Design, synthesis, and cocrystal structure of a nonpeptide Src SH2 domain ligand.

The specific association of an SH2 domain with a phosphotyrosine (pTyr)-containing sequence of another protein precipitates a cascade of intracellular molecular interactions (signals) which effect a wide range of intracellular processes. The nonreceptor tyrosine kinase Src, which has been associated with breast cancer and osteoporosis, contains an SH2 domain. Inhibition of Src SH2-phosphoprotein interactions by small molecules will aid biological proof-of-concept studies which may lead to the development of novel therapeutic agents. Structure-based design efforts have focused on reducing the size and charge of Src SH2 ligands while increasing their ability to penetrate cells and reach the intracellular Src SH2 domain target. In this report we describe the synthesis, binding affinity, and Src SH2 cocrystal structure of a small, novel, nonpeptide, urea-containing SH2 domain ligand.

Hydrophobic D-amino acids in the design of peptide ligands for the pp60src SH2 domain.

Src homology-2 (SH2) domains, containing approximately 100 amino acid residues, are noncatalytic motifs involved with intracellular signal transduction. These domains can be found on nonreceptor kinases, phosphatases, and in regulatory adapter proteins among others. SH2 domains bind proteins containing phosphotyrosine (pTyr) residues in a sequence specific manner. Our efforts have focused on designing peptide mimetic ligands for the SH2 domain of the nonreceptor tyrosine kinase pp60src. We employed the cocrystal structure of the 11mer Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-IIe-Tyr-Leu IC50 = 800 nM as a starting point for our design efforts. These efforts have resulted in the discovery of tripeptide ligands containing D-amino acids that are only 2-fold less potent than the 11mer.

PD 90780, a non peptide inhibitor of nerve growth factor's binding to the P75 NGF receptor.

Nerve growth factor is a peptide that supports the survival and differentiation of discrete neuronal populations in the peripheral and central nervous systems. NGF binds to both trkA, a tyrosine kinase receptor, and to the p75 nerve growth factor receptor, a protein lacking a consensus signalling sequence. We have identified a substituted pyrazoloquinazolinone, PD 90780, which inhibits binding of nerve growth factor to the p75 receptor. This inhibition of binding occurs due to PD 90780 binding to nerve growth factor, not to the p75 receptor. This compound may be useful in identifying the region(s) of nerve growth factor involved in binding to the p75 receptor and in clarifying the role of p75 receptor in the actions of the neurotrophins.

Structure-activity relationships for enhancement of adenosine A1 receptor binding by 2-amino-3-benzoylthiophenes.

The structural requirements for stimulation of adenosine A1 agonist binding by 2-amino-3-benzoylthiophenes and related compounds were investigated. Slowing of the dissociation of [3H]N6cyclohexyladenosine binding was used as a specific measure of the allosteric effects of these compounds. The thiophene ring could be replaced with benzene but not with several nitrogen-containing heterocycles. The 2-amino group was required, and at least one hydrogen on the amino group appeared to be necessary for activity. The keto carbonyl was also essential. Alkyl substitution at the 4-position of the thiophene ring increased activity, whereas 5-position substitution appeared to have little effect. Activity was also increased by various substitutions on the phenyl ring, with 3-(trifluoromethyl) showing optimal activity. The phenyl ring could be replaced with cyclohexyl without major loss of activity. 1-Aminofluoren-9-one, a conformationally locked derivative, was active. Based in part in the latter observation, the active conformation is proposed to have an intramolecular hydrogen bond between the amino nitrogen and the carbonyl oxygen. Because the 2-amino-3-benzoylthiophenes showed competitive adenosine antagonism as well as allosteric enhancement, their affinities as competitive inhibitors of [3H]8-cyclopentyl-1,3-dipropylxanthine binding to A1 receptors were also assessed. Structure-activity relationships for competitive antagonism were distinct from those for allosteric enhancement, with ratios between the two activities varying by more than 1000-fold. Of the analogs tested, (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone (PD 81,723) had the most favorable ratio of enhancement to antagonism.

Allosteric enhancement of adenosine A1 receptor binding and function by 2-amino-3-benzoylthiophenes.

