Basiliximab or antithymocyte globulin for induction therapy in kidney transplantation: a meta-analysis.

OBJECTIVE: To compare efficacy and safety of basiliximab versus antithymocyte globulin (ATG) for induction therapy in kidney transplantation. METHODS: A literature search of the MEDLINE, EMBASE, CBMdisc, and Cochrane databases was used to identify randomized controlled trials that compared basiliximab and ATG for induction therapy in kidney transplantation. Inclusion criteria comprised: prospective randomized controlled clinical trials, follow-up time >or=12 months, randomized comparisons of ATG versus basiliximab as induction therapy in kidney transplantation. Meta-analytical techniques were applied to identify differences in outcomes between the two agents. RESULTS: A total of six studies involving 853 patients were identified. No differences between ATG and basiliximab were seen in terms of biopsy-proven rejection (relative risk [RR] 1.15, 95% confidence interval [CI] 0.88-1.52, P = .31), delayed graft function (RR 1.02, 95% CI 0.69-1.51, P = .93), graft loss (RR 1.15, 95% CI 0.73-1.80, P = .55), and patient death (RR 1.22, 95% CI 0.65-2.30, P = .54). But basiliximab had a lower incidence of infection (RR 0.87, 95% CI 0.78-0.97, P = .02) and neoplasm (RR 0.29, 95% CI 0.09-0.97, P = .04). CONCLUSIONS: Basiliximab is as effective as ATG for induction therapy in kidney transplantation, whereas basiliximab has a lower incidence of infection. Basiliximab may be a safer and preferable option for induction therapy in kidney transplantation.

Discovery of macrocyclic hydroxamic acids containing biphenylmethyl derivatives at P1', a series of selective TNF-alpha converting enzyme inhibitors with potent cellular activity in the inhibition of TNF-alpha release.

SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.

Design, synthesis, and structure-activity relationships of macrocyclic hydroxamic acids that inhibit tumor necrosis factor alpha release in vitro and in vivo.

To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of

Aggrecanase. A target for the design of inhibitors of cartilage degradation.

In arthritic diseases there is a gradual erosion of cartilage that leads to a loss of joint function. Aggrecan, which provides cartilage with its properties of compressibility and elasticity, is the first matrix component to undergo measurable loss in arthritis. This loss of aggrecan appears to be due to an increased rate of degradation, that can be attributed to proteolytic cleavage of the core protein within the interglobular domain (IGD). Two major sites of cleavage have been identified within the IGD. One, between the amino acids Asn341-Phe342, where the matrix metalloproteinases (MMPs) have been shown to clip; and the other, between Glu373-Ala374, which is attributed to a novel protease, "aggrecanase." We have generated aggrecanase in conditioned media from IL-1-stimulated bovine nasal cartilage and have used an enzymatic assay to evaluate this proteinase activity. In these studies we follow the generation of aggrecanase and MMPs in response to IL-1 in this system and examine the contribution of these enzymes in aggrecan degredation. Our data suggest that aggrecanase is a key enzyme in cartilage aggrecan degradation that represents a novel target for cartilage protection therapy in arthritis.

P1, P2'-linked macrocyclic amine derivatives as matrix metalloproteinase inhibitors.

A novel series of 13- and 14-membered macrocyclic amines was developed by linking the P1 and P2' groups. The synthesis entails stereoselective Frater alkylation to install the anti-succinate configuration and macrocyclic amination via nucleophilic displacement. This strategy resulted in a new class of conformationally constrained inhibitors that are potent and selective for MMP-8 and 9 over MMP-1 and 3.

Macrocyclic hydroxamate inhibitors of matrix metalloproteinases and TNF-alpha production.

Several macrocyclic, hydroxamate derivatives were synthesized and evaluated as inhibitors of matrix metalloproteinases (MMPs) and tumour necrosis factor-alpha (TNF-alpha) production. These macrocycles are anti-succinate based inhibitors linked from P1 to P2'. A variety of functionality was installed at the P1-P2' linkage, which gave inhibitors that displayed excellent MMP inhibition and good TNF-alpha suppression.

Synthesis and antiplatelet effects of an isoxazole series of glycoprotein IIb/IIIa antagonists.

Despite the excellent in vitro potency of a series of benzamide glycoprotein IIb/IIIa antagonists, which have been reported previously, poor in vivo potency in the inhibition of platelet aggregation was observed when the most potent inhibitor XU057 was dosed intravenously to dogs. In this communication, we report that replacement of the benzamide in XU057 with an isoxazolecarboxamide resulted in significant improvement in in vivo potency. More importantly, the analogue XU065 showed an excellent oral antiplatelet effect in dogs.

