Optimization of Methionyl tRNA-Synthetase Inhibitors for Treatment of Cryptosporidium Infection.

Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most needy populations of malnourished children and HIV-infected persons. We describe here the discovery and early optimization of a class of imidazopyridine-containing compounds with potential for treating Cryptosporidium infections. The compounds target the Cryptosporidium methionyl-tRNA synthetase (MetRS), an enzyme that is essential for protein synthesis. The most potent compounds inhibited the enzyme with Ki values in the low picomolar range. Cryptosporidium cells in culture were potently inhibited with 50% effective concentrations as low as 7 nM and >1,000-fold selectivity over mammalian cells. A parasite persistence assay indicates that the compounds act by a parasiticidal mechanism. Several compounds were demonstrated to control infection in two murine models of cryptosporidiosis without evidence of toxicity. Pharmacological and physicochemical characteristics of compounds were investigated to determine properties that were associated with higher efficacy. The results indicate that MetRS inhibitors are excellent candidates for development for anticryptosporidiosis therapy.

Transmembrane signaling in human polymorphonuclear neutrophils: 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid modulates receptor agonist-triggered cell activation.

15(S)-Hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) exerted a time- and concentration-dependent inhibition of superoxide anion (O2-) production and exocytosis of both azurophil and specific granule constituents from human polymorphonuclear neutrophils (PMN) stimulated with the receptor-specific agonists, N-formylmethionylleucylphenylalanine (FMLP), platelet-activating factor, and leukotriene B4, but not that elicited by phorbol 12-myristate 13-acetate. 15-HETE did not alter the binding of FMLP to its specific receptors on PMN but, rather, appeared to interfere with a subsequent process in signal transduction. Receptor-coupled production of inositol 1,4,5-trisphosphate (InsP3) and increases in cytosolic free calcium elicited with FMLP, platelet-activating factor, and leukotriene B4 were suppressed by 15-HETE. 15-HETE did not, however, inhibit the mobilization of 45Ca from intracellular stores elicited by the addition of InsP3 to permeabilized PMN. 15-HETE suppressed O2- production and increases in intracellular [Ca2+] induced when cell-surface receptors were bypassed and the PMN were activated directly by the guanine nucleotide-binding protein (G protein) activators aluminum fluoride (AlF4-) and mastoparan. 15-HETE, however, did not perturb all G protein functions because cAMP production in FMLP-activated PMN was essentially unaffected by 15-HETE. These data support the proposition that 15-HETE modulates receptor-triggered activation of PMN either by uncoupling G protein stimulation of phospholipase C or by directly inhibiting phospholipase C, thus inhibiting the InsP3-dependent rise in intracellular [Ca2+] that is prerequisite for PMN responsiveness to receptor agonists.

Induction of neutral proteinase and prostanoid production in bovine nasal chondrocytes by interleukin-1 and tumor necrosis factor alpha: modulation of these cellular responses by interleukin-6 and platelet-derived growth factor.

We have previously reported that recombinant human interleukin-1 (IL-1) stimulates matrix erosion in bovine nasal cartilage explants (R. J. Smith et al., Inflammation 13, 367-382, 1989). This action of IL-1 is believed to be caused by matrix-degrading neutral proteinases produced by activated chrondrocytes. Accordingly, we investigated the effects of recombinant human interleukin-1 alpha (IL-1 alpha), recombinant human interleukin-1 beta (IL-1 beta), and recombinant human tumor necrosis factor alpha (TNF alpha) on bovine nasal chondrocyte (BNC) responsiveness. IL-1 alpha and IL-1 beta stimulated a time (0-72 hr) and concentration-dependent (0.01-10 ng/ml) production of collagenase, gelatinase, caseinase, and prostaglandin E2 (PGE2) in BNC monolayer cultures. Neutral proteinase and PGE2 production by BNC was also induced by TNF alpha (0.2-200 ng/ml) in a time-dependent (0-72 hr) manner. Recombinant human interleukin-6 (IL-6) caused a concentration-dependent (6-200 ng/ml) potentiation of IL-1-stimulated neutral proteinase and PGE2 production by BNC. However, recombinant human platelet-derived growth factor homodimer BB suppressed BNC responsiveness to IL-1. A recombinant human IL-1 receptor antagonist protein inhibited BNC activation by IL-1 but not TNF alpha.

