for a series of thiadiazole urea matrix metalloproteinase (MMP) inhibitors are described. A broad screening effort was utilized to identify several thiadiazoles which were weak inhibitors of stromelysin. Optimization of the thiadiazole leads to include an alpha-amino acid side chain with variable terminal amide substituents provided a series of ureas which were moderately effective stromelysin inhibitors, with Ki's between 0.3 and 1.0 microM. The most effective analogues utilized an L-phenylalanine as the amino acid component. In particular, unsubstituted 46 had a Ki of 710 nM, while the p-fluoro analogue 52 displayed increased potency (100 nM). Stromelysin inhibition was further improved using a pentafluorophenylalanine substituent which resulted in 70, a 14 nM inhibitor. While gelatinase inhibition was generally poor, the use of 1-(2-pyridyl)piperazine as the amide component usually provided for enhanced activity, with 71 inhibiting gelatinase with a Ki of 770 nM. The combination of this heterocycle with a p-fluorophenylalanine substituent provided the only analogue, 69, with collagenase activity (13 microM). The SAR for analogues described within this series can be rationalized through consideration of the X-ray structure recently attained for70 complexed to stromelysin. Uniquely, this structure showed the inhibitor to be completely orientated on the left side of the enzyme cleft. These results suggest that thiadiazole urea heterocycles which incorporate a substituted phenylalanine can provide selective inhibitors of stromelysin. Careful selection of the amide substituent can also provide for analogues with modest gelatinase inhibition.

Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity.

The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.

Synthetic matrix metalloproteinase inhibitors and tissue inhibitor of metalloproteinase (TIMP)-2, but not TIMP-1, inhibit shedding of tumor necrosis factor-alpha receptors in a human colon adenocarcinoma (Colo 205) cell line.

The solubilization of plasma membrane receptors through proteolytic cleavage of the ligand binding domain at the cell surface is an important mechanism for regulating cytokine function and receptor signaling. The inhibition of the shedding of a variety of receptors by synthetic inhibitors of the matrix metalloproteinases (MMPs) implicates metalloproteinases in this regulatory event. We examined the effects of two naturally occurring tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, and several synthetic MMP inhibitors (MMPIs) on the shedding of both tumor necrosis factor alpha receptor type I (TNFalpha-RI; Mr 55,000) and TNFalpha-RII (Mr 75,000) by the Colo 205 human colon adenocarcinoma cell line. Culture of Colo 205 cells for 48 h resulted in the shedding of both TNFalpha-RI and TNFalpha-RII, as determined by ELISA. The shedding of TNFalpha receptors was not affected by TIMP-1 or protease inhibitors aprotinin, pepstatin, or leupeptin but was inhibited in a dose-dependent manner by the following synthetic MMPIs: batimastat and marimastat (BB-94 and BB-2516, respectively, British Biotech, Inc.); CT1418 (Celltech Therapeutics); CGS27023A (Novartis Pharmaceuticals); and RO31-9790 (Roche), with IC50s ranging from 3.2 to 38.0 microM. Similarly, TIMP-2 from two different sources reproducibly inhibited the shedding of both TNFalpha-RI and TNFalpha-RII in a dose-dependent manner (IC50 = 286 +/- 33 nM for TNFalpha-RI shedding and 462 +/- 52 nM for shedding of TNFalpha-RII). The inhibition of TNFalpha-RI shedding was confirmed in the SW626 human ovarian adenocarcinoma cell line. The synthetic MMPIs and TIMP-2, but not TIMP-1, also caused a dose-dependent increase in the number of TNFalpha receptors retained on the surface of Colo 205 cells, as determined by flow cytometry. Inhibition of TNFalpha receptor shedding with TIMP-2 occurs at molar concentrations 10-100 times less than those required with low molecular weight, synthetic MMPIs but at concentrations greater than those required to inhibit collagen degradation. Modulation of TNFalpha receptor shedding by TIMP-2 could have important implications for the pleiotropic effects of TNFalpha in both normal and malignant cells and for the pharmacological activity of synthetic MMPIs.

Modulation of bovine microvascular endothelial cell proteolytic properties by inhibitors of angiogenesis.

