Kinases are involved in a broad range of essential cellular functions, and are regulated in vivo by adopting different conformations. Disruption of kinase conformational regulation through protein fusions, deletions, or missense mutations can lead to pathologic conditions, including cancer. We have initiated a broad-based approach to kinase inhibition through identification of key amino acid residues that are critical for the fluxing of kinases between conformational states, and the design of inhibitors that bind to such residues.
In ABL1, R386 is a critical anchoring partner for stabilizing pY393 in the active conformation. In the inactive Type II conformation, R386 and Y393 separate from each other, with Y393 moving to occupy the substrate pocket, and R386 shifting to the interior of the kinase towards E282 (Figure S1C
). This alternative disposition of E282/R386 provides a basis for rational inhibitor design. Here, we have demonstrated the utility of this E282/R386 anchor for design of ABL1 inhibitors, culminating in the discovery of DCC-2036. Prototype inhibitor compounds 1
(), which orient a THIQ or quinoline basic ring system toward the E282-R386 inhibitory anchor, exhibited potencies in the 57-140 nM range for ABL1native
, whereas close structural analogs lacking functionality for interaction with E282 or R386 were inactive (Figure S1E
). It is noteworthy that this level of inhibition, which is equivalent to the potency of imatinib for ABL1native
, is achieved without extending inhibitor binding into the ATP hinge region. Additionally, compounds 1
also exhibit potencies in the 700 nM range for the gatekeeper mutant ABL1T315I
. A similar level of potency was realized by compound 3
, which anchors into the ATP hinge but does not extend to the switch E282-R386 salt bridge.
Combining both an ATP hinge binding anchor and an E282/R386 binding anchor within a single inhibitor led to compound 4 and DCC-2036, which exhibit much greater potency vs. ABL1native (IC50 of 9 and 0.8 nM, respectively) and against mutant ABL1T315I (17 and 4 nM, respectively). While compound 3 is equipotent against ABL1native and ABL1T315I and must therefore avoid steric clash with substitutions at the gatekeeper residue, the ability to bind both the ATP hinge and the switch control regions is what affords DCC-2036 the potency (100-fold decrease in ABL1native IC50 vs. compound 3) required for a clinical drug candidate. A derivative of DCC-2036 lacking the 2-fluorine substituent on the phenyl ring maintained full potency for inhibiting ABL1native and ABL1T315I (IC50 1 nM and 7 nM, respectively), demonstrating that the orientation of this fluorine is not critical for inhibition of the gatekeeper mutant by DCC-2036 (data not shown). The structure of DCC-2036 with u-ABL1native also revealed an ideal hydrogen bond (2.76 Å) between the carboxylate side chain of E282 and the guanidinium side chain of R386. We speculate that this Type II switch state E282/R386 hydrogen bond induced by DCC-2036 contributes to its high potency vs. u-ABL1native (IC50 0.8 nM).
DCC-2036’s mode of binding provides a durable mode of Type II inhibition that affords a prolonged off-rate, resiliency to high ATP concentrations, and overcomes the clinical problem of conformational escape resistance by making interactions within the Type II state () that override the effect of point mutations that drive ABL1 towards the active Type I state. We refer to this type of inhibition as conformational control inhibition
. DCC-2036 inhibits both ABL1T315I
values of 4 nM and 1.4 nM, respectively), which are known to exist predominantly in the Type I active conformation (Azam et al., 2008
; Young et al., 2006
). Structural data () suggest that DCC-2036 may, in part, lead to conformational control of ABL1 by invoking an inhibitor-participating hydrophobic spine (Azam et al., 2008
; Kornev et al., 2006
) in which a portion of DCC-2036 substitutes for phenylalanine in the third spine position while also making interactions with the second and fourth spine residues, M290 and H361 ().
DCC-2036 exhibits high potency against cell lines expressing mutant forms of BCR-ABL1, including T315I and H396P, that collectively account for more than 85% of TKI-resistant CML patients in whom ABL1 kinase mutations are identified (Shah et al., 2002
; Shah et al., 2007
). ABL1 mutants involving the P-loop residue E255 (E255V/K) were relatively less sensitive to DCC-2036 (), and are also refractory to the type II inhibitors imatinib and nilotinib. In the native ABL1 structure, the acidic side chain of E255 makes an electrostatic interaction with a lysine residue on the other arm of the loop, and disruption of this interaction might distort the nearby ATP hinge region into which DCC-2036 binds. A Ba/F3 cell-based mutagenesis screen for resistance to DCC-2036 recovered no BCR-ABL1 mutations at higher drug concentrations, while mutations at Y253 and E255 emerged at lower concentrations (T. O’Hare, personal communication). However, pharmacokinetic data from the phase 1 clinical trial have demonstrated that DCC-2036 can achieve plasma levels well above these concentrations (data not shown). There were no mutations recovered that are known to destabilize the inactive conformation of BCR-ABL1 towards its active, Type I conformation (Azam et al., 2003
; Sherbenou et al., 2010
), nor were there mutations identified in the switch control amino acids E282/R386, which may be required for assembly of the catalytically active state (Kornev et al., 2006
). Together, these observations suggest that acquired resistance to DCC-2036 therapy in CML may be less frequent than for the three ATP-competitive ABL1 inhibitors in current clinical use.
DCC-2036 inhibits BCR-ABL1, STAT5, and CrkL phosphorylation in BCR-ABL1-expressing cells. In a Ba/F3 cell allograft model, oral administration of DCC-2036 resulted in sustained inhibition of phosphorylation of both BCR-ABL1T315I and STAT5, a significant reduction in leukemic burden, and prolonged survival (). Oral administration of DCC-2036 at doses of 60-100 mg/kg/d prolonged survival and reduced circulating leukemia cells in physiologically relevant mouse models of CML-like myeloproliferative neoplasia and Ph+ B-cell acute lymphoblastic leukemia induced by BCR-ABL1T315I () but had no effect on hematopoiesis in normal mice (data not shown). Whereas plasma concentrations of DCC-2036 exceeding 50 μM were observed in recipient mice, after accounting for protein binding (97-99%) the active in vivo drug concentrations were in the range of 100-1000 nM (data not shown). Tested against primary patient cells in vitro, DCC-2036 suppressed Ph+ myeloid colony formation and inhibited BCR-ABL1T315I kinase activity and phosphorylation of STAT5 and CrkL at these concentrations but did not significantly inhibit growth of normal BM progenitors at concentrations up to 2 μM (). Together, these results demonstrate a differential inhibitory effect of DCC-2036 against BCR-ABL1-expressing vs. normal hematopoietic cells at clinically relevant concentrations.
Based on its composite properties and these positive preclinical results, DCC-2036 was selected for clinical development. Correlative studies from the Phase 1 clinical trial of DCC-2036 have demonstrated sustained inhibition of phospho-BCR-ABL1, phospho-STAT5, and phospho-CrkL in refractory CML patients. In summary, DCC-2036 represents a potential therapeutic option for patients with Ph+ leukemia who have relapsed on or are refractory to conventional TKIs. Exploiting the diversity of switch control mechanisms among different kinases is a promising strategy to develop molecularly targeted therapies for hematologic neoplasms, solid tumors, and non-malignant diseases.