Postreceptor events associated with human neutrophil activation by interleukin-8.

Recombinant human monocyte-derived interleukin-8 (IL-8M), recombinant human endothelium-derived IL-8 (IL-8E), and a recombinant human truncated form of IL-8 (IL-8T) stimulated a time-dependent (t 1/2 approximately 2-3 s) and concentration-dependent (0.1-100 nM) release of azurophil (myeloperoxidase) and specific (vitamin B12 binding protein, gelatinase) granule constituents from cytochalasin B-treated human neutrophils (HNs) wherein IL-8T = IL-8M greater than IL-8E. An increase in the cytosolic free calcium concentration ([Ca2+]i) was greater in IL-8T- than in IL-8M- or IL-8E-activated HNs, and IL-8T was more potent than either IL-8M or IL-8E in sequentially desensitizing the HNs to the effects of the other IL-8 forms. IL-8 induced a time- and concentration-dependent (0.1-100 nM) increase in the production of inositol 1,4,5-trisphosphate (IP3) in HNs. U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione), a potent inhibitor of phospholipase C-catalyzed events in HNs, suppressed IL-8-triggered IP3 production, increased [Ca2+]i and granule exocytosis in HNs. The membrane-associated activity of the alpha and beta subtypes of protein kinase C was significantly enhanced in IL-8-activated cells.

Platelet-derived growth factor potentiates cellular responses of articular chondrocytes to interleukin-1.

Recombinant human interleukin-1 alpha (IL-1 alpha) induced a time-dependent (0-72 hours) and concentration-dependent (0.01-10 ng/ml) production of metalloproteinases (collagenase, gelatinase, stromelysin) and prostaglandin E2 (PGE2) in rabbit articular chondrocytes (RAC). Exposure of RAC to recombinant human platelet-derived growth factor homodimer BB (PDGF-BB; 2-200 ng/ml) in the presence of stimulatory and substimulatory concentrations of IL-1 alpha resulted in a marked augmentation of metalloproteinase and PGE2 production. PDGF-BB exerted no agonist effects on RAC responsiveness. PDGF-BB up-regulated the number of IL-1 receptors per chondrocyte but had no effect on receptor affinity. Cycloheximide and actinomycin D caused a concentration-dependent suppression of the PDGF-BB-mediated potentiation of radiolabeled IL-1 alpha binding to RAC and cell responsiveness to IL-1 alpha. Similarly, IL-1 increased the number of PDGF receptors on RAC without changing receptor affinity. These data are discussed within the context of cytokine-growth factor interactions as components of the pathogenesis of arthritic diseases.

Biologic effects of an interleukin-1 receptor antagonist protein on interleukin-1-stimulated cartilage erosion and chondrocyte responsiveness.

Recombinant human interleukin-1 alpha (IL-1 alpha) and recombinant human IL-1 beta stimulate matrix proteoglycan degradation and inhibit glycosaminoglycan synthesis in bovine nasal cartilage explants. A 17-kd human recombinant IL-1 receptor antagonist protein (IRAP) caused a concentration-dependent (0.2-200 ng/ml) suppression of the effects of IL-1 alpha and IL-1 beta in cartilage organ cultures. IRAP inhibited the binding of radiolabeled IL-1 alpha to rabbit articular chondrocytes. Matrix metalloproteinase (collagenase, gelatinase, and stromelysin) and prostanoid production by IL-1-activated rabbit articular chondrocytes was also suppressed by IRAP. These results could have potential significance in the development of a new antiarthritis therapy based on an IRAP.

Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness.

