Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.

Small molecule antagonists of the bradykinin B1 receptor.

Screening Pharmacopeia's encoded combinatorial libraries has led to the identification of potent, selective, competitive antagonists at the bradykinin B1 receptor. Libraries were screened using a displacement assay of [3H]-des-Arglo-kallidin ([3H]-dAK) at IMR-90 cells expressing an endogenous human B1 receptor (Bmax = 20,000 receptors/cell, K(D) = 0.5+/-0.1 nM) or against membranes from 293E cells expressing a recombinant human B1 receptor (Bmax = 8,000 receptors/cell, K(D) = 0.5 +/- 0.3 nM). Compound PS020990, an optimized, representative member from the class of compounds, inhibits specific binding of 3H-dAK at IMR-90 cells with a KI of 6 +/- 1 nM. The compound inhibits dAK-induced phosphatidyl inositol turnover (K(Bapp) = 0.4 +/- 0.2 nM) and calcium mobilization (K(Bapp) = 17 +/- 2 nM) in IMR-90 cells. Compounds from the lead series are inactive at the B2 receptor and are > 1000-fold specific for B1 vs. a variety of other receptors, ion channels and enzymes. PS020990 and other related chemotypes therefore offer an excellent opportunity to explore further the role of B1 receptors in disease models and represent a potential therapeutic avenue.

A tacrolimus-related immunosuppressant with biochemical properties distinct from those of tacrolimus.

BACKGROUND: Tacrolimus (FK506) is an immunosuppressive drug 50-100 times more potent than cyclosporine (CsA), the current mainstay of organ transplant rejection therapy. Despite being chemically unrelated, CsA and tacrolimus exert their immunosuppressive effects through the inhibition of calcineurin (CaN), a critical signaling molecule during T-lymphocyte activation. Although numerous clinical studies have proven the therapeutic efficacy of drugs within this class, tacrolimus and CsA also have a strikingly similar profile of unwanted side effects. METHOD: Our objective has been to identify a less toxic immunosuppressant through the modification of ascomycin (FK520). Quantitative in vitro immunosuppression and toxicity assays have demonstrated (see the accompanying article, p. 18) that we achieved our goal with L-732,531 (indolyl-ascomycin; indolyl-ASC), a 32-O-(1-hydroxyethylindol-5-yl) ascomycin derivative with an improved therapeutic index relative to tacrolimus. RESULTS: We report that the attributes of indolyl-ASC may result from its distinctive biochemical properties. In contrast to tacrolimus, indolyl-ASC binds poorly to FK506 binding protein 12 (FKBP12), the major cytosolic receptor for tacrolimus and related compounds. However, the stability of the interaction between the FKBP12-indolyl-ASC complex and CaN is much greater than that of the FKBP12-tacrolimus complex. These distinguishing properties of indolyl-ASC result in the potent inhibition of CaN within T lymphocytes but may lower the accumulation of the drug at sites of toxicity. CONCLUSIONS: Indolyl-ASC may define those properties needed to increase the therapeutic efficacy of a macrolactam immunoregulant for treating both human autoimmune disease and organ transplant rejection.

A tacrolimus-related immunosuppressant with reduced toxicity.

