Myristoyl Binding Pocket Research Articles

Elvn-001, a Next Generation ATP-Competitive ABL1 Tyrosine Kinase Inhibitor for the Treatment of Chronic Myeloid Leukemia

Of the six small-molecule ABL1 tyrosine kinase inhibitors (TKIs) that have received FDA-approval for the treatment of BCR-ABL1-driven Chronic Myeloid Leukemia (CML), five (Imatinib, Nilotinib, Dasatinib, Ponatinib, Bosutinib) target the ATP-binding site in the kinase domain. While each of these TKIs provides added value to the arsenal of treatments for CML, these agents suffer from selectivity issues, inhibiting a number of off-target kinases that drive an array of dose-limiting toxicities (DLTs) which constrain their ability to fully engage ABL1 and drive maximal efficacy. In addition, the emergence of on-target resistance against these TKIs, such as the highly prevalent T315I mutation at the gatekeeper position of the active site, is common and represents a significant clinical challenge. The recently approved ABL1 TKI Asciminib is an allosteric inhibitor that interacts with the unique myristoyl binding pocket of ABL1 and therefore possesses high kinome selectivity and an improved tolerability profile. Despite its unique binding mode, clinical activity against the T315I mutant requires a dose five times higher than that approved for the broader third-line or greater patient population, which leads to issues with tolerance. In addition, emergent on-target resistance remains a challenge, particularly with respect to mutations within the myristoyl pocket as well as select active site mutants. Here we report the discovery of ELVN-001, a potent ATP-competitive small-molecule ABL1 TKI which, unlike the majority of currently approved ABL1 TKIs, is highly selective in both biochemical and cell-based assays versus the kinome in general as well as the key off-target kinases KDR, c-KIT, PDGFR and SRC, which are associated with a variety of DLTs including myelosuppression, edema, pleural effusions, hypertension, and certain cardiovascular events. ELVN-001 exhibited potent pharmacodynamic (phosphorylated CRKL) and anti-proliferative activity in vitro against BCR-ABL1-driven CML cell lines with comparable or superior activity to the approved therapies. Evaluation of ELVN-001 against Ba/F3 cell lines expressing a panel of clinically relevant BCR-ABL1 TKI-resistant mutants demonstrated activity of ELVN-001 against many of these mutants, including T315I as well as two myristoyl pocket mutants (A334P, P465S) which are highly resistant to asciminib. Cell-based mutagenesis screens using Ba/F3 BCR-ABL1 cells confirmed a concentration-dependent reduction in outgrowth of resistant clones, which generally narrowed to a select subset of P-loop mutations. Consistent with previously reported findings for ponatinib, while ELVN-001 showed little to no activity against cells expressing a subset of T315I-inclusive BCR-ABL1 compound mutants, the addition of Asciminib resulted in varying degrees of re-sensitizing these mutants to ELVN-001. Importantly, ELVN-001 possessed a favorable absorption, distribution, metabolism, elimination, and toxicity profile, as determined from both in vitro and in vivo studies, that readily translated into favorable pharmacokinetic properties in all preclinical species evaluated. Accordingly, administration of doses of ELVN-001 corresponding to well-tolerated free drug exposures in rats and non-human primates readily translated to mouse in vivo tumor xenograft models of efficacy, including a model of T315I CML. These anti-tumor activities also compared favorably to currently approved therapies when evaluated head-to-head in these studies. Together, these findings support ELVN-001 as an extremely selective, novel ATP-competitive inhibitor of ABL1 kinase with superior pharmacokinetic and tolerability properties compared to currently approved TKIs and which retains activity against T315I and other key on-target resistance mutations, warranting its further investigation in patients with TKI-resistant/intolerant CML.

Imatinib disassembles the regulatory core of Abelson kinase by binding to its ATP site and not by binding to its myristoyl pocket.

It was recently reported (Xie et al., 2022) that the Abelson tyrosine kinase (Abl) ATP-site inhibitor imatinib also binds to Abl's myristoyl binding pocket, which is the target of allosteric Abl inhibitors. This was based on a crystal structure of a truncated Abl kinase domain construct in complex with imatinib bound to the allosteric site as well as further isothermal titration calorimetry (ITC), NMR, and kinase activity data. Although imatinib's affinity for the allosteric site is significantly weaker (10 M) than for the ATP site (10 nM), imatinib binding to the allosteric site may disassemble the regulatory core of Abl, thereby stimulating kinase activity, in particular for Abl mutants with reduced imatinib ATP-site affinity. It was argued that the previously observed imatinib-induced opening of the Abl regulatory core (Skora et al., 2013; Sonti et al., 2018) may be caused by the binding of imatinib to the allosteric site and not to the ATP site. We show here that this is not the case but that indeed imatinib binding to the ATP site induces the opening of the regulatory core at nanomolar concentrations. This agrees with findings that other type-II ATP-site inhibitors (nilotinib, ponatinib) disassemble the regulatory core despite demonstrated negligible binding to the allosteric site.