1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]- 1H-pyrrole-2,5-dione (U-73122), an inhibitor of phospholipase C (PLC)-dependent processes in human platelets, was found to be a potent inhibitor of human polymorphonuclear neutrophil (PMN) activation by structurally unrelated receptor-specific agonists. U-73122 caused a time- and concentration-dependent (0.1-1 microM) inhibition of myeloperoxidase and vitamin B12-binding protein release from PMNs exposed to N-formyl-methionyl-leucyl-phenylalanine, recombinant human C5a, leukotriene B4 and platelet-activating factor. Activation of the respiratory burst, as measured by superoxide anion production, in PMNs stimulated with these agonists was also suppressed by U-73122. These data suggested that U-73122 inhibited a component of signal transduction that was common to the mechanisms of action of these stimuli. Production of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol and the rise in the cytosolic free calcium concentration, which are early postreceptor events in PMN activation, were all suppressed in U-73122-treated PMNs stimulated with the agonists. These signal transduction events require activation of PLC. Receptor-coupled activation of PLC in membranes isolated from PMNs was potently inhibited by U-73122. U-73122, however, had no direct effect on PMN protein kinase C activity. 1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl] -2,5- pyrrolidine-dione (U-73343), a close analog of U-73122 that does not suppress PLC activity, did not inhibit receptor-specific agonist-induced PMN responsiveness. U-73122, therefore, is a novel reagent that is useful in investigating PLC function in receptor-mediated PMN activation.

Recombinant human granulocyte-macrophage colony-stimulating factor induces granule exocytosis from human polymorphonuclear neutrophils.

Exposure of human polymorphonuclear neutrophils (PMNs) to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in a time- and concentration-dependent (3-100 units/ml) extracellular release of a specific (vitamin B12-binding protein) but not azurophil granule constituent (myeloperoxidase). Negligible granule exocytosis occurred if PMNs were not preincubated with cytochalasin B prior to contact with GM-CSF. The extent of degranulation elicited with GM-CSF was reduced but not abolished when PMNs were incubated with EGTA in calcium-free medium. GM-CSF did not stimulate a rise in the cytosolic-free calcium concentration ([Ca2+]i), and it had no effect on PMN protein kinase C (PKC) activity.

Recombinant human interleukin-1 alpha and recombinant human interleukin-1 beta stimulate cartilage matrix degradation and inhibit glycosaminoglycan synthesis.

Recombinant human interleukin-1 alpha (rhIL-1 alpha) and recombinant human interleukin 1 beta (rhIL-1 beta) stimulated the time- and concentration-dependent release of glycosaminoglycan (GAG) from bovine nasal cartilage explants. Maximum GAG release occurred during six to eight days of cartilage exposure to either species of rhIL-1; and rhIL-1 alpha was consistently more potent than rhIL-1 beta. In addition to inducing cartilage matrix resorption, rhIL-1 alpha and rhIL-1 beta also inhibited the incorporation of [35SO4]sulfate into cartilage, which is a reflection of the suppression of GAG synthesis. IL-1 had no capacity to stimulate GAG relase from or inhibit GAG synthesis by dead cartilage. Cycloheximide, an inhibitor of protein synthesis, and 1, 10-phenanthroline, a metalloproteinase inhibitor, suppressed rhIL-1-stimulated cartilage matrix resorption. Polyclonal antisera to rhIL-1 alpha and rhIL-1 beta specifically neutralized the respective cytokines.

Function and stimulus-specific effects of phorbol 12-myristate 13-acetate on human polymorphonuclear neutrophils: autoregulatory role for protein kinase C in signal transduction.

Exposure of polymorphonuclear neutrophils (PMNs) to phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent (1-10 ng/ml) inhibition of granule exocytosis induced with the receptor-specific ligands, N-formyl-methionyl-leucyl-phenylalanine (FMLP), pepstatin A, 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), and acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC). PMA exerted a marginal inhibitory effect on calcium ionophore A23187-induced PMN degranulation, and the PMA analog, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), was inactive. However, PMA potentiated AGEPC, pepstatin A, FMLP, LTB4, and A23187-stimulated superoxide anion (O2-) production. The mobilization of intracellular sequestered calcium (Ca2+) by the receptor-specific ligands, as reflected by a rise in the cytosolic-free Ca2+ concentration ([Ca2+]i) in PMNs loaded with the Ca2+-sensitive dye, Fura-2, was suppressed by PMA. A protein kinase C (PKC) inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) reversed the PMA-mediated inhibition of PMN degranulation and intracellular CA2+ mobilization. However, another, but less potent PKC inhibitor, N-(2-guanidino-ethyl)-5-isoquinolinesulfonamide (HA1004), had no effect on the inhibition of PMN activation by PMA.