BACKGROUND: Tacrolimus (FK506) has potent immunosuppressive properties reflecting its ability to block the transcription of lymphokine genes in activated T cells through formation of a complex with FK506 binding protein-12, which inhibits the phosphatase activity of calcineurin. The clinical usefulness of tacrolimus is limited, however, by severe adverse effects, including neurotoxicity and nephrotoxicity. Although this toxicity, like immunosuppression, appears mechanistically related to the calcineurin inhibitory action of the drug, a large chemistry effort has been devoted to search for tacrolimus analogs with reduced toxicity but preserved immunosuppressive activity that might have enhanced therapeutic utility. METHODS: Here, we report on the identification of such an analog, which was synthetically derived from ascomycin (ASC), the C21 ethyl analog of tacrolimus, by introducing an indole group at the C32 position. The profile of biological activity of indolyl-ASC was characterized in rodent models of immunosuppression and toxicity. RESULTS: Indolyl-ASC was found to exhibit an immunosuppressive potency equivalent to that of tacrolimus in T-cell activation in vitro and in murine transplant models, even though indolyl-ASC bound about 10 times less to intracellular FK506 binding protein-12 than tacrolimus or ASC. Further evaluation of indolyl-ASC revealed that it is threefold less potent than tacrolimus in inducing hypothermia, a response that may reflect neurotoxicity, and in causing gastrointestinal transit alterations in mice. Moreover, indolyl-ASC was at least twofold less nephrotoxic than tacrolimus upon 3-week oral treatment in rats. CONCLUSIONS: Altogether, these data indicate a modest but definite improvement in the therapeutic index for indolyl-ASC compared with tacrolimus in rodent models.

Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.

The voltage activated K+ channel (Kv1.3) has recently been identified as the molecule that sets the resting membrane potential of peripheral human T lymphoid cells. In vitro studies indicate that blockage of Kv1.3 inhibits T cell activation, suggesting that Kv1.3 may be a target for immunosuppression. However, despite the in vitro evidence, there has been no in vivo demonstration that blockade of Kv1.3 will attenuate an immune response. The difficulty is due to species differences, as the channel does not set the membrane potential in rodent peripheral T cells. In this study, we show that the channel is present on peripheral T cells of miniswine. Using the peptidyl Kv1.3 inhibitor, margatoxin, we demonstrate that Kv1.3 also regulates the resting membrane potential, and that blockade of Kv1.3 inhibits, in vivo, both a delayed-type hypersensitivity reaction and an Ab response to an allogeneic challenge. In addition, prolonged Kv1.3 blockade causes reduced thymic cellularity and inhibits the thymic development of T cell subsets. These results provide in vivo evidence that Kv1.3 is a novel target for immunomodulation.

New technologies for high-throughput screening.

To screen efficiently the millions of compounds that are synthesized using combinatorial and automated methods, dramatically improved assay technologies are currently needed. In 96-well microtiter plates, nonradioactive techniques (primarily fluorimetric) and cell-based functional methods have moved to the cutting edge, while clever assays that extract information from large bead-based combinatorial libraries have begun to show considerable promise. In the future, miniaturized assays that break out of the 96-well format will be enabled by innovative technologies for high-throughput screening.

A paradigm for drug discovery employing encoded combinatorial libraries.

Very large combinatorial libraries of small molecules on solid supports can now be synthesized and each library element can be identified after synthesis by using chemical tags. These tag-encoded libraries are potentially useful in drug discovery, and, to test this utility directly, we have targeted carbonic anhydrase (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1) as a model. Two libraries consisting of a total of 7870 members were synthesized, and structure-activity relationships based on the structures predicted by the tags were derived. Subsequently, an active representative of each library was resynthesized (2-[N-(4-sulfamoylbenzoyl)-4'-aminocyclohexanespiro]-4-oxo-7 -hydroxy- 2,3-dihydrobenzopyran and [N-(4-sulfamoylbenzoyl)-L-leucyl]piperidine-3-carboxylic acid) and these compounds were shown to have nanomolar dissociation constants (15 and 4 nM, respectively). In addition, a focused sublibrary of 217 sulfamoylbenzamides was synthesized and revealed a clear, testable structure-activity relationship describing isozyme-selective carbonic anhydrase inhibitors.

Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation.

The role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca(2+)-activated potassium channels (PK,Ca) and voltage-gated potassium channels (PK,V) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (IL-2 and interferon gamma) are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,Ca and PK,V, we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for PK,V. These studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca(2+)-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

A single administration of LFA-1 antibody confers prolonged allograft survival.

