Robert L. Panek

Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain.

Angiogenesis, the sprouting of new blood vessels from pre‐existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3‐d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP‐binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation.

Endothelin and structurally related analogs distinguish between endothelin receptor subtypes

The endothelin (ET) analog ET-1[1,3,11,15-Ala] was compared with ET-1, ET-2, ET-3 and sarafotoxins (SRTX) S6b and S6c for receptor binding and function. All the peptides exhibited high affinity binding and contracted rabbit pulmonary artery with near equal potency. In rat aorta both ET-3 and ET-1 [1,3,11,15-Ala] bound with much lower affinity than ET-1 while ET-3 displayed weak contractile potency and ET-1 [1,3,11,15-Ala] and SRTX-c were inactive. In rat left atria, ET-1 [1,3,11,15-Ala] and SRTX-c were weak inhibitors of binding and were also functionally inactive, whereas ET-1, ET-2, ET-3, and SRTX-b were equipotent in producing contractile responses. The data support the idea of there being a predominance of ETA receptors in rat aorta and ETB receptors in rabbit pulmonary artery. In rat left atria, the ET receptor could not be readily classified into ETA or ETB and suggests the existence of a new receptor subtype.

discovery and structure-activity studies of a novel series of pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors

Abstract The inhibition of tyrosine kinase-mediated signal transduction pathways represents a therapeutic approach to the intervention of proliferative diseases such as cancer, atherosclerosis, and restenosis. A novel series of pyrido[2,3-d]pyrimidine inhibitors of the PDGFr, bFGFr, and c-Src tyrosine kinases was developed from compound library screening and lead optimization.1 In addition, highly selective inhibitors of the FGFr tyrosine kinase were also discovered and developed from this novel series of pyrido[2,3-d]pyrimidines. The syntheses, biological evaluation, and structure-activity relationships of this series are reported.

Progress in the Development of Inhibitors of SH2 Domains

SH2 domains are discrete structural motifs common to a variety of critical intracellular signaling proteins. Inhibitors of specific SH2 domains have become important therapeutic targets in the treatment and/or prevention of restenosis, cancers (including small cell lung), cardiovascular disease, osteoporosis, apoptosis among others. Considering the social and economic impact of these diseases significant attention has been focused on the development of potent and selective inhibitors of specific SH2 domains. In particular, considerable research has been performed on Src, PI 3-kinase, Grb2 and more recently, Lck. In this review, we will focus on progress in the development of inhibitors for these specific SH2 domains and evaluate potential future targets.

Subclasses of cyclic GMP-specific phosphodiesterase and their role in regulating the effects of atrial natriuretic factor.

Two subclasses of cyclic guanosine monophosphate (GMP)-specific phosphodiesterases were identified in vascular tissue from several beds. The activity of one subclass (phosphodiesterase IB) was stimulated severalfold by calmodulin and selectively inhibited by the phosphodiesterase inhibitor TCV-3B. The activity of the other subclass (phosphodiesterase IC) was not stimulated by calmodulin and was selectively inhibited by the phosphodiesterase inhibitor M&B 22,948. To assess the involvement of both subclasses in regulating cyclic GMP-dependent responses, the ability of TCV-3B and M&B 22,948 to potentiate the in vitro and in vivo responses to the endogenous guanylate cyclase stimulator atrial natriuretic factor (ANF) was evaluated. Both TCV-3B and M&B 22,948 relaxed isolated rabbit aortic and pulmonary artery rings and also potentiated the relaxant effect of ANF. In addition, both inhibitors produced small increases in urine flow and sodium excretion in anesthetized rats and potentiated the diuretic and natriuretic responses to exogenous ANF. M&B 22,948 (30 micrograms/kg/min) produced a threefold increase in the natriuretic response to simultaneously administered ANF, and TCV-3B (10 micrograms/kg/min) produced a twofold increase in the response to ANF. The results of the present experiments suggest that both the calmodulin-sensitive and calmodulin-insensitive subclasses of cyclic GMP-specific phosphodiesterase play a role in regulating the in vitro and in vivo response to ANF.

Inhibition of FGF-1 receptor tyrosine kinase activity by PD 161570, a new protein-tyrosine kinase inhibitor.