A tightly controlled increase in extracellular proteolysis, restricted both in time and space, is an important component of the angiogenic process, while anti-proteolysis is effective in inhibiting angiogenesis. By focussing on the plasminogen activator (PA)-plasmin system, the objective of the present studies was to assess whether previously described inhibitors of angiogenesis modify bovine microvascular endothelial cell proteolytic properties. We demonstrate that although synthetic angiostatic steroids (U-24067 and U-42129), heparin, suramin, interferon alpha-2a, and retinoic acid are all inhibitors of in vitro angiogenesis, each of these agents has distinct effects on the plasminogen-dependent proteolytic system. Specifically, angiostatic steroids and interferon alpha-2a reduce urokinase-type PA (u-PA) and PA inhibitor-1 activity, while heparin and retinoic acid increase u-PA activity. Suramin reduces cell-associated u-PA activity and greatly increases PAI-1 production at doses which induce monolayer disruption. These findings demonstrate that a spectrum of alterations in extracellular proteolysis is associated with anti-angiogenesis, and that anti-angiogenesis and anti-proteolysis are not necessarily correlated. A reduction in extracellular proteolysis would be expected to reduce invasion, whereas an increase in proteolysis might modulate the activity of inhibitory cytokines, which in turn could reduce endothelial cell proliferation and migration and inhibit angiogenesis. The spectrum of effects on different elements of the PA system observed in response to the agents assessed suggests that the role of modulations in extracellular proteolytic activity in anti-angiogenesis is likely to be varied and complex.

Recombinant truncated thrombospondin-1 monomer modulates endothelial cell plasminogen activator inhibitor 1 accumulation and proliferation in vitro.

The angiogenic and malignant phenotypes of hamster tumor cells are inversely correlated with the expression of an amino terminally truncated thrombospondin (TSP) subunit. In the present study, we have constructed a truncated TSP subunit from a human fibroblast cDNA library (rt-TSP1) and expressed it in Chinese hamster ovary (CHO) cells. Increased concentrations of plasminogen activator inhibitor-1 (PAI-1) were detected in endothelial cell conditioned medium following treatment with rt-TSP1. This rt-TSP1-induced increase in PAI-1 was neutralized by monoclonal antibodies to both TSP and TGF beta. rt-TSP1 also inhibits the proliferation of endothelial cells and this response is also neutralized by TSP and TGF beta antibodies. Serine and cysteine proteases inhibitors were used to determine if rt-TSP1 activated the latent TGF beta. However, these protease inhibitors did not neutralize the effect of rt-TSP1. The data indicate that the anti-angiogenic properties of TSP may be due to inhibition of the pericellular proteolysis required for endothelial cell migration and endothelial cell proliferation.

Visualization of histamine binding to nuclei.

The binding of histamine to cultured microvascular endothelial cells and glycol methacrylate embedded ovarian tissue sections has been localized using fluorescein-albumin-histamine conjugate. Histamine conjugate was bound to the plasma membranes and nuclei of luteal, endothelial, and ovarian stromal cells. An apparent increase in the binding of histamine to nuclei was observed in the presence of cimetidine but the plasma membrane staining was still evident. Unlike cimetidine, pyrilamine completely inhibited the binding of histamine to the plasma membrane. Instead, in the presence of pyrilamine, histamine bound exclusively to the nuclei of endothelial, germinal epithelial, granulosa, and stromal cells. However, the nuclei of terminally differentiated luteal cells and oocytes were not labeled. The functional significance of these nuclear histamine binding sites remains to be determined.

Isolation and characterization of microvascular endothelial cells from developing corpus luteum.

We have isolated and characterized microvascular endothelial cells from the developing rabbit corpus luteum. The isolated cells express Factor VIII-related antigen and angiotensin-converting enzyme, internalize acetylated low-density lipoprotein, and form capillary-like tubules in collagen gel cultures. Of the mitogens tested, only basic fibroblast growth factor stimulated the proliferation of these cells. Transforming growth factor-beta 1 and tumor necrosis factor-alpha strongly inhibited the proliferation of these endothelial cells. Platelet-derived growth factor, epidermal growth factor, insulin-like growth factor-1, histamine, prostaglandins, sex steroids, and interleukin-6 (interferon-beta 2) had no effect on the proliferation of these microvascular endothelial cells from the corpus luteum, whereas interleukin-1 alpha and 1 beta were mildly inhibitory. Endothelial cells are an essential component of corpus luteum physiology. Therefore, the availability of these cells will allow us to investigate the potential interactions between endothelial cells and luteal cells in vitro.

