Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein–protein interactions.

Identifying protein-ligand binding interactions is a key step during early-stage drug discovery. Existing screening techniques are often associated with drawbacks such as low throughput, high sample consumption, and dynamic range limitations. The increasing use of fragment-based drug discovery (FBDD) demands that these techniques also detect very weak interactions (mM K(D) values). This paper presents the development and validation of a fully automated screen by mass spectrometry, capable of detecting fragment binding into the millimolar K(D) range. Low sample consumption, high throughput, and wide dynamic range make this a highly attractive, orthogonal approach. The method was applied to screen 157 compounds in 6 h against the anti-apoptotic protein target Bcl-x(L). Mass spectrometry results were validated using STD-NMR, HSQC-NMR, and ITC experiments. Agreement between techniques suggests that mass spectrometry offers a powerful, complementary approach for screening.

The synthesis of a range of novel bidentate and hexadentate ligands containing the chelating moiety 3-hydroxy-2(1H)-pyridinone is described. The pKa values of the ligands and the stability constants of their iron(III) complexes have been determined. The stability constant of the iron(III) complex of one of the hexadentate ligands is comparable to that of desferrioxamine B. The distribution coefficients of the ligands and their iron(III) complexes were also determined. One of the novel hexadentate compounds has been shown to markedly enhance iron(III) excretion from both hepatocytes and iron-overloaded mice.

A series of antagonists of gonadotropin-releasing hormone (GnRH) homologous to azaline B ([Ac-DNal1,DCpa2,DPal3,Aph5(Atz),DAph6+ ++(Atz),ILys8,DAla10]GnRH) was synthesized, characterized, and tested in a rat antiovulatory assay (AOA). Selected analogues were also tested in both an in vitro dispersed rat pituitary cell culture assay for inhibition of GnRH-stimulated luteinizing hormone release and an in vitro histamine release assay. The duration of action of some of the most potent and safest analogues in those assays was also determined in the castrated male rat in order to measure the extent (efficacy and duration of action) of inhibition of luteinizing hormone release. Structurally, this series of analogues has novel substitutions (X and Y) in the structure of the azaline B precursor: [Ac-DNal1,DCpa2,DPal3,-Aph5(X),DAph6(Y),++ +ILys8,DAla10]GnRH. These substitutions were designed to confer increased hydrophilicity as compared to that of azaline B (determined by relative retention times on a C18 reverse phase column using a triethylammonium phosphate buffer at pH 7.3) or to make them more easily accessible synthetically. Some bulky substituents were introduced in order to probe the spatial limitations of the receptor's cavity. These substitutions include acylated 4-aminophenylalanine at positions 5 and/or 6 (29 analogues), N alpha-methylated backbone substitutions (six analogues), N omega-isopropylaminophenylalanine at position 8, and hydrophilic amino acids at position 1. Out of 20 novel analogues tested for long duration of action in this series, only seven ([Ac-DNal1,DCpa2,DPal3,Aph5,DAph6,ILys8 ,DAla10]GnRH, [Ac-DNal1,DCpa2,DPal3,Aph5(For),DAph6(For) ,ILys8,DAla10]GnRH, [Ac-DNal1,DCpa2,DPal3,Aph5(Ac),DAph6(Ac),- ILys8,DAla10]GnRH (acyline), [Ac-DNal1,DCpa2,DPal3,Aph5(Pio),DAph6++ +(Pio),ILys8,DAla10]GnRH, [Ac-DNal1,DCpa2,DPal3,Aph5(Atz),DAph6++ +(Ac),ILys8,DAla10]GnRH, [Ac-DNalDCpa2,DPal3,Aph5(Atz-beta Ala),DAph6(Atz-beta Ala),ILys8, DAla10]GnRH, [Ac-DNal1,DCpa2,DPal3,Aph5(Atz-Gab), DAph6(Atz-Gab),ILys8,DAla10]GnRH) had relative potencies and/or duration of action comparable to those of azaline B. The others were one-half to one-tenth as effective as azaline B. N alpha-Methylated backbone substitutions at position 5 yielded analogues that were significantly more hydrophilic presumably because of the breakage of the NH alpha-Tyr5 to Arg8-CO hydrogen bond reported to stabilize a beta-turn encompassing residues 5-8 and which favored beta-sheet formation as shown earlier by Haviv et al. This substitution resulted, however, in an increased potency in the histamine release assay and in significantly shorter duration of action. Similarly, attempts at replacing isopropyllysine in position 8 by either isopropyl-4-aminophenylalanine or isopropyl-4-(aminomethyl)phenylalanine resulted in loss of potency in the AOA. Changes in chirality at position 1 or 10 resulted in analogues that were one-tenth and one-half as potent, respectively, as acyline.(ABSTRACT TRUNCATED AT 400 WORDS)