Through direct synthetic efforts we discovered a small molecule which is a 40 nanomolar inhibitor of the human FGF-1 receptor tyrosine kinase. 1-Tert-butyl-3-[6-(2,6-dichloro-phenyl)-2-(4-diethylamino-butylamino)-py rido[2,3-d]pyrimidin-7-yl]-urea (PD 161570) had about 5- and 100-fold greater selectivity toward the FGF-1 receptor (IC50 = 40 nM) compared with the PDGFbeta receptor (IC50 = 262 nM) or EGF receptor (IC50 = 3.7 microM) tyrosine kinases, respectively. In addition, PD 161570 suppressed constitutive phosphorylation of the FGF-1 receptor in both human ovarian carcinoma cells (A121(p)) and Sf9 insect cells overexpressing the human FGF-1 receptor and blocked the growth of A121(p) cells in culture. The results demonstrate a novel synthetic inhibitor with nanomolar potency and specificity towards the FGF-1 receptor tyrosine kinase.

Structure-activity studies of the C-terminal region of the endothelins and the sarafotoxins

Peptides corresponding to the C-terminal 16-21 hexapeptide of the endothelins (-His-Leu-Asp-Ile-Ile-Trp) and sarafotoxins (a-c) (-His-Gln-Asp-Val-Ile-Trp) were prepared to study the role of the individual amino acids in receptor recognition and activation. Receptor binding in rabbit aorta, rabbit pulmonary artery, and rat heart ventricle is reported for all analogues. In addition, selected C-terminal hexapeptides have been evaluated functionally in two tissues (rabbit pulmonary artery and rat left atria). The C-terminal carboxylate, indole nitrogen, and nature of the aromatic residue are all important for receptor binding, but N-terminal acetylation has no effect. L-Amino acids are required in positions 19 and 21, whereas D-amino acids are tolerated in 17 and 18. D-Amino acids in positions 16 and 20 enhance the binding affinity of the hexapeptide in all three tissues. The nature of the basic residue at position 16 is important. Glu and Asn are acceptable substitutions for Asp18, although Ala leads to a substantial loss in binding. The binding of the C-terminal hexapeptide of SRTX-a, -b, and -c is less than ET[16-21] and this appears to be primarily due to the substitution of Gln for Leu17. None of the 16-21 hexapeptides showed any functional activity in the tissues studied.

1-oxo-3-aryl-1H-indene-2-carboxylic acid derivatives as selective inhibitors of fibroblast growth factor receptor-1 tyrosine kinase

Abstract Fibroblast growth factor receptor (FGFr) mediated signal transduction is implicated in vascular proliferative diseases and some cancers. We have identified methyl 1-oxo-3-phenyl-1H-indene-2-carboxylic ester as a small molecule inhibitor of the tyrosine kinase activity of FGFr-1, (IC50 = 5.1 μM). We report here the synthesis and structure-activity studies about this template core. Additionally, screening of this series against a panel of tyrosine kinases shows selective inhibition of FGFr.

Importance of secondary structure for endothelin binding and functional activity

ET-1[16-Phe] and ET-1[12-Pro] were prepared in order to investigate the importance of secondary structure of ET-1 for receptor binding and function. ET-1[16-Phe] displayed the greatest binding and contractile potency of the ET-analogs tested in rabbit pulmonary artery, rat aorta, and rat left atria. ET-1[12-Pro] also exhibited low nanomolar binding in these tissues but showed less contractile activity than ET-1[16-Phe] or ET-1. The results indicate that the helical region between residues Lys9 and Cys15 of ET-1 is not critical for receptor binding and functional activity. However, replacement of His16 with Phe altered the charge characteristics of the C-terminal region of ET-1 producing the most potent ET-1 analog yet reported.

Development of a High Renin Model of Hypertension in the Cynomolgus Monkey

Hypertension was produced in cynomolgus monkeys by reducing blood flow to the left kidney by 60% via renal artery stenosis (2-kidney, 1-clip). Significant increases in mean arterial blood pressure (MABP) were observed within two to three weeks. Maximum increase (from 95 +/- 6 mmHg to 130 +/- 7 mmHg) occurred at about four to six weeks following renal artery stenosis and was sustained for more than 24 weeks. Plasma renin activity (PRA) was elevated concomitantly with the increase in MABP. PRA was raised to 42 +/- 3 ng angiotensin I/ml/hr six weeks after renal artery stenosis from a control PRA of 3 +/- 0.7 ng angiotensin I/ml/hr. At six months post renal artery stenosis, PRA was 33.4 +/- 4.2 ng angiotensin I/ml/hr. The angiotensin II (AII) receptor antagonist saralasin, the angiotensin I converting enzyme inhibitor captopril, and the renin inhibitor CGP 38,560 produced sustained reductions in MABP. The antihypertensive response to the renin inhibitor CGP 38,560 was associated with a reduction in PRA of greater than 99%, and a greater than 90% reduction in immunoreactive AII. These studies demonstrate that high-renin hypertension can be induced in the cynomolgus monkey. This pathological model provides a useful method for investigating the antihypertensive effects of agents which antagonize the renin-angiotensin system in a nonhuman primate.