Pd173955

Figure 2 First-generation Bcr-Abl inhibitors.

7.08.5.2.2 Inhibitors of Bcr-Abl

The first Bcr-Abl inhibitor to enter clinical development, and the only marketed drug in this area is Novartis's imatinib (1). The discovery of imatinib was a result of judicious optimization of a series of phenylaminopyrimidines that had been identified as inhibitors of the serine/threonine kinase PKC-a.82 Further profiling of compound 2 showed that it inhibited a number of other serine/threonine and tyrosine kinases, including Bcr-Abl, platelet-derived growth factor receptor b (PDGFR-b), and Src.83,84 Refinement of the substituent at the 4-position of the pyrimidine ring, and introduction of the 'flag methyl group' to the 6-position of the phenyl ring gave compounds with significantly improved potency against both Bcr-Abl and PDGFR-b.85 Compounds such as 3 also showed no inhibitory activity toward a range of serine/threonine kinases, such as PKC-a, but were typically insoluble and lacked oral bioavailability. Exploiting an observation made in the quinolinone antibiotic area,86 an N-methyl piperazinyl group was introduced to the benzoic acid moiety, and gave imatinib (1), which was selected for clinical development.

Imatinib is an ATP-competitive inhibitor of Bcr-Abl (IC50 = 188 nM, Ki = 85 nM).70'87 When tested against a panel of kinases (recombinant kinase domains), it also showed activity against c-Kit (IC50 = 400 nM) and PDGFR-b (IC50 = 400 nM), but had no activity against Src, PKC-a, and eight other kinases.85 Autophosphorylation of Bcr-Abl in cells was inhibited (250 nM),88 as was signal output due to PDGFR, c-Kit, and MAP kinase activation.89,90 Imatinib blocks proliferation of Bcr-Abl-positive CML and ALL cell lines, causing apoptosis.91,92 In vivo activity was demonstrated after three-times daily oral dosing (160mgkg_ 1) to a KU812 (Bcr-Abl-positive) xenograft in nude mice, causing complete inhibition of tumor growth over the duration of dosing.93

7.08.5.2.3 Mechanistic understanding of imatinib

To rationalize the kinase selectivity of imatinib, retrospective modeling studies were carried out,85 building on the published structure of PD173074 (4) bound to the tyrosine kinase domain of FGFR1.94 This analysis suggested that the aminopyrimidine group bound to the nucleotide binding site, and that the terminal N-piperazine lay outside the ATP-binding site in a solvent-accessible region.95 In a key paper, Kuriyan reported the crystal structure of the imatinib analog 3, with the inhibitor bound to an inactive form of Bcr-Abl.96 In this inactive from, Tyr393 in the activation loop is not phosphorylated, leaving it free to hydrogen bond to Asp363, a conserved residue that is essential for kinase activity.

It appears that imatinib specifically recognizes this particular inactive conformation of Bcr-Abl, which is presumed to be in a dynamic equilibrium with a catalytically active conformation. While imatinib specifically targets an inactive from of Bcr-Abl, PD173955 (5) has been demonstrated to bind to a conformation of Bcr-Abl in which the activation loop adopts an orientation similar to that found in many active kinases. PD173955 is significantly more potent against Bcr-Abl than imatinib, and is also active against the Src family of kinases.98 Thus, the different selectivity profile between imatinib and PD173955 can be rationalized in terms of the former targeting an inactive form of Bcr-Abl that is structurally distinct from the inactive conformations of the Src kinases. As has been discussed earlier, this detailed understanding of the molecular mechanism of the action of imatinib has stimulated considerable interest in examining whether 'activation inhibitors' can be found for other kinases.

7.08.5.2.4 Bcr-Abl inhibitors in the clinic

Phase I trials with imatinib (1) began in June 1998 in CML patients who had failed interferon therapy with dosing in the range 25-1000 mg day_ 1.99 The compound was well absorbed, showed dose-proportional exposure, and had a plasma half-life of between 13 and 16h. In general, imaitinib was well tolerated; the MTD was not reached, and the most common adverse events were nausea, myalgias, edema, and diarrhea.100 Encouragingly, hematological responses were seen in all patients receiving doses of 140 mg day_ 1 or more, and 53 of 54 patients who received doses of 300 mg day_ 1 or greater (for a period of at least 4 weeks) showed complete hematological response. Of these 54 patients, 54% showed cytogenic responses, with 13% having a complete cytogenic response. Phase II studies, starting in 1999, focused on CML patients with either interferon refractory disease or with myeloid blast crisis, and also patients with Ph + ALL.101 Imatinib was approved by the US Food and Drugs Administration (FDA), following accelerated review, in May 2001 for the treatment of CML refractory to treatment with interferon.