Specific inhibition of endothelial cell proliferation by thrombospondin.

Angiogenesis is a multi-step event involving endothelial cell migration, attachment, and proliferation. A thrombospondin (TSP)-like protein has recently been described as a naturally-occurring inhibitor of angiogenesis. We now report that human platelet TSP inhibits the in vitro proliferation of endothelial cells from the rabbit corpus luteum, bovine adrenal cortex and pulmonary artery, and human umbilical vein. The antiproliferative effect of TSP was neutralized by monoclonal antibodies against TSP. The growth arrest seen with TSP was specific for endothelial cells since TSP actually stimulated the growth of vascular smooth muscle cells and human foreskin fibroblasts. These results imply that the angiogenesis-inhibiting effect of TSP is mediated through an inhibition of endothelial cell proliferation. Elucidation of the mechanism of action of TSP on endothelial cell proliferation may lead to potential therapeutic approaches for the control of neovascular diseases.

Aprotinin antagonizes the binding of human chorionic gonadotropin to its receptor.

Aprotinin caused a dose-dependent inhibition of [125I]hCG binding to its receptor in plasma membranes prepared from luteinized rat ovaries. Displacement of [125I]hCG from its receptor by aprotinin was temperature dependent, with an ID50 of 300 microM at 4 C and an ID50 of 3700 microM at 22 C. Equilibrium binding data showed a decrease in the Ka for hCG in the presence of increasing concentrations of aprotinin; aprotinin had no effect on the number of binding sites. The rate of association of hCG with its receptor was markedly reduced in the presence of aprotinin, but aprotinin had little effect on the rate of dissociation. Control experiments established that aprotinin did not inhibit the binding of [125I]hCG to its receptor due to its lectin properties, an ionic effect or some irreversible impairment of the receptor or hormone. Aprotinin did not inhibit hCG binding when the receptor was solubilized out of ovarian membranes with Triton X-100. Aprotinin inhibited the hCG-mediated activation of adenylate cyclase in rat ovarian plasma membranes in a dose-dependent manner. Similarly, aprotinin antagonized the hCG-stimulated biosynthesis of progesterone by dispersed rat luteal cells. The maximal agonistic activity of hCG was attenuated in these two bioassays. The data are compatible with a hypothesis that aprotinin sterically hinders the entry of hCG into its receptor site by binding to a protease in the plasma membrane that is closely associated with the hCG receptor. This proposal is consistent with the emerging concept that integral membrane proteases are modulators of receptor function.

Inhibition of gonadotropin-stimulated adenylate cyclase by dansyl-arginyl-(4'-ethyl)piperidine amide (DAPA).

Protease inhibitors are known to suppress basal, fluoride-, and hormone-stimulated adenylate cyclase activities. The thrombin inhibitor, dansyl-arginyl-(4'-ethyl)piperidine amide (DAPA), also specifically inhibits the binding of gonadotropins to their receptors. Our studies were undertaken to find a concentration of DAPA that would specifically inhibit gonadotropin-stimulated adenylate cyclase without significantly altering basal, fluoride-, isoproterenol-, or prostaglandin E1-stimulated cyclase. Basal adenylate cyclase activity was not inhibited by DAPA in either human chorionic gonadotropin (hCG)- or follicle-stimulating hormone (FSH)-responsive rat ovarian plasma membranes. Human chorionic gonadotropin-stimulated cyclase was completely inhibited by DAPA at a concentration of 2.96 mM; the ID50 was 1.32 mM. Follicle-stimulating hormone-stimulated cyclase was completely inhibited by a DAPA concentration of 4.44 mM, and the ID50 was 1.75 mM. Dansyl-arginyl-(4'-ethyl)piperidine amide (2.96 mM) inhibited isoproterenol-, prostaglandin E1-, and fluoride-stimulated cyclase in hCG-responsive membranes by 11%, 28%, and 35%, respectively. Dansyl-arginyl-(4'-ethyl)piperidine amide (4.44 mM) inhibited fluoride- and prostaglandin-stimulated cyclase in FSH-responsive membranes by 10% and 11%, respectively. The data show that appropriate concentrations of DAPA can antagonize gonadotropin-stimulated adenylate cyclase while only minimally affecting fluoride- and other receptor-activated cyclase activities.