Diacylglycerols modulate human polymorphonuclear neutrophil responsiveness: effects on intracellular calcium mobilization, granule exocytosis, and superoxide anion production.

The synthetic diacylglycerols (DG), sn-1,2-dihexanoylglycerol (diC6), sn-1,2-dioctanoylglycerol (diC8), and 1-oleoyl-2-acetylglycerol (OAG) stimulated the release of granule constituents from and superoxide anion (O2-) generation by human polymorphonuclear neutrophils (PMN). The DGs did not induce a rise in the cytosolic-free calcium concentration ([Ca2+]i), as monitored by the fluorescence of PMNs loaded with the fluorescent CA2+ indicator, Fura-2. DiC6, diC8, and OAG inhibited PMN degranulation elicited with the receptor-specific ligands, N-formyl-methionyl-leucyl-phenylalanine (FMLP), acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), and 5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans eicosatetraenoic acid (LTB4) and the calcium ionophore, A23187. In contrast to their inhibitory effects on granule exocytosis, diC6, diC8 and OAG enhanced FMLP-, AGEPC-, LTB4 and A23187-stimulated O2- production. Activation of the respiratory burst with phorbol 12-myristate 13-acetate (PMA) was unaffected by the DGs. DiC8 inhibited the rise in [Ca2+]i elicited with FMLP, LTB4, and AGEPC; this effect, as well as the DG-mediated suppression of degranulation, could be reversed with the protein kinase C (PKC) inhibitor, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride (H-7). These data indicate that in addition to possessing the intrinsic capacity to activate PMNs, DG may function in a PKC-mediated autoregulatory mode to influence PMN activation in a response-specific manner by affecting certain components of receptor-coupled and receptor-independent signal transduction systems in a stimulus-specific manner.

Effects of a protein kinase C inhibitor, K-252a, on human polymorphonuclear neutrophil responsiveness.

We investigated the capacity of K-252a, an inhibitor of rat brain protein kinase C (PKC), to influence polymorphonuclear neutrophil (PMN) PKC and PMN activation with chemically and structurally dissimilar agonists. K-252a inhibited PMN PKC (IC50 = 0.58 microM), and caused a concentration-dependent (0.1-10 microM) inhibition of degranulation elicited with the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), the lipid agonists, 5(S), 12(R)-dihydroxy-5,14-cis-8,10-trans eicosatetraenoic acid (LTB4) and acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), and phorbol 12-myristate 13-acetate. Superoxide anion (O2-) production by PMNs exposed to these stimuli as well as sn-1,2-dioctanoylglycerol (diC8) was also suppressed by K-252a. PMN PKC activity was inhibited with concentrations of K-252a which suppressed PMN responsiveness. Therefore, K-252a appears to be a useful probe for examining the role of PKC in the underlying pathway(s) of PMN activation.

Human neutrophil activation with interleukin-1. A role for intracellular calcium and arachidonic acid lipoxygenation.

Human monocyte-derived interleukin-1 (IL-1) stimulated the selective extracellular release of cytoplasmic granule-associated elastase from human neutrophils. Although extracellular calcium (Ca2+) enhances but is not required for the expression of granule exocytosis, IL-1 did induce the mobilization of previously sequestered intracellular Ca2+ as measured with the highly selective fluorescent Ca2+ indicator, Quin 2. Further, IL-1 stimulated the mobilization of cell membrane-associated Ca2+ as monitored by a decrease in fluorescence of chlorotetracycline (CTC)-loaded neutrophils. W-7, a calmodulin antagonist, and TMB-8[8(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride], an intracellular Ca2+ antagonist, inhibited the Quin 2 fluorescent response by neutrophils to IL-1. TPCK (N-alpha-p-tosyl-L-lysine chloromethylketone), a serine protease inhibitor, suppressed IL-1-induced Quin 2 and CTC fluorescence. Exposure of neutrophils to IL-1 resulted in a concentration-dependent production of the 5-lipoxygenase product, LTB4 [5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid] which was enhanced in the presence of arachidonic acid (AA). LTB4 production by IL-1-activated neutrophils was suppressed by the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and piriprost potassium [6,9,deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin l1], and a cyclooxygenase/lipoxygenase inhibitor, 5,8,11,14-eicosatetraynoic acid (ETYA), whereas a cyclooxygenase inhibitor, flurbiprofen, was inactive. These data indicate that cytosolic free Ca2+ ([Ca2+]i) and a metabolite(s) of AA lipoxygenation mediate granule exocytosis elicited with IL-1.