C57BL/6 (B6) thyroid gland transplanted to the left kidney capsule of an allogeneic (BALB/c) host was typically rejected in 14 days. A single administration of 500 micrograms of an antibody to the adhesion molecule, leucocyte function-associated antigen (LFA-1, CD11a), prevented all thyroid allograft rejection for at least 70 days. Fifty percent of the treated recipients retained intact allografts for 470 days. However, the same treatment with anti-CD11a could not protect a sensitized BALB/c mouse from rejecting a second B6 thyroid allograft. Production of donor-specific alloantibodies elicited by allograft rejection was also inhibited in this system. In this transplant model, the Ab therapy is more efficacious than that of FK506, administered daily for 14 days at 15 mg/kg. These results demonstrate the remarkable effect of an anti-LFA-1 antibody in promotion of allograft survival.

Recent advances in the mechanism of action of cyclosporine and FK506.

The immunosuppressants cyclosporine and FK506 (tacrolimus) are extremely potent inhibitors of T-lymphocyte activation. Recent studies have shown that these agents are actually prodrugs that become active only when bound to specific members of the cyclophilin or FK506 binding protein receptor gene families. The cyclosporine-cyclophilin or FK506-FK506 binding protein receptor complexes interact with a key component of the T-cell antigen receptor signal transduction pathway, the calcium-calmodulin-dependent phosphoprotein phosphatase calcineurin. The drug-receptor complexes inhibit the phosphatase activity of calcineurin and thereby prevent transcriptional activation of the interleukin-2 gene.

FK-506 and cyclosporin A: immunosuppressive mechanism of action and beyond.

Cyclosporin A and FK-506 are important therapeutic agents that have found widespread use in preventing graft rejection during tissue transplantation. Research efforts aimed at elucidating the molecular mechanism of action of these drugs have, in addition to defining their immunosuppressive functions, led to the identification of two new gene families whose products may function as components of several diverse signal transduction pathways. In the presence of the immunosuppressive drugs, some members of the receptor families interact with the Ca2+/calmodulin-dependent protein phosphatase 2B, also known as calcineurin. Inhibition of phosphatase activity may effect several downstream biochemical processes. In this way, cyclosporin A and FK-506 have proved to be useful probes of signaling events in both lymphocytic and other cell types.

Isolation of a human cDNA encoding a 25 kDa FK-506 and rapamycin binding protein.

Recently, the nearly complete peptide sequence of a 25 kDa rapamycin and FK-506 binding protein that had been isolated from calf thymus, brain, and spleen was reported (1). Based upon the amino acid sequence of this bovine protein, bFKBP25, we have isolated from a JURKAT cDNA library the cDNA encoding the human homolog, hFKBP25. Translation of the open reading frame contained within this cDNA clone yields a sequence that, in its C-terminal half, is 41% identical to the major human FK-506 binding protein, hFKBP12, and 43% identical to hFKBP13. The N-terminal half of hFKBP25 is unrelated to any known protein.

Analogs of cyclosporin A modified at the D-Ala8 position.

The conversion of [2-deutero-3-fluoro-D-Ala8]cyclosporin A (1) to a dehydroalanine analog [delta-Ala8]cyclosporin A (2) was achieved with lithium diisopropylamide in THF at low temperature. This dehydro compound is a useful intermediate for the preparation of position 8 analogs of cyclosporin A formed from it by the conjugate addition of thiol compounds. NMR conformational studies have provided evidence for the restoration of D-stereochemistry in the modified Ala8 residues. The preparation of several of these cyclosporin analogs and their bioactivities are described.

Cyclosporin A, FK-506, and rapamycin: pharmacologic probes of lymphocyte signal transduction.