Relationship between progesterone receptor binding and progestin biological activity.

The binding affinities of a series of steroidal compounds for the hamster uterine progesterone receptor were determined using two sets of incubation conditions. These competitive binding conditions were designed to deduce the relative rates of ligand dissociation from the progesterone receptor. The progestin activity of these compounds was also determined in a bioassay employing the measurement of diamine oxidase in the traumatized hamster uterus. Steroids could be classified into two categories based on either an increase or decrease in relative binding affinity (RBA) with increasing time of competitive incubation. The mean (+/- SEM) progestin biopotency for the compounds having an increase in RBA was 120 +/- 18 (progesterone = 100), while the biopotency for compounds having a decrease in RBA was only 44 +/- 17. This difference was significant (P less than 0.01). Linear regression analyses revealed significant correlations between the RBAs and progestin biopotencies. Compounds showing a decrease in RBA with increasing time of incubation did not have antiprogestin activity. Kinetic studies of this type should be useful for selecting compounds with potent agonistic activity, but cannot unequivocally predict antihormonal activity.

Additional studies on pregnancy termination and inhibition of the monkey corpus luteum with 5-oxa-17-phenyl-18,19,20-trinor-PGF1 alpha methyl ester and structurally related prostaglandins.

Oral administration of 5-oxa-17-phenyl-18,19,20-trinor-PGF1 alpha methyl ester (PGF-analog) resulted in a consistent and dose-dependent inhibition of corpus luteum progesterone production in nonpregnant rhesus monkeys concomitantly treated with human chorionic gonadotropin. Similarly, vaginal suppositories containing PGF-analog also inhibited the monkey corpus luteum. Side effects by the oral route of administration were minimal, whereas side effects following vaginal treatment with PGF-analog were higher. Five prostaglandins with structural similarity to PGF-analog were studied for their ability to inhibit the monkey corpus luteum, but none showed an advantage over the parent molecule. PGF-analog did not synergize with 9-deoxo-16,16-dimethyl-9-methylene-PGE2 for the inhibition of the monkey corpus luteum, nor did it synergize with (15S)-15-methyl-PGF2 alpha methyl ester for the interruption of early pregnancy in the monkey. 9-Deoxo-9-methylene-5-oxa-17-phenyl-18,19-20-trinor-PGE1 methyl ester did not terminate early gestation in the monkey at doses of 8 or 24 mg.

Biologic activity of human chorionic gonadotropin following reversed-phase high-performance liquid chromatography.

Human chorionic gonadotropin (hCG) was analyzed by reversed-phase high-performance liquid chromatography (HPLC) using mobile phases previously described in the literature, as well as newly developed solvent systems. Fractions of hCG collected following reversed-phase HPLC were bioassayed by activation of adenylate cyclase to determine their biologic potencies. hCG retained only 10-60% of its biologic activity following reversed-phase HPLC, depending on the chromatographic conditions employed. A portion of the reduced biologic activity was attributed to dissociation of the alpha- and beta-subunits of hCG at the low pH of the mobile phases, since neutralization of the pH prior to lyophilization and bioassay increased the biologic potency of the chromatographed hormone. The remaining loss in biologic activity is presumably due to organic solvent denaturation.

Inhibition of folliculogenesis in the monkey following early follicular phase administration of an LRH agonist.