Discovery of an orally active series of isoxazoline glycoprotein IIb/IIIa antagonists.

Using isoxazoline XR299 (1a) as a starting point for the design of highly potent, long-duration GPIIb/IIIa antagonists, the effect of placing lipophilic substituents at positions alpha and beta to the carboxylate moiety was evaluated. Of the compounds studied, it was found that the n-butyl carbamate 24u exhibited superior potency and duration of ex vivo antiplatelet effects in dogs. Replacement of the benzamidin-4-yl moiety with alternative basic groups, elimination of the isoxazoline stereocenter, and reversal of the orientation of the isoxazoline ring resulted in lowered potency and/or duration of action.

Design, synthesis, and in vitro activities of benzamide-core glycoprotein IIb/IIIa antagonists: 2,3-diaminopropionic acid derivatives as surrogates of aspartic acid.

In an effort to discover novel nonpeptide glycoprotein IIb/IIIa (GPIIb/IIIa, alpha IIb/beta 3) inhibitors, we investigated RGD mimetics featuring a 3-substituted benzoic acid as the core, benzamidine as the basic moiety, and a series of beta- and alpha-substituted beta-alanine derivatives as aspartic acid surrogates. It was found that the use of beta-methyl beta-alanine slightly improved the anti-aggregant potency in human platelet-rich plasma over the unsubstituted beta-alanine compound, while beta-substitution with a trifluoromethyl group resulted in considerable loss in activity. Significant enhancement (up to 100-fold) in potency was obtained when the beta-alanine was replaced with N2-substituted 1-2,3-diaminopropionic acid derivatives. Among the three types of alpha-substituents (carbamate, amide, and sulfonamide) investigated, no apparent preference was observed with respect to in vitro potency. However, alkyl groups were more favorable than arylalkyl groups (Cbz) in the carbamate analogues. We also investigated piperidine, piperazine, and N-formamidinopiperidine as replacements for the benzamidine moiety. The former two replacements led to a drop in potency while the latter replacement resulted in maintenance of activity as compared with the corresponding benzamidine analogue.

Novel nonpeptide antiplatelet glycoprotein IIb/IIIa receptor antagonist, DMP754: receptor binding affinity and specificity.

OBJECTIVE: To define the antiplatelet efficacy and specificity of the glycoprotein IIb/IIIa complex (GPIIb/IIIa) antagonist prodrug DMP754 and its free acid form, XV459. METHODS AND MATERIALS: DMP754 has an IC50 > 1 mumol/l, and, upon its conversion with esterases to its free acid form, demonstrated high potency (IC50 20-45 nmol/l) in inhibiting human platelet aggregation induced by 10 mumol/l adenosine diphosphate, 20 micrograms/ml collagen, 1 mmol/l epinephrine, 10 mumol/l platelet activating factor or 0.5 IU/ml thrombin. The in-vitro rate of hydrolysis of DMP754 or XV459 is much faster with human or canine liver esterases (t 1/2 = 2.4-23 min) than with plasma esterases (t 1/2 = 5.5-7.6 h). Platelet GpIIb/IIIa integrin binding affinity and specificity for XV459 were determined using cell binding/adhesion assays. RESULTS: The range of IC50 values of XV459 in inhibiting platelet aggregation in platelet-rich plasma obtained from 12 subjects was 0.035-0.069 mumol/l with a mean IC50 of 0.050 +/- 0.003 mumol/l. Additionally, XV459 inhibited platelets obtained from mongrel dogs, baboons, sheep, guinea pigs, and mice with IC50 in the range 0.024-0.06 mumol/l, and IC50 in the range 0.16-5.8 mumol/l in pigs, rabbits, and rats. XV459 inhibited [125I]-fibrinogen binding to activated human platelets with an IC50 of 0.011 +/- 0.003 mumol/l. XV459 demonstrated a high degree of selectivity in specifically inhibiting fibrinogen binding to the platelet integrin, GPIIb/IIIa (IC50 = 0.00025 +/- 0.00005 mumol/l) compared with inhibiting other integrins (alpha v beta 3, IC50 > 10 mumol/l; or alpha v beta 5, alpha 5 beta 1, or alpha 4 beta 1, for which the IC50 exceeded 100 mumol/l). CONCLUSION: DMP754 is a potent antiplatelet agent in inhibiting platelet aggregation, and has a high specificity and affinity for human platelet GPIIb/IIIa receptors.