Clinical experience with imatinib has shown that the outcome is critically dependent on which phase of CML (chronic, accelerated, or blast crisis) the patient is in.102 While 95% of patients in the chronic phase show complete hematological remission, the majority of patients in blast crisis quickly relapse.99,103 Resistance to imatinib in blast phase CML is believed to be due to secondary mutations in the kinase rather than Bcr-Abl amplification or targeted drug efflux.104,105 An analysis of 11 patients who relapsed during imatinib treatment showed that all 11 had re-activated Bcr-Abl signaling rather than acquiring Bcr-Abl-independent growth.106

Further analysis of clinical material led to the discovery of a number of mutations in Bcr-Abl, including three (T351I and Y255F/H) that are not significantly inhibited by imatinib.107 Of the known mutations, six (T315I, Y235F/H, E255V/K, and M351T) occur in 60% of blast phase patients, with a mutational frequency of 30-90%. The functional consequence of these mutations is highly variable; many mutated kinases remain effectively inhibited by imatinib (e.g., E355G and M244V). The significance of mutations such as T315I and E255Vis that they cause a greater than 200-fold decrease in biochemical sensitivity to imatinib.

7.08.5.2.5 Future directions

While imatinib has revolutionized the treatment of CML, there remains a real need to identify second-generation compounds that target key Bcr-Abl mutations. As many of the mutations seem to prevent the kinase adopting the specific inactive form that imatinib binds to, there is renewed interest in inhibitors that target activated Bcr-Abl.108 A number of mixed Bcr-Abl and Src kinase inhibitors are in early clinical development, and are showing promising results, although inhibition of the T315I mutated form (the 'gate keeper' residue) remains challenging.108 Recent publications from various groups have described the activity of compounds such as PD166326 (6) ( ),

PD180970 (7),111 AP23464 (8),112 and AG1024 (39)113 (see Figure 9) against imatinib-resistant cell lines. BMS354825 (9) (Sprycell, Dasatanib) is a mixed Src:Brc-Abl kinase inhibitor which shows good in vivo activity against all Bcr-Abl mutants other than T315I and has recently been approved for the treatment of CML.114'115 Recent publications from the Novartis group have described the activity of AMN107 (10), which is structurally related to imatinib (1) but has activity against a number of the mutated forms of Bcr-Abl, although it too is inactive against the T315I form.116-117

7.08.5.3 Src Family Kinases 7.08.5.3.1 Src family kinases in cancer

Identification of the Rous sarcoma virus (RSV) in chickens demonstrated that some cancers could be of infectious rather than endogenous in origin.118-121 In 1978, v-Src (the protein product of the v-src gene)122,123 was shown to be a protein tyrosine kinase.124-126 In parallel, a normal gene homolog, c-src, had been identified,127 suggesting that the c-src sequence had been captured by the virus through recombination at both sides of the c-src gene.128 It is now known that c-src is a proto-oncogene,129 and that its reduced transforming ability, compared with v-src, is due to the presence of a C-terminal regulatory domain (absent from v-src).130 Phosphorylation of Tyr527 within the regulatory domain of c-Src, by Csk,131 acts to modulate Src activity.132

The Src family of non-RTKs currently consists of nine members (Src, Yes, Fgr, Yrk, Fyn, Lyn, Hck, Lck, and Blk), all of which share a common structural motif. The molecules comprise an N-terminal myristoylation sequence, a proline-rich linker region, SH3 and SH2 protein interaction domains, a kinase domain, and a C-terminal regulatory domain.133 The crystal structures of human Src,134 chicken Src,135 and Hck136'137 have been published, and show that the SH2 and SH3 domains lie at the back of the kinase domain, with the SH2 domain interacting with phosphotyrosine 527. This interaction, along with an interaction between the SH3 domain and the polyproline linker (which forms a type II helix), maintains the kinase in an inactive form, and explains how the SH2 and SH3 domain ligands activate Src family kinases.138

Increased Src activity has been demonstrated in prostate,139 ovarian,140 breast,141 and colorectal cancers,142 with some studies suggesting that Src plays a significant role in invasion and metastasis, with the kinase often highly expressed in metastatic tissue.143 The only normal tissues with high levels of Src are platelets, neural cells, and osteoclasts; gene knockout studies in mice are characterized by osteopetrosis.144 In cancer cells, Src kinase activity is localized to the cell membrane, and is associated with adhesion and cytoskeletal changes, which promote a motile, invasive phenotype.145 Src activity is critical in the turnover of focal adhesions,146 again promoting cell motility. With clear links between Src and the well-known growth factor signaling pathways,147 it may be surprising that a clear link between elevated Src kinase activity and a proliferative cellular phenotype has not yet been made.129

7.08.5.3.2 Src family kinase inhibitors

Among the earliest Src family inhibitors were the pyrrolo[2,3-d]pyrimidines,148 exemplified by Pfizer's PP1 (11) and PP2 (12), which show nanomolar potency against Lck and Fyn (Figure 4).149 Starting with the related

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