Development of asciminib, a novel allosteric inhibitor of BCR-ABL1.

Chronic myeloid leukemia (CML) is driven by a translocation event between chromosomes 9 and 22, leading to the formation of a constitutively active BCR-ABL1 oncoprotein. Approved tyrosine kinase inhibitors (TKIs) for CML inhibit BCR-ABL1 by competitively targeting its adenosine triphosphate (ATP)-binding site, which significantly improves patient outcomes. However, resistance to and intolerance of TKIs remains a clinical challenge. Asciminib is a promising investigational agent in development that allosterically targets BCR-ABL1 in a non-ATP-competitive manner. It binds to the ABL1 myristoyl-binding pocket and is effective against most ABL1 kinase domain mutations that confer resistance to ATP-competitive TKIs, including the T315I mutation. This review discusses unmet needs in the current CML treatment landscape, reports clinical data from asciminib trials that support the use of single-agent asciminib as third-line therapy and beyond, and explores the potential benefit of asciminib in combination with approved TKIs in earlier lines.

Exploiting LY3009120 and Asciminib Combination to Target TKI-Resistant CML

The oncogenic BCR-ABL1 tyrosine kinase is the driver of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). Tyrosine kinase inhibitors (TKIs) targeting ABL kinase are generally effective, but subsets of patients treated with single-agent TKIs develop resistance due to mutations in BCR-ABL1 that impair TKI binding. We have previously reported that BCR-ABL1 compound mutants (exhibiting two mutations within the same BCR-ABL molecule) that include the T315I gatekeeper mutation confer a high degree of resistance to all clinical ABL TKIs used as single agents, including ponatinib and the allosteric inhibitor asciminib. However, combining asciminib with ponatinib provides an effective strategy for overcoming compound mutation-based resistance (Eide et al. Cancer Cell 2019). As the clinical utility of ponatinib is limited by cardiovascular toxicity, including arterial occlusive events (AOEs), we decided to search for alternative molecules for use in combination with asciminib.To identify functional ponatinib analogs, we performed Quantum Similarity Modeling (QSM) on the reported crystal structure of T315I mutant ABL1 kinase in complex with nilotinib and asciminib (5MO4) (Wylie et al. Nature 2017) to search for other molecules. Compared to conventional computational modeling, QSM identifies novel classes of structurally distinct compounds that are comparable on a quantum level by precisely defining their interaction with the target. Affinity inferred by close complementarity with the shared ligand-protein surface in the region of the surveyed binding site is mapped, using multiple weak local associations. Our in silico QSM platform combines quantum methods with machine learning to investigate extensive chemical spaces. We screened several million compounds against BCR-ABL1 and identified 51 potential candidates predicted effectively to block T315I mutant BCR-ABL1 when combined with asciminib. To prioritize potent and non-toxic drug combinations for further development against compound mutants, we initially profiled all 51 compounds for their efficacy against Ba/F3 BCR-ABL T315I cells, alone and in combination with asciminib (1 nM). Of 51 compounds, LY3009120, a pan-RAF inhibitor that is currently in phase I clinical development for advanced solid malignancies (Sullivan et al. Mol Cancer Ther 2020), showed strong activity against BCR-ABL T315I when combined with asciminib. These data provided proof of principle for the QSM approach.We next tested the efficacy of all 51 candidates ± asciminib against Ba/F3 cells harboring T315I-inclusive BCR-ABL1 compound mutants, including Y253H/T315I, E255V/T315I, H396R/T315I, G250E/T315I, and T315L as the most resistant mutants. Neither single agent showed any effect. However, LY3009120 strongly inhibited BCR-ABL1 compound mutants when combined with asciminib. No toxicity was observed against Ba/F3 parental cells, confirming that the effects of the combinations are mediated by inhibition of BCR-ABL1. Synergy quantification of the dose-response matrix for the LY3009120/asciminib combination using the Zero Interaction Potency model demonstrated highly synergistic interactions (Synergy score > 10) between the two inhibitors. To directly assess the binding affinity of LY3009120 to the ABL1 kinase domain, we used the cell-based NanoBRET intracellular ABL1 kinase assay on HEK-293 cells expressing luciferase-tagged ABL1. The NanoBRET assay uses energy transfer to quantify the affinity of test compounds by competitive displacement of a cell-permeable fluorescent tracer that is reversibly bound to an ABL1-luciferase fusion protein. We found that LY3009120 competes off the fluorescent tracer at a low micromolar range (EC 50 = 0.75 μM), confirming direct binding of LY3009120 to the kinase domain of ABL1. We hypothesize that the binding of LY3009120 to the ABL1 kinase domain induces a conformational change that re-establishes asciminib binding to the myristoyl binding pocket, allowing for synergy. Studies to quantify the binding affinity of LY3009120 and asciminib to BCR-ABL1 mutants are underway, and data will be presented.In summary, our findings validate QSM as a novel in silico approach to identify TKI combinations. Combining LY3009120 with asciminib may be an effective, low-risk strategy to target BCR-ABL1 compound mutants in patients with clinical TKI resistance. DisclosuresDeininger: SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Novartis: Consultancy, Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding.