Probing the functional conformation of the tridecapeptide mating pheromone of Saccharomyces cerevisiae through study of disulfide-constrained analogs.

Analogs of the Saccharomyces cerevisiae alpha-mating factor, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, where Lys7 and Gln10 were replaced with Cys, Cys(CH3), or Ser, were synthesized using solid-phase procedures on a phenylacetamidomethyl resin. Cyclo7,10[Cys7,X9,Cys10,Nle12]alpha-factor , where X=D-Val, D-Ala, L-Ala and Gly, were prepared by on-resin cyclization using thallic trifluoroacetate in yields of 20-30%. Linear sulfhydryl-containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys7 and Gln10 with a cysteine residue resulted in an over 100-fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5-10-fold more active than their sulfhydryl-containing homologs, and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] alpha-factor was 50-fold more potent than linear analogs containing Ser or Cys(CH3) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the alpha-factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5-aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form beta-turns spanning residues 7-10 than their less active linear counterparts. The results provide strong evidence that this beta-turn is important for optimal signal transduction by alpha-factor.

Molecular determinants of bioactivity of the Saccharomyces cerevisiae lipopeptide mating pheromone.

The a-factor of Saccharomyces cerevisiae (YIIKGVF-WDPAC(Farnesyl)-OCH3) is a peptide pheromone in which post-translational modification with a farnesyl isoprenoid and carboxyl methyl group is required for export and bioactivity. Truncated and carboxyl-terminal modified analogs of the a-factor were synthesized in order to determine the effect of such modifications on bioactivity. Bioactivity studies on carboxyl-terminal analogs in which the chirality, the cysteine thioether, and the carboxyl ester were varied in an attempt to study the influence of topology on a-factor activity indicate that the hydrophobicity conferred by the farnesyl moiety and not its specific spatial orientation is a key determinant of a-factor potency. Analyses on truncated a-factors suggest that sequential removal of NH2-terminal residues leads to a gradient of potency loss, with some amino acids exhibiting a slightly greater contribution to bioactivity than others. Random oligonucleotide-targeted mutagenesis of the gene encoding a-factor was coupled to a biological screen to identify altered a-factor peptides which are secreted yet exhibit a loss of a-factor bioactivity. Transformants exhibiting this phenotype were examined to identify codon changes presumably responsible for the altered phenotype, thus indicating residues that may contribute significantly to a-factor bioactivity.

NMR investigation of cyclo7,10[C7,X9,C10,Nle12] analogues of the alpha-factor from Saccharomyces cerevisiae.

The cyclo7,10[Cys7,Cys10,Nle12], cyclo7,10[Cys7,D-Ala9,Cys10,Nle12], and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] analogues of the alpha-factor mating pheromone (WHWLQLKPGQPMY) of the yeast Saccharomyces cerevisiae were studied in DMSO/water (80:20) and aqueous solution by nmr spectroscopy. In addition, the cyclo7,10[Cys7,D-Val9,Cys10,Nle12]alpha-fa ctor was examined in DMSO/water. Nuclear Overhauser effect (NOE) and NH d delta/dT data indicate that the cyclo7,10[Cys7,D-Val9,Cys10,Nle12]alpha-fa ctor adopts a type II beta-turn in DMSO/water and that the cyclo7,10[Cys7,D-Ala9,Cys10,Nle12]- and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12]alpha-fa ctor analogues adopt type II and type I/III beta-turns, respectively, in both DMSO/water and aqueous solutions. In aqueous solution, residues 8 and 9 of the cyclo7,10[Cys7,Nle12] alpha-factor appear to adopt at least two distinct conformations, one of these being identified as a type I/III beta-turn. In contrast, the cyclo7,10[Cys7,Cys10,Nle12] alpha-factor appears to adopt predominately a type II beta-turn in DMSO/water. Quantitative NOE measurements of the cyclo7,10[Cys7,Cys10,Nle12]-, cyclo7,10[Cys7,D-Val9,Cys10,Nle12]-, and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] alpha-factors in DMSO/water were used to derive three-dimensional structures of the cyclo7,10[Cys7,Pro8,X9,Cys10] portion of these analogues.

Chemical synthesis of the M-factor mating pheromone from Schizosaccharomyces pombe.

