AP24534 is a next-generation ABL kinase inhibitor optimized using structure-based drug design to bind to the inactive, DFG-out conformation of ABL and ABLT315I. The key structural feature of the molecule is a carbon-carbon triple bond linkage that makes productive hydrophobic contact with the side chain of I315, allowing inhibition of the T315I mutant. The triple bond also acts as an inflexible connector that enforces correct positioning of the two binding segments of AP24534 into their established binding pockets. AP24534 maintains an extensive hydrogen-bonding network and occupies a region of the kinase that overlaps significantly with the imatinib binding site.
A key design feature of AP24534 underlying its pan-BCR-ABL inhibitor profile is incorporation of multiple contact points to confer very high potency and to balance and distribute the overall binding affinity. While each of the hydrogen-bonding and contact residue interactions contribute substantially to the inhibitor’s affinity for its target, mutation-based disruption of one element of the binding network or distortion of a sub-region within the binding pocket results in only a slight reduction in affinity. As a consequence, AP24534 also retains potency against other imatinib-resistant ABL mutants in addition to ABLT315I. While mutations that destabilize the inactive conformation of ABL to which AP24534 binds, including T315I and E255V, result in modest reductions in binding affinity, substantial reductions would be expected to require at least two changes at non-proximal residues – a prediction consistent with findings from our mutagenesis screen.
Kinase selectivity studies showed that AP24534 does not inhibit Aurora kinases, clearly distinguishing it from other T315I inhibitors in development. These studies also revealed inhibition of SRC, LYN, PDGFRα, and c-KIT with <10-fold selectivity compared to ABLT315I
. Several of these kinases are important clinical targets of imatinib, nilotinib and/or dasatinib, although only dasatinib has been reported to inhibit SRC family kinases. Although assay differences preclude direct comparison of the kinase profiles of AP24534 and dasatinib, a comprehensive kinase interaction map for dasatinib was recently reported (Karaman et al., 2008
). In general, the linearity of the triple bond in AP24534 is predicted to minimize steric clash between the inhibitor and hydrophobic gatekeeper residues. This feature probably contributes to the relatively broad kinase specificity profile of AP24534, which includes VEGFR and FGFR family kinases, receptors not inhibited by the three currently approved BCR-ABL drugs. As SRC, VEGFR, FGFR, and PDGFR family kinases are potential targets in a variety of other malignancies, this supports the potential testing of AP24534 in a wider range of cancers.
Evaluation of AP24534 in cellular proliferation assays confirmed its potent pan-BCR-ABL inhibition against cells expressing native or mutant BCR-ABL, including BCR-ABLT315I
, while retaining a high degree of selectivity (>1000-fold) for Ph-positive cells. Among the BCR-ABL mutants tested, the E255V mutant, which confers high-level resistance to imatinib and intermediate-level resistance to nilotinib and dasatinib (O’Hare et al., 2007
), was most resistant to AP24534. Notably, AP24534 potently inhibited mutants at residues Y253 and F359 (which have been reported in patients failing nilotinib (Cortes et al., 2007
; Kantarjian et al., 2007
)), as well as F317 (implicated in clinical resistance to dasatinib (Burgess et al., 2005
; Cortes et al., 2007
; Khorashad et al., 2008
; Shah et al., 2007
; Talpaz et al., 2006
)). While clinically achievable and effective doses will need to be determined, the sizeable selectivity for BCR-ABL-expressing cells (regardless of mutational status) over normal cells suggests the potential for efficacy with minimal toxicity.
In clinical studies of BCR-ABL inhibitors, pharmacodynamic evaluation of target inhibition is an important component of dose optimization. In the preclinical studies reported here we monitored phosphorylation of CrkL, a direct substrate of native and mutant BCR-ABL, by immunoblot analysis. In both Ba/F3 cells and primary CML BCR-ABLT315I
cells, treatment with AP24534 resulted in a marked reduction in phosphorylated CrkL, while imatinib, dasatinib, and nilotinib had no effect. This assay was recently used to monitor BCR-ABL activity in patients treated with nilotinib; values of percent phosphorylated CrkL from serially collected peripheral blood samples were consistent with BCR-ABL kinase domain mutation status and matched closely with other measures of response, including BCR-ABL transcript levels and white cell counts (La Rosee et al., 2008
). Given its extensive validation in the clinic, this assay is being employed to monitor the pharmacodynamic effects of AP24534 in its phase 1 evaluation.