Pathogenic variants causing ABL1 malformation syndrome cluster in a myristoyl-binding pocket and increase tyrosine kinase activity

ABL1 is a proto-oncogene encoding a nonreceptor tyrosine kinase, best known in the somatic BCR-ABL fusion gene associated with chronic myeloid leukaemia. Recently, germline missense variants in ABL1 have been found to cause an autosomal dominant developmental syndrome with congenital heart disease, skeletal malformations and characteristic facies. Here, we describe a series of six new unrelated individuals with heterozygous missense variants in ABL1 (including four novel variants) identified via whole exome sequencing. All the affected individuals in this series recapitulate the phenotype of the ABL1 developmental syndrome and additionally we affirm that hearing impairment is a common feature of the condition. Four of the variants cluster in the myristoyl-binding pocket of ABL1, a region critical for auto-inhibitory regulation of the kinase domain. Bio-informatic analysis of transcript-wide conservation and germline/somatic variation reveals that this pocket region is subject to high missense constraint and evolutionary conservation. Functional work to investigate ABL1 kinase activity in vitro by transient transfection of HEK293T cells with variant ABL1 plasmid constructs revealed increased phosphorylation of ABL1-specific substrates compared to wild-type. The increased tyrosine kinase activity was suppressed by imatinib treatment. This case series of six new patients with germline heterozygous ABL1 missense variants further delineates the phenotypic spectrum of this condition and recognises microcephaly as a common finding. Our analysis supports an ABL1 gain-of-function mechanism due to loss of auto-inhibition, and demonstrates the potential for pharmacological inhibition using imatinib.

Bcr-Abl Allosteric Inhibitors: Where We Are and Where We Are Going to.

The fusion oncoprotein Bcr-Abl is an aberrant tyrosine kinase responsible for chronic myeloid leukemia and acute lymphoblastic leukemia. The auto-inhibition regulatory module observed in the progenitor kinase c-Abl is lost in the aberrant Bcr-Abl, because of the lack of the N-myristoylated cap able to bind the myristoyl binding pocket also conserved in the Bcr-Abl kinase domain. A way to overcome the occurrence of resistance phenomena frequently observed for Bcr-Abl orthosteric drugs is the rational design of allosteric ligands approaching the so-called myristoyl binding pocket. The discovery of these allosteric inhibitors although very difficult and extremely challenging, represents a valuable option to minimize drug resistance, mostly due to the occurrence of mutations more frequently affecting orthosteric pockets, and to enhance target selectivity with lower off-target effects. In this perspective, we will elucidate at a molecular level the structural bases behind the Bcr-Abl allosteric control and will show how artificial intelligence can be effective to drive the automated de novo design towards off-patent regions of the chemical space.

CML-121: Efficacy and Safety of Asciminib in Heavily Pretreated Patients with Philadelphia Chromosome-Positive (Ph+) Chronic Myeloid Leukemia in Chronic Phase (CML-CP) with Sensitivity to Tyrosine Kinase Inhibitors (TKIs)

Context There is a need for new therapies that will improve molecular responses in patients with Ph+ CML lacking deep response to TKIs. Asciminib is a novel, specific BCR-ABL1 inhibitor that binds the myristoyl-binding pocket and is expected to result in improved safety/tolerability vs. available ATP-binding TKIs. Objective To evaluate the safety and efficacy of asciminib monotherapy in heavily pretreated patients with baseline BCR-ABL1IS ≤ 1%. Design A phase I, dose-escalation and -expansion study for asciminib monotherapy or combination therapies ( NCT02081378 ). Patients Eligible adults with Ph+ CML in CP or accelerated phase who were relapsed/refractory to or intolerant of ≥2 TKIs were enrolled. In the current analysis, patients in the monotherapy cohorts (any dose) with BCR-ABL1IS ≤ 1%, regardless of the reason for TKI discontinuation, without a T315I mutation at baseline were included. Results Of 48 patients with CML-CP in 9 dose cohorts; 42 (87.5%) are still on treatment and 36 (75.0%) achieved major molecular response (MMR) or better at the data cutoff (30 Aug 2019). The most common grade 3/4 AEs (>10%), regardless of study drug relationship, were lipase increase (27.1%) and hypertension (12.5%). Serious AEs, regardless of study drug relationship, occurred in 16/48 pts (33.3%), with myocardial infarction being the most common (n=2; 4.2%). Among patients without MMR, MR4, or MR4.5 at baseline, 18/24 (75.0%), 16/38 (42.1%), and 18/42 (42.9%) achieved MMR, MR4, and MR4.5, respectively. All 18 patients who achieved MMR maintained MMR or better for ≥2 y, except 3 whose BCR-ABL1IS fluctuated around 0.1% but who were still in MMR at ≈ 60 mo. Among the 6 patients who did not achieve MMR by the cutoff date, 1 had BCR-ABL1IS > 1%, and 5 remained between BCR-ABL1IS 0.1% and 1%. Among patients who achieved MR4 or MR4.5, 9/16 (56.3%) and 10/18 (55.6%), respectively, maintained the response for ≥2 y. Conclusion Asciminib monotherapy was well tolerated and efficacious in patients with baseline BCR-ABL1IS ≤ 1%. Asciminib should be further investigated in patients lacking optimal outcomes and in those that have discontinued TKIs. Study sponsored by Novartis Pharmaceuticals Corporation. This abstract was accepted and previously published at the 25th EHA Annual Congress.

Combining the Allosteric ABL1 Inhibitor Asciminib (ABL001) with Ponatinib Suppresses Emergence of and Restores Efficacy Against Highly Resistant BCR-ABL1 Compound Mutants

Combining the Allosteric ABL1 Inhibitor Asciminib (ABL001) with Ponatinib Suppresses Emergence of and Restores Efficacy Against Highly Resistant BCR-ABL1 Compound Mutants

Combining the Allosteric Inhibitor Asciminib with Ponatinib Suppresses Emergence of and Restores Efficacy against Highly Resistant BCR-ABL1 Mutants

BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph+) leukemia is effectively managed with several approved drugs, including ponatinib for BCR-ABL1T315I-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants. We demonstrate that combining asciminib with ATP site TKIs enhances target inhibition and suppression of resistant outgrowth in Ph+ clinical isolates and cell lines. Inclusion of asciminib restores ponatinib's effectiveness against currently untreatable compound mutants at clinically achievable concentrations. Our findings support combining asciminib with ponatinib as a treatment strategy for this molecularly defined group of patients.

S126 COMBINATION OF ASCIMINIB (ABL001) WITH ATP‐COMPETITIVE TYROSINE KINASE INHIBITORS TARGETS EARLY CML PROGENITOR CELLS.

Background:The BCR‐ABL1 chimeric oncoprotein drives chronic myeloid leukaemia (CML) pathogenesis. The introduction of tyrosine kinase inhibitors (TKIs) has transformed clinical outcomes for patients with CML, with over 80% of those treated with imatinib (IM) surviving for more than 10 years. Second and third‐generation TKIs are more potent and can impede the emergence of resistance, inducing deep molecular responses in both untreated and IM‐resistant CML patients. Despite the success of TKIs, up to 35% of patients discontinue their TKI due to intolerance or develop resistance and this remains a key area of unmet clinical need. Asciminib (formerly ABL001) is a potent and selective allosteric inhibitor of ABL1 that binds to the myristoyl binding pocket of ABL1 to hold BCR‐ABL1 in an inactive conformation. Preclinical studies have shown that asciminib selectively inhibits the growth of BCR‐ABL1 positive (+) cells regardless of the presence of BCR‐ABL1 point mutations1. Clinical trials for patients with CML or Ph+ acute lymphoblastic leukaemia, testing asciminib alone and in combination with TKIs are currently underway and preliminary results are promising.Aims:To assess if dual inhibition of BCR‐ABL1 leads to improved treatment outcomes in preclinical studies.Methods:Here, we assess the effects of asciminib, alone and in combination with ATP‐competitive TKIs (IM, nilotinib (NIL) and ponatinib (PON)) in CML cell lines and primary CD34+ chronic phase (CP)‐CML stem/progenitor cells (HSPC). We assessed synergy using resazurin readouts using CompuSyn Software. We performed cell counts, apoptosis, cell‐cycle and proliferation assays to determine the effect of drug combinations in CML cell lines and primary samples; and confirmed effects on primitive cells using colony‐forming cell (CFC) and long‐term culture‐initiating cell (LTC‐IC) assays in vitro.Results:Dose‐response studies using the resazurin assays in CML cell lines (Bv173, K562, KCL22) indicate that asciminib is potent at low nanomolar concentration, even in cells that express the BCR‐ABL1 T315I point mutation (KCL22). Apoptosis and cell cycle assays’ assessed by FACS showed that the inhibitory effects of asciminib were maintained in KCL22T315I‐expressing cells when asciminib was used in combination with PON. Washout studies with asciminib in KCL22WT/T315I‐expressing cells demonstrated a prolonged phenotypic response using low‐nanomolar doses of asciminib as the cells failed to regrow and had irreversible cell‐cycle damage.Primary CD34+ CML HSPCs demonstrated proliferation arrest and increased apoptosis (70–100% increase relative to control; p < 0.001) when treated for up to 72 hours with asciminib, alone and in combination with IM or NIL. LTC‐IC and CFC assays, determining the functional activity of primitive CML HSPCs in vitro, demonstrated that the combination of asciminib with IM or NIL reduced colony outputs (60–90% decrease relative to controll, p < 0.001), beyond that achieved with each drug alone (40–80% relative to control, p < 0.001), and in separate experiments, with minimal effect on normal HSPC.Summary/Conclusion:These results suggest that asciminib represents a novel therapeutic approach with effects on primitive CP‐CML HSPCs both as a single agent and in combination with TKI and has efficacy in cells expressing the multi‐TKI resistant T315I mutation. We are now investigating the mechanism of action for asciminib, alone and in combination with NIL, by RNA‐sequencing.

Identification and characterization of activating ABL1 1b kinase mutations: impact on sensitivity to ATP-competitive and allosteric ABL1 inhibitors.

Although pathologically activated ABL1 fusion kinases represent well-validated therapeutic targets, tumor genomic sequencing has identified numerous point mutations in the ABL1 proto-oncogene of unclear significance. Here we describe ten novel ABL1 1b point mutations, including two from clinical isolates, that cause constitutive kinase activation and cellular transformation. All mutants retained sensitivity to ATP-competitive tyrosine kinase inhibitors (TKIs). Several substitutions cluster near the myristoyl-binding pocket, the target of ABL001, a novel clinically active allosteric kinase inhibitor that mimics the autoinhibitory myristoyl group, and likely activate the kinase by relieving physiologic autoinhibition. In addition, several mutations activate the kinase and confer resistance to allosteric inhibition despite a lack of proximity to this region. We demonstrate that BCR-ABL1 and ABL1 1b point mutations can co-exist in a proportion of clinical cases as a consequence of the chromosome 9 breakpoint location. Collectively, our findings support clinical investigation of ATP-competitive TKIs in malignancies harboring ABL1 point mutations, and sequencing of BCR-ABL1 and ABL1 1b in patients with acquired resistance to allosteric ABL1 inhibitors.

Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles

GCAPs are neuronal Ca2+-sensors playing a central role in light adaptation. GCAPs are N-terminally myristoylated membrane-associated proteins. Although, the myristoylation of GCAPs plays an important role in light adaptation its structural and physiological roles are not yet clearly understood. The crystal-structure of GCAP-1 shows the myristoyl moiety inside the hydrophobic core of the protein, stabilizing the protein structure; but 2H-solid-state NMR investigations on the deuterated myristoyl moiety of GCAP-2 in the presence of liposomes showed that it is inserted into the lipid bilayer. In this study, we address the question of the localization of the myristoyl group of Ca2+-bound GCAP-2, and the influence of CHAPS-, DPC-micelles and DMPC/DHPC-bicelles on the structure, and on the localization of the myristoyl group, of GCAP-2 by solution-state NMR. We also carried out the backbone assignment. Characteristic chemical shift differences have been observed between the myristoylated and the non-myristoylated forms of the protein. Our results support the view that in the absence of membrane forming substances the myristoyl moiety is buried inside a hydrophobic pocket of GCAP-2 similar to the crystal structure of GCAP-1. Addition of CHAPS-micelles and DMPC/DHPC-bicelles cause specific structural changes localized in and around the myristoyl binding pocket. We interpret these changes as an indication for the extrusion of the myristoyl moiety from its binding pocket and its insertion into the hydrophobic interior of the membrane mimic. On the basis of the backbone chemical shifts, we propose a structural model of myristoylated GCAP-2 in the presence of Ca2+ and membrane mimetics.

Structural basis for Arl3-specific release of myristoylated ciliary cargo from UNC119

Access to the ciliary membrane for trans-membrane or membrane-associated proteins is a regulated process. Previously, we have shown that the closely homologous small G proteins Arl2 and Arl3 allosterically regulate prenylated cargo release from PDEδ. UNC119/HRG4 is responsible for ciliary delivery of myristoylated cargo. Here, we show that although Arl3 and Arl2 bind UNC119 with similar affinities, only Arl3 allosterically displaces cargo by accelerating its release by three orders of magnitude. Crystal structures of Arl3 and Arl2 in complex with UNC119a reveal the molecular basis of specificity. Contrary to previous structures of GTP-bound Arf subfamily proteins, the N-terminal amphipathic helix of Arl3·GppNHp is not displaced by the interswitch toggle but remains bound on the surface of the protein. Opposite to the mechanism of cargo release on PDEδ, this induces a widening of the myristoyl binding pocket. This leads us to propose that ciliary targeting of myristoylated proteins is not only dependent on nucleotide status but also on the cellular localization of Arl3.

Nonnative interactions regulate folding and switching of myristoylated protein

We present an integrated experimental and computational study of the molecular mechanisms by which myristoylation affects protein folding and function, which has been little characterized to date. Myristoylation, the covalent linkage of a hydrophobic C14 fatty acyl chain to the N-terminal glycine in a protein, is a common modification that plays a critical role in vital regulated cellular processes by undergoing reversible energetic and conformational switching. Coarse-grained folding simulations for the model pH-dependent actin- and membrane-binding protein hisactophilin reveal that nonnative hydrophobic interactions of the myristoyl with the protein as well as nonnative electrostatic interactions have a pronounced effect on folding rates and thermodynamic stability. Folding measurements for hydrophobic residue mutations of hisactophilin and atomistic simulations indicate that the nonnative interactions of the myristoyl group in the folding transition state are nonspecific and robust, and so smooth the energy landscape for folding. In contrast, myristoyl interactions in the native state are highly specific and tuned for sensitive control of switching functionality. Simulations and amide hydrogen exchange measurements provide evidence for increases as well as decreases in stability localized on one side of the myristoyl binding pocket in the protein, implicating strain and altered dynamics in switching. The effects of folding and function arising from myristoylation are profoundly different from the effects of other post-translational modifications.

Role of N-Myristoylation of Camp-Dependent Protein Kinase a in Recognition and Phosphorylation of Membrane-Bound Substrates

The catalytic subunit of cAMP-dependent protein kinase A (PKA-C), controls the activation and deactivation of over 100 unique cytosolic and membrane bound substrates. Nearly all the PKA-C in mammals is subjected to myristoylation and myristoylation can be dependent on cell/tissue type. Although the function of myristoylation is not known, it has been hypothesized to mediate PKA-C/membrane and PKA-C/membrane protein interactions. Here, we developed an in vitro myristoylation method to obtain homogeneously myristoylated PKA-C (myrPKA-C) in quantities suitable for NMR studies. Using NMR spectroscopy, the interactions of membrane mimicking isotropic bicelles (DMPC/DHPC) with 15N-labeled myrPKA-C or 15N-labeled non-myristoylated PKA-C was monitored by [1H-15N]-TROSY-HSQC spectroscopy. Significant chemical shift