Arachidonic acid and 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid modulate human polymorphonuclear neutrophil activation by monocyte derived neutrophil activating factor.

Exposure of human polymorphonuclear neutrophils (PMN) to human monocyte derived neutrophil activating factor(s) (NAF) resulted in a concentration-dependent extracellular release of granule constituents. NAF also induced the generation of 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid [Leukotriene B4 (LTB4)] by PMNs which was enhanced in the presence of exogenous arachidonic acid (AA). In contrast to its enhancing effect on LTB4 production, AA inhibited NAF-stimulated PMN degranulation. 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE), a product of the 15-lipoxy-genation of AA in PMNS, caused a concentration-dependent suppression of degranulation and LTB4 generation by PMNs in contact with NAF. 15-HETE also inhibited the rise in cytosolic-free calcium [( Ca2+]i) observed in NAF activated PMNs. These data suggest that AA and a 15-lipoxygenase product modulate the NAF-associated activation pathway in human PMNs.

Human polymorphonuclear neutrophil activation with arachidonic acid.

The capacity of arachidonic acid (AA) to stimulate granule exocytosis from human polymorphonuclear neutrophils (PMNs) was investigated. AA induced the selected extracellular release of azurophil (myeloperoxidase, lysozyme) and specific (lysozyme, vitamin B12 binding protein) granule constituents from human PMNs in a time- and concentration-dependent manner. Cytochalasin B (CB) enhanced but was not required for PMN activation with AA. Although extracellular calcium had no effect on granule exocytosis, AA did stimulate the mobilization of intracellular sequestered Ca2+ which resulted in an increase in cytosolic-free Ca2+ ([Ca2+]i) as reflected by increased fluorescence of Fura-2-treated cells. AA stimulated Ca2+/phospholipid-dependent protein kinase C (PK-C) activity in PMNs. 4,4'-Diisothiocyano-2,2'-disulphonic acid stilbene (DIDS), an anion channel blocker, caused a concentration-dependent inhibition of granule enzyme release. Activation of PMNs with AA was unaffected by the lipoxygenase/cycle-oxygenase inhibitors, 5,8,11, 14-eicosatetraynoic acid (ETYA) and benoxaprofen, a lipoxygenase inhibitor, 6, 9, deepoxy-6,9-(phenylimino) delta 6,8-prostaglandin 1(1) (piriprost potassium) or a pure cyclo-oxygenase inhibitor, flurbiprofen. These data define the properties of AA as a secretory stimulus for human PMNs.

Requirement for cell-bound proteases in the mechanism of human neutrophil activation with various stimuli.

A cell membrane-associated protease/esterase has been implicated in the mechanism of "stimulus-secretion coupling" described for human neutrophil degranulation. In this regard, a broad spectrum of protease inhibitors were evaluated for their effects on granule enzyme release from neutrophils exposed to soluble, surface-active stimuli. The serine protease inhibitors, L-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK) and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK) and a thiol protease inhibitor, p-hydroxymercuribenzoate (PHMB), caused a concentration-related suppression of neutrophil degranulation elicited with 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), ionophore A23187, phorbol myristate acetate (PMA), and 5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4). However, other inhibitors, such as aprotinin and p-hydroxyphenylpyruvic acid, were inactive. The maximum inhibitory effect with TPCK, TLCK, and PHMB was observed when neutrophils were exposed to these inhibitors prior to contact with the respective stimuli. In addition, the magnitude of inhibition increased in proportion to the preincubation time of protease inhibitor with stimulus. The results of these studies implicate proteases in the sequence of events underlying activation of the human neutrophil secretory process in response to structurally and chemically dissimilar stimuli.