The oral bioavailability of AP24534 was confirmed in mouse pharmacology studies, where concentrations above the IC50s for all tested mutants could be safely sustained following daily oral dosing. AP24534 demonstrated potent activity after daily oral administration in a series of mouse models of CML driven by native BCR-ABL or BCR-ABLT315I. In a survival model using Ba/F3 cells expressing native BCR-ABL, AP24534 significantly prolonged survival at low doses of 2.5 and 5 mg/kg and demonstrated similar efficacy to dasatinib. In an analogous model using BCR-ABLT315I cells, AP24534 significantly extended survival whereas dasatinib, as expected, was inactive. AP24534 was also active in a subcutaneous BCR-ABLT315I tumor model, where tumor stasis or regression occurred at doses of 30 and 50 mg/kg, and suppression of BCR-ABL signaling was demonstrated using the shift CrkL phosphorylation assay. AP24534 was well tolerated at all dose levels used in these studies. Thus, AP24534 is orally bioavailable, inhibits its molecular target, and has a wide therapeutic range (5–50 mg/kg) in BCR-ABLT315I dependent CML animal models.
Mutation-mediated resistance to clinical ABL inhibitors is the main route of BCR-ABL signaling reactivation, particularly in chronic phase disease. As AP24534 advances into clinical evaluation, anticipating potential resistance liabilities, especially compared to those of nilotinib and dasatinib, will be important for prospective treatment decisions. Several mutations have been reported in association with clinical resistance to nilotinib (L248V, Y253F/H, E255K/V, T315I, F359C/V; (Cortes et al., 2007
; Kantarjian et al., 2007
)) or dasatinib (V299L, T315A/I, F317I/L; (Cortes et al., 2007
; Khorashad et al., 2008
; Shah et al., 2007
)) which are largely consistent with our in vitro profiling (Bradeen et al., 2006
). In our accelerated mutagenesis screens for AP24534, we found a concentration-dependent reduction in both the percentage of wells with outgrowth and in the range of mutations observed. Although at 10 nM AP24534 we observed 16 different substitutions across 13 different residues, the only resistant subclones recovered at 20 nM harbored either a T315I or E255V mutation, and at 40 nM AP24534 and above complete suppression of outgrowth was observed. Depending on achievable plasma levels, our data suggest that AP24534 may have the potential to overcome single-mutation-based resistance in the clinical setting. This result has been previously achieved in this assay only with combinations of nilotinib or dasatinib and a pre-clinical T315I inhibitor (O’Hare et al., 2008
). To our knowledge no other ABL kinase inhibitor has been shown to have this potential as a single agent.
As compound (multiple) mutations of BCR-ABL represent a rare but challenging scenario clinically, we carried out additional accelerated mutagenesis screens starting with cells expressing either of the two individually most resistant mutants, BCR-ABLT315I
. This predictive assay implicated certain compound mutations, especially those involving any two of Y253H, E255V, and T315I in moderate to high-level resistance to AP24534. Among these, Y253H/T315I and E255V/T315I are predicted to be the most resistant pairings, although high concentrations of AP24534 still prevented these mutations emerging. Thus, AP24534 has the capability to eliminate compound mutations involving T315I and E255V predicted to be highly resistant to all other inhibitors. Currently, the number of clinically documented compound mutations within the kinase domain of BCR-ABL associated with treatment failure is low (Table S5
). Nonetheless, they represent a formidable problem for those patients harboring them, and incidence may increase with the prolonged survival of CML patients and with more patients undergoing sequential ABL kinase inhibitor treatment (Shah et al., 2007
). Overall, although no mutagenesis screen can be completely exhaustive, our data indicate AP24534 has the potential to address this currently unmet clinical issue.
Our pre-clinical profiling indicates that AP24534 has potential as an important option for controlling resistance in CML. The combined results of our biochemical, cell-based, and in vivo studies suggest that AP24534 exhibits sufficient activity against native BCR-ABL and all tested BCR-ABL mutants to warrant consideration for single-agent use as a pan-BCR-ABL inhibitor. Moreover, our results indicate that AP24534 holds promise for controlling compound mutants involving T315I, while raising awareness that it is advantageous to eliminate resistant subclones at the single-mutation stage. In the longer term, this may advocate for the potential future use of a pan-BCR-ABL inhibitor such as AP24534 in a first-line therapeutic capacity.
Clinical use of a pan-BCR-ABL inhibitor active against T315I could make long-term remissions an achievable goal at least for some patients with advanced CML. A phase 1 clinical trial evaluating oral AP24534 in patients with refractory CML and other hematologic malignancies is ongoing (NCT00660920, www.clinicaltrials.gov