Several 2-amino-3-benzoylthiophenes were found to increase the binding of [3H]N6-cyclohexyladenosine to A1 adenosine receptors in rat brain membranes. Concentration-response curves were bell-shaped, with up to 45% stimulation of binding at 10 microM followed by inhibition at higher concentrations. Because these compounds originated from a series of nonxanthine adenosine antagonists, the inhibition of binding was attributed to the presence of interfering adenosine antagonist activity. The compounds stimulated binding of several A1 agonist ligands but only inhibited binding of the A1 antagonist ligand [3H]8-cyclopentyl-1,3-dipropylxanthine, indicating that enhancement was specific for the agonist conformation of the receptor. The enhancement was also specific for the A1 receptor, because agonist binding to A2 adenosine, M2 muscarinic, alpha 2 adrenergic, and delta opiate receptors showed little or no enhancement. Uncoupling of the A1 receptor from the inhibitory guanine nucleotide-binding protein did not prevent enhancement. The enhancers slowed the dissociation of [3H]N6-cyclohexyladenosine from the A1 receptor, implying an allosteric mechanism of action. The inhibition of forskolin-stimulated cyclic AMP accumulation in FRTL-5 cells was employed as a functional index of A1 receptor activation. The enhancers caused up to 19-fold leftward shifts in the concentration-response curve for N6-cyclopentyladenosine and also caused up to 55% inhibition of cyclic AMP accumulation in the absence of agonist. The binding and functional results are consistent with a model in which the enhancers bind preferentially to the agonist conformation of the A1 receptor, thereby shifting the receptor equilibrium in favor of agonist binding. Adenosine enhancers may be useful for ischemia and other conditions involving local energy deficits. More generally, allosteric enhancers may provide a means for strengthening physiological control circuits in a variety of receptor systems.

Solubilities of adenosine antagonists determined by radioreceptor assay.

The practical use of many adenosine receptor antagonists is limited by poor aqueous solubility. In some cases, solubilities are so low that they are difficult to measure by conventional means. To determine solubilities of adenosine antagonists, a sensitive radioreceptor method has been developed. Solubilities in Tris buffer (pH 7.7) ranged from 141 nM for 8-(2-amino-4-chlorophenyl)-1,3-dipropylxanthine to 945 microM for the amino-substituted xanthine PD 113,297. Ratios between solubility and adenosine receptor affinity varied from 15.8 for the A2-selective antagonist HTQZ to 169,000 for PD 113,297. From literature data on functional activity, it is apparent that useful adenosine antagonist activity in-vivo is only seen in compounds with solubility/affinity ratios greater than 100.

Binding of the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes.

8-Cyclopentyl-1,3-dipropylxanthine (PD 116,948) is a very potent, very A1-selective adenosine antagonist, with a Ki of 0.46 nM in 3H-CHA binding to A1 receptors in rat whole brain membranes and 340 nM in 3H-NECA binding to A2 receptors in rat striatal membranes. Its 740-fold A1-selectivity is the highest reported for an adenosine antagonist. 3H-PD 116,948 (117 Ci/mmol) was prepared by reduction of the diallyl analog. 3H-PD 116,948 bound to a single site in rat whole brain membranes, with a Bmax of 46 pmol/g wet weight and Kd of 0.42 nM. Nonspecific binding was extremely low, amounting to about 3% of total binding under standard conditions and less than 1% when higher tissue concentrations were used. Affinities of compounds for inhibition of 3H-PD 116,948 binding were highly consistent with an A1 adenosine receptor. Antagonists were equally potent in 3H-PD 116,948 binding and in 3H-CHA binding, while agonists were consistently about 12-fold more potent in 3H-CHA binding. Hill coefficients were 1.0 for antagonists and about 0.65 for agonists. 3H-PD 116,948 should be a useful antagonist ligand for adenosine A1 receptors.

PD 115,199: an antagonist ligand for adenosine A2 receptors.

PD 115,199, N-[2-(dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3- dipropyl-1H-purin-8-yl)benzenesulfonamide, was found to have high affinity for the A2 adenosine receptor labeled by 3H-NECA in rat striatal membranes (Ki 15.5 nM). Unlike other potent adenosine antagonists, which always showed some degree of selectivity for the A1 receptor, PD 115,199 had equal affinity at A1 and A2 receptors (Ki in 3H-CHA binding to A1 receptors 13.9 nM). 3H-PD 115,199 (126 Ci/mmol) was prepared by reduction of the diallyl analog, and binding experiments were performed with 0.5 nM 3H-PD 115,199 at 25 degrees C in rat striatal membranes. By nonlinear least-squares analysis of the concentration-inhibition curve for the highly A1-selective adenosine antagonist PD 116,948 (8-cyclopentyl-1,3-dipropylxanthine), it could be demonstrated that about 11% of specific 3H-PD 115,199 binding was to A1 receptors, and the remainder to A2 receptors. A 20 nM concentration of PD 116,948 was included in subsequent experiments to eliminate the A1 component of binding. The remaining binding had a Kd of 2.6 nM and Bmax of 56 pmol/g wet weight. Specific binding was about 79% of total binding. Affinities of compounds in the 3H-PD 115,199 assay were consistent with binding to a high-affinity A2 receptor: antagonists were consistently about three times more potent in 3H-PD 115,199 binding than in 3H-NECA binding, whereas agonists were consistently about fivefold less potent.(ABSTRACT TRUNCATED AT 250 WORDS)