Conjugation in the fission yeast Schizosaccharomyces pombe is controlled by the reciprocal action of mating pheromones. We recently showed that M-factor, the pheromone released by cells of the cellular mating type Minus, is a nonapeptide in which the C-terminal cysteine residue is carboxyl-methylated and S-alkylated, probably with a farnesyl residue (Davey, 1992): Tyr-Thr-Pro-Lys-Val-Pro-Tyr-Met-Cys(S-farnesyl)- OCH3. Here we describe the chemical synthesis of this modified peptide and show that it exhibits all of the properties of the native pheromone. These results confirm the structure of the M-factor while the production of relatively large amounts of pure pheromone will be invaluable for studying the mating response in this yeast.

Role of prenylation in the interaction of the a-factor mating pheromone with phospholipid bilayers.

We have studied the interaction between phospholipids and a-factor (YIIKGVFWDPAC-[Farn]OMe), S-alkylated forms of a-factor with the farnesyl group substituted by methyl, hexadecanyl, or benzyl groups, and truncated forms of this lipopeptide. Circular dichroism studies suggest that, despite its lack of farnesylation, S-methyl-a-factor is incorporated into vesicles of dimyristoylphosphatidylcholine in a conformation similar to that which a-factor adopts in this membrane. However, studies of the intrinsic fluorescence of the Trp residues of these peptides indicate that this residue is more deeply imbedded into the bilayer in the case of the farnesylated peptide. The a-factor is more effective in raising the bilayer to the hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine than is the S-methyl-a-factor. This bilayer-stabilizing ability is also reflected in a-factor inhibiting leakage from vesicles of N-methyldioleoylphosphatidylethanolamine. Studies on a-factor analogs permit the conclusion that the bilayer-stabilizing effect of a-factor is not solely a consequence of its greater partitioning into the membrane but is also a consequence of the degree of penetration into the bilayer and the specific conformation of the peptide at the membrane interface. These results indicate that the farnesyl group alone, in the absence of cellular factors, bestows a particular physical interaction with membranes.

Studies on the yeast alpha-mating factor: a model for mammalian peptide hormones.

Small peptides initiate sexual conjugation in the yeast Saccharomyces cerevisiae and this phenomenon is an ideal paradigm for studying the mode of action of mammalian peptide hormones. 1H-nmr spectroscopy was used to examine the conformation of linear and cyclic analogues of the alpha-factor (WHWLQLKPGQPMY) in aqueous solution. In all cases peptides that exhibit nmr parameters expected for a type II beta-turn have higher biological activities than those that do not appear to assume this conformation. Based on a simple model for the interaction of the pheromone with its receptor, we prepared fragments of the alpha-factor. Several of these fragments either antagonize or potentiate the activity of the alpha-factor. The latter represent the first example of peptide fragments that synergize the activity of the parent pheromone.

Antagonistic and synergistic peptide analogues of the tridecapeptide mating pheromone of Saccharomyces cerevisiae.

Biologically inactive, truncated analogues of the Saccharomyces cerevisiae alpha-mating factor (WHWLQLKPGQPMY) either antagonized or synergized the activity of the native pheromone. An amino-terminal truncated pheromone [WLQLKPGQP(Nle)Y] had no activity by itself, but the analogue acted as an antagonist by competing with binding and activity of the mating factor. In contrast, a carboxyl-terminal truncated pheromone [WHWLQLKPGQP] was not active by itself nor did the peptide compete with alpha-factor for binding to the alpha-factor receptor, but it acted as a synergist by causing a marked increase in the activity of alpha-factor. The observation that residues near the amino terminus may be involved in signal transduction whereas those near the carboxyl terminus influence binding allows us to separate binding and signal transduction in the yeast pheromone response pathway. If found for other hormone-receptor systems, synergists may have potential as therapeutic compounds.

The conformation of a-factor is not influenced by the S-prenylation of Cys12.

Two-Dimensional NMR was used to examine the solution conformation of the lipopeptide a-factor, YIIKGVFWDPAC (S-farnesyl) OCH3, from the yeast Saccharomyces cerevisiae and five analogues containing various S-alkylated cysteines in DMSO-d6. NOESY data, NH temperature coefficients, and 3J alpha NH coupling constants indicate that the a-factor is a predominantly unstructured peptide in DMSO. Similar results were obtained for the other peptides indicating that S-prenylation of Cys12 does not affect the conformation of these peptides.