CsA, FK-506, and rapamycin are microbial products with potent immunosuppressive properties that result primarily from a selective inhibition of T lymphocyte activation. Although chemically unrelated, CsA and FK-506 affect a similar subset of calcium-associated signaling events involved in the regulation of lymphokine gene expression, activation-driven T-cell death and exocytosis. Rapamycin has structural similarity with FK-506 but suppresses T-cell activation at a different level, mainly through inhibition of proliferation induced by growth-promoting lymphokines. CsA interacts with an abundant 17 kDa protein, termed cyclophilin, that possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity. Additional, minor cyclophilin-like molecules have been identified. Both FK-506 and rapamycin interact with FKBP, a 12 kDa protein, which, although unrelated to cyclophilin, is also abundant and ubiquitous, has a similar enzymatic activity, and is a member of a larger family of FKBPs. All three immunosuppressants inhibit the PPIase activity of their respective binding proteins. However, nonimmunosuppressive analogs of CsA and FK-506 are also inhibitory, indicating that inhibition of PPIase activity is not directly implicated in immunosuppression. Moreover, only a small fraction of the cellular pool of the major forms of cyclophilin or FKBP needs to be occupied by the drugs in order to achieve maximal immunosuppression. These observations suggest that complexes formed between the drugs and their major binding proteins may affect the function of other, unidentified, molecules or, alternatively, that minor binding proteins may play a role in the drugs' action. Further characterization of the biochemical processes altered by CsA, FK-506, and rapamycin should yield important insights into the signal transduction pathways involved in T-cell activation and should help in the development of novel immunosuppressive agents.

FKBP, the binding protein for the immunosuppressive drug, FK-506, is not an inhibitor of protein kinase C activity.

Recently, the amino acid sequence of a 12 Kd endogenous protein inhibitor of protein kinase C (PKC-I 2) has been shown to be identical to that of the 12 KDa receptor for the immunosuppressive drug, FK-506. In view of this observation we examined the effects of recombinant and native human FKBP on protein kinase C (PKC) activity. FKBP, at molar concentrations up to 1900-fold over that of PKC, failed to inhibit PKC phosphorylation of histone H1 and failed to block the auto-phosphorylation of PKC. Interestingly, FKBP is phosphorylated by PKC in these reactions. The phosphorylation of FKBP by PKC appears to be specific since the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate the binding protein. Our results fail to support a role for FKBP as an inhibitor of protein kinase C.

FK-506 and cyclosporin A: selective inhibition of calcium ionophore-induced polymorphonuclear leukocyte degranulation.

This paper investigates the abilities of FK-506 and cyclosporin A (CsA) to inhibit human polymorphonuclear leukocyte (PMNL) degranulation. PMNLs, purified from human blood, were stimulated in vitro with A23187, ionomycin, the complement derived peptide C5a, formylmethionylleucinylphenylalanine (FMLP) or phorbol myristate acetate (PMA). Degranulation was assessed by measuring the release of either lactoferrin or N-acetyl-beta-D-glucosaminidase (NAG). Both FK-506 and CsA produced a concentration-related inhibition of degranulation induced by either A23187 or ionomycin but did not affect C5a-, FMLP- or PMA-induced degranulation. The IC50 values for inhibition of degranulation (approximately 0.7 nM for FK-506 and 33.7 nM for CsA) are very close to the published values for inhibition of human T-cell proliferation. Removal of calcium from the incubation medium with ethyleneglycolbis(aminoethylether)tetra-acetate (EGTA) totally inhibited calcium ionophore-induced degranulation but had no effect against C5a-, FMLP- or PMA-induced degranulation. Preincubation of PMNLs with actinomycin D or cycloheximide did not affect either A23187- or PMA-induced degranulation. Non-immunosuppressive analogs of CsA were ineffective at inhibiting degranulation. Rapamycin, a macrolide structurally related to FK-506, did not inhibit degranulation but it did antagonize the inhibition produced by FK-506. Given the similar profiles of activity of FK-506 and CsA in neutrophils and T cells, we conclude that similar activation or signal transduction pathways may be present in both T cells and neutrophils. Because A23187-induced PMNL degranulation was not sensitive to either actinomycin D or cycloheximide, it is apparent that the signal transduction pathways ultimately control different cellular functions.