Design of Potent IGF1‐R Inhibitors Related to Bis‐azaindoles

Abstract

From an azaindole lead, identified in high throughput screen, a series of potent bis-azaindole inhibitors of IGF1-R have been synthesized using rational drug design and SAR based on a in silico binding mode hypothesis. Although the resulting compounds produced the expected improved potency, the model was not validated by the cocrystallization experiments with IGF1-R.Key words: drug discovery, enzyme structure, kinase, phosphatase, structure-based drug design Insulin-like growth factor (IGF) promotes growth and mediates metabolic signals (1). There is evidence that links IGF to cancer or tumorigenesis, and examination of the literature (2-5) from the last decade suggests that agents capable of inhibiting IGF receptor would have potential in the treatment of such malignancies (6). To date, the first results from clinical trials on antibodies or small molecules targeting IGF-1 receptor are starting to be reported and confirm these hypotheses (7). We now wish to report our own findings in this area.We initiated a high throughput screen (HTS) on IGF1-R using an Homogeneous Time-resolved Fluorescence (HTRF) assay relying on the receptor autophosphorylation. This led to the identification of a very potent hit 1 belonging to an azaindole chemical series from a Syk kinase inhibitor program (8).Me 1 IC 50 = 55 nMAs 1 was already quite potent on the target, we decided to evaluate it in an extensive panel of biological, Absorption, Distribution, Metabolism, Elimination (ADME), drug metabolism and Pharmacokinetics assays to anticipate potential liabilities and set the objectives of our optimization program.We first evaluated 1 in a series of secondary assays related to IGF function. The potency in an ELISA IGF1-R autophosphorylation assay was comparable to the one obtained in the HTRF assay. Compound 1 was also able to inhibit the IGF1-induced proliferation in two different cell lines, a Mouse Embryonic Fibroblasts (MEF) cell line engineered to over express IGF1-R and an MCF7 breast tumor cell line, with IC 50 's of 1.6 and 4.3 lM, respectively (Table 1).Selectivity against a panel of kinases was also evaluated. As expected because of its origin, 1 was a potent Syk inhibitor ( Table 2). As expected also because of its high homology, there was no selectivity versus insulin receptor kinase (IRK). Inhibitors of IRK would be expected to induce insulin resistance (hyperglycemia). It was hypothesized that for a short treatment period (about 4 months), there should not be any major metabolic disturbances (compared with the physiological insulin resistance during late state of pregnancy). In addition, some dual inhibitors of IGF1-R and IR-A such as 1H-Benzoimidazol-2-yl)-1H-pyridin-2-one BMS-536924 (9) are undergoing preclinical studies and could be advantageous for the treatment of tumours expressing both IGF1-R and IR-A (10). Finally, some inhibitors in development, such as the pyrrolo[2,3-d]pyrimidine NVP-AEW541 (11) displayed good selectivity for IGF1-R versus IR in intact cells in spite of no enzymatic selectivity for the kinas...

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

“…We identified a previously undescribed autophosphorylation complex of IGF1R [PDB: 3LVP (21)] in which Tyr 1166 of the activation loop of chain B in the asymmetric unit is in the active site of chain C (Fig. 5A).…”

“…The newly identified autophosphorylation events include: (i) the activation loop Tyr of human nonreceptor tyrosine kinase LCK [PDB: 2PL0 (20)], which is similar to the IGF1R structure (10); (ii) a second tyrosine autophosphorylation site (Tyr 1166 ) in the activation loop of human IGF1R [PDB: 3LVP (21)]; (iii) a Tyr in the N-terminal juxtamembrane region of human colony stimulating factor 1 receptor (CSF1R) [PDB: 3LCD (22)] that is homologous to the tyrosine observed in the c-KIT complex (7); (iv) a Tyr in the N-terminal juxtamembrane region of Ephrin receptor A2 (EPHA2) [PDB: 4PDO (23)] that represents a phosphorylation site near but distinct from that in the c-KIT and CSF1R complexes; and (v) a Ser in the C-terminal tail of human CDC2/CDC28-like kinase 2 (CLK2) [PDB: 3NR9 (24)]. We also identified several additional structures of autophosphorylation complexes of PAK1: PDB: 4O0R, 4O0T (25); 4P90 (26); 4ZY4, 4ZY5, 4ZY6 (27); and 4ZLO, 4ZJI, and 4ZJJ (28).…”

Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth

“…We also used the available crystal structures that, although truncated below amino acid 1256, encompass the SFYYS motif (7,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and enabled us to explore the structural basis for this motif in regulating IGF-1R activity. Our findings indicate that a direct regulatory interaction of the C terminus with the kinase domain can be controlled by phosphorylation of Ser-1248.…”

“…To do this, we examined crystal structures of the IGF-1R kinase that, although C-terminally truncated after amino acid 1256, encompass the SFYYS motif. These include crystal structures with the kinase A-loop in various states of phosphorylation (from unphosphorylated to fully phosphorylated) and variously complexed with ATP mimetics, inhibitors, and phospho-acceptor peptide substrates (7,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). In these structures, the unphosphorylated SFYYS motif universally adopts a conformation tightly packed against helices ␣I and ␣E of the kinase C-lobe (Fig.…”

7-Azaindole: A Versatile Scaffold for Developing Kinase Inhibitors

“…The observation that both "normal" and "flipped" binding modes are possible in Janus kinase 2 (JAK2; TK), c-Met (Met; TK), and apoptosis signalregulating kinase 1 (ASK1; STK) kinases suggests that the binding mode is not dependent on kinase structural features. Rather, it would depend on the ligand structure, because 2-substituted derivatives of the azaindole give only "flipped" binding mode (compounds 7-11 [19][20][21][22][23] in Fig. 4).…”