This study was undertaken to determine if early follicular phase administration of a synthetic luteinizing hormone releasing hormone (LRH) agonist would produce luteal phase defects in the monkey. [D-His(im-Bzl)6,Pro9]LRH N-ethylamide was administered to groups of rhesus monkeys on days 1-3 of the menstrual cycle. Two responses were observed: a) anovulatory menstrual cycles of less than 14 days duration, and b) ovulatory menstrual cycles characterized by unusually long follicular phases. All 4 monkeys with shortened menstrual cycles had prominent increases in serum gonadotrophin and oestradiol concentrations during treatment with the LRH agonist; early menses in these animals was attributed to uterine bleeding upon oestrogen withdrawal. Serum FSH concentrations declined, serum LH concentrations were unaltered, and only 2 of 8 monkeys had elevations in serum oestradiol during ovulatory menstrual cycles. The mean interval from cessation of treatment with the LRH agonist to the next preovulatory gonadotrophin surge was 21.5 +/- 3.2 days in ovulatory menstrual cycles. Corpus luteum function was normal following treatment with the LRH agonist in ovulatory cycles. The results indicate that both the long and short menstrual cycles observed following early follicular phase administration of the LRH agonist to monkeys can be attributed to a profound inhibition in follicle recruitment. [D-His(im-Bzl)6,Pro9]LRH N-ethylamide did not alter corpus luteum function in the monkeys.

Interruption of early pregnancy in the monkey with mestranol and 5-oxa-17-phenyl-18,19,20-trinor prostaglandin F1 alpha methyl ester.

Prior work has shown that 5-oxa-17-phenyl-18,19,20- trinor prostaglandin F1 alpha methyl ester (PGF-analog) inhibits luteal progesterone secretion, but does not shorten menstrual cycles in human chorionic gonadotropin (hCG)-treated, nonpregnant monkeys. This report demonstrates that a combination treatment of PGF-analog and mestranol not only reduces blood progesterone concentrations in the hCG-treated monkey, but also results in a significant shortening of menstrual cycles. The corpus luteum-inhibiting activity of PGF-analog in hCG-treated, nonpregnant monkeys was not enhanced by simultaneous administration of nonestrogenic steroids (norethisterone, oxymetholone, azastene , 17 alpha-allyl-3-methoxy-1,3,5(10)- estratrien-17 beta-ol). Most importantly, pregnancy was interrupted in 11 or 12 monkeys when PGF-analog and mestranol were administered on Day 28 of fertile menstrual cycles; this abortifacient activity of the prostaglandin-estrogen treatment was not prevented by concomitant administration of progesterone. Administration of PGF-analog and mestranol in the third trimester terminated pregnancy in only 1 of 3 monkeys. The data indicate that a combination treatment of PGF-analog and mestranol is highly effective for the termination of early pregnancy in the monkey. Although PGF-analog and mestranol clearly inhibit the monkey corpus luteum, it is unlikely that this activity is essential for the abortifacient activity of the prostaglandin-estrogen treatment.

Inhibition of the primate ovarian cycle by a porcine follicular fluid protein(s).

Monkeys received twice daily intramuscular injections of 3 mg of purified porcine follicular fluid protein(s) for the first 14.5 days of the menstrual cycle. Two of five treated monkeys had anovulatory menstrual cycles. Three monkeys had cycles characterized by long follicular phases, low follicular and luteal phase serum estradiol concentrations, and subnormal luteal progesterone production. Serum gonadotropin concentrations were not affected by the follicular fluid protein(s). The data demonstrate in the nonhuman primate that porcine follicular fluid contains a protein(s) that acts at the ovarian level to inhibit gonadotropin action.

Arrest of folliculogenesis and inhibition of ovulation in the monkey following weekly administration of progestins.

Studies were undertaken in the rhesus monkey to determine whether development of a dominant ovarian follicle could be repeatedly arrested by the administration of a progestin on day 7 of the menstrual cycle, and then every 7 days thereafter regardless of menstrual bleeding history. Progesterone (7.5 mg), norethisterone (1.5 mg), and 17 alpha-ethinyl-17 beta-methoxy-7 alpha-methyl-4-estren-3-one (1.0 or 1.5 mg) effectively inhibited ovulation when injected intramuscularly once a week for 8 weeks. Orally administered STS 557 (17 alpha-cyanomethyl-17 beta-hydroxy-4,9-estradien-3-one, 1.0 mg) also inhibited ovulation. Two structurally related steroids (17 beta-methoxy-4-estren-3-one, 1.0 mg; and 17 beta-methoxy-7 alpha-methyl-4-estren-3-one, 1.5 mg) did not inhibit ovulation when given intramuscularly at the indicated doses. Although weekly administration of certain progestins effectively arrested follicular development and inhibited ovulation in the primate, the treatment was accompanied by disturbances in menstrual bleeding patterns.