In order to be used as fertility regulators in humans, gonadotropin releasing hormone (GnRH) antagonists must be extremely potent and long acting and exhibit negligible side effects such as stimulating histamine release. To this aim, we have recently synthesized a series of analogues with the standard Ac-DNal1-DCpa2-DPal3 substitutions, where the N omega-amino function of ornithine, lysine, or p-aminophenylalanine (Aph) was converted to the aminotriazolyl (atz) derivatives at positions 5 and 6 with further modifications at positions 7 and 10. The analogues were tested for their ability to bind to pituitary cell membranes, to release histamine in a mast cell assay, to inhibit luteinizing hormone (LH) secretion by castrated male rats or cultured pituitary cells, and to interfere with the ovulation in intact female rats. While the subcutaneous (sc) injection of 50 micrograms of Azaline A (7, [Ac-DNal1,DCpa2,DPal3,Lys5(atz),DLys6++ +(atz),ILys8,DAla10]GnRH) dissolved in 0.2 mL of an aqueous media significantly inhibited LH release in the castrated male rat for 24 h, the same dose of Azaline B (11), [Ac-DNal1,DCpa2,DPal3,Aph5(atz),DAph6++ +(atz),ILys8,DAla10]GnRH, inhibited LH release for 72 h. A similar long duration of action was observed for Antide ([Ac-DNal1,DCpa2,DPal3,Lys5(Nic),DLys6(Nic ),ILys8,DAla10]GnRH) but not for Nal-Glu ([Ac-DNal1,DCpa2,DPal3,Arg5,4-(pmethoxybenzoy l)-D-2-Abu6,DAla10]GnRH). In the same paradigm, a 5-fold dilution of the peptide (50 micrograms in 1 mL) and the use of three injection sites rather than one resulted in significantly shorter duration of action for most of the peptides tested. This suggested that long duration of action might be the result of slow release from the injection site(s). In order to investigate this possibility, Nal-Glu and Azaline B were injected intravenously (i.v.) at three doses (10, 50, 250 micrograms) to castrated male rats. At all doses, both peptides significantly lowered LH levels for 8 h. By 24 h, Nal-Glu (250 micrograms) and Azaline B (50 and 250 micrograms) still measurably inhibited LH secretion. Finally, only Azaline B (250 micrograms) was still active at 48 h. These findings demonstrate that subtle structural modifications will yield peptides with different half-lives after iv administration. These findings led us to investigate the effects of other structural modifications on duration of action. We observed that systematic substitutions at positions 7 (NMeLeu) and 10 (Pro9-NHEt, and Gly-NH2) were found to be deleterious. Of interest was the observation that only the DAla10-NH2 substitution led to long duration of action and enzymatic stability under the conditions tested.(ABSTRACT TRUNCATED AT 400 WORDS)

In order to minimize the adverse effect of histamine release in the rat of some gonadotropin releasing hormone (GnRH) antagonists, such as [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH, new structures with modifications at positions 1, 2, 3, 5, 6, 7, and 10 were synthesized and tested in several biological systems. In vitro: the affinity for the pituitary GnRH receptor was measured as was the ability of the analogues to inhibit GnRH-stimulated release of luteinizing hormone (LH) by dispersed anterior pituitary cells in culture and to release histamine from rat mast cells. In vivo: inhibition of ovulation in the cycling rat was determined after subcutaneous (sc) injection of the peptides at noon on the day of proestrus; the duration of action of the peptides was evaluated by measuring LH levels in the castrated male rat after sc injection of some selected analogues. [Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,D-4-p-methoxy benzoyl-2-aminobutyric acid6,DAla10]-GnRH was found to be one of the most potent analogues of this series, causing a 100% inhibition of ovulation at 5 micrograms/kg or less. Release of histamine was observed at doses 10-25 times that required for [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]-GnRH. Thus, introduction of arginine in position 5 with a hydrophobic amino acid in position 6 is compatible with high potency in several biological systems and results in compounds with lowered potency to release histamine compared to homologous peptides with tyrosine in position 5 and D-arginine in position 6.

In the course of our studies toward the development of novel analogs of the decapeptide gonadotropin releasing hormone (GnRH), we have examined a hexapeptide that is an antagonist of endothelin (ET). It was found that this peptide, Ac-D-Trp-Leu-Asp-Ile-Ile-Trp (peptide 1), binds specifically to the pituitary GnRH receptor. Moreover, peptide 1 exhibits a GnRH agonistic activity (i.e., it induces luteinizing hormone release from rat pituitary). This activity is mediated directly by the GnRH receptor and is suppressed by a GnRH antagonist. Removal of the acetyl group of peptide 1 results in a hexapeptide (peptide 2) with binding properties similar to those of GnRH but with a diminished affinity toward the ET receptor. Several other ET antagonists were screened for a potential interaction with the GnRH receptor. Two of these, the hexapeptide PD145065 and the cyclic pentapeptide BQ-123, expressed GnRH agonistic activity at micromolar concentrations in vitro. BQ-123, previously approved for trials on humans as an ET antagonist, is demonstrated to act in vivo as a GnRH agonist, in a dose that was demonstrated previously as the minimal required dose for significant ET antagonism. The GnRH agonistic activity of ET antagonists may therefore result in interference with the physiological control of the reproductive system. Such effects may be most severe when ET antagonists are used chronically. Thus, the major practical message of this study is the need to circumvent the potential side effects of ET antagonist-based drugs.

We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide. The sequences of the two peptides Pca-Cys-Gly-Gln-Ala-Trp*-Cys-NH 2 were determined using a combination of mass spectrometry, amino acid, and chemical sequence analyses, where Pca ‫؍‬ pyroglutamic acid, Hyp ‫؍‬ hydroxyproline, Gla ‫؍‬ ␥-carboxyglutamate, and Trp* ‫؍‬ L-6-bromotryptophan. The precise structure and stereochemistry of the modified residue were determined as L-6-bromotryptophan by synthesis, co-elution, and enzymatic hydrolysis experiments. To our knowledge this is the first documentation of tryptophan residues in peptides/proteins being modified in a eukaryotic system and the first report of halogenation of tryptophan in vivo. SEQUENCE 1 andPolypeptides encoded by genes are primarily made up of the 20 common amino acids that are directly translated using the genetic code. However, many of these amino acids can be further modified post-translationally to yield a set of additional amino acids that contribute to the function of the mature protein. Together the 20 primary amino acids and these "secondary" amino acids which are found in proteins comprise the set of proteinogenous amino acids.These modified amino acids include amino acid conjugates where the side chain is linked to a glycosyl, phosphate, or sulfate group and amino acids such as 5-hydroxylysine, 4-hydroxyproline, and ␥-carboxyglutamate. One notable set of modified amino acids are halogenated derivatives of tyrosine and histidine. Naturally occurring halogenated tyrosine and histidine residues have previously been identified from proteins (1-4). A particularly well documented example of in vivo posttranslational halogenation of an amino acid residue in a protein is afforded by thyroglobulin (5). After iodination of several tyrosine residues, selective cleavages release the iodinated thyroid hormones thyroxine (3,5,3Ј,5Ј-tetraiodothyronine or T 4 ) 1 ; 3,5,3Ј-triiodothyronine (T 3 ), and 3,3Ј-di-iodothyronine. The presence of free T 3 and T 4 has also been shown in protochordates, suggesting a primitive thyroid function exists in tunicates (2).Of the 20 common amino acids, alanine, glycine, isoleucine, leucine, and valine, which lack side chain functional groups, have not been implicated in post-translational modifications (6). In 1907 it was proposed that hydroxytryptophan was a proteinogenous amino acid (7), but this proposal has not been verified (1), and chemical oxidation of tryptophan is likely to be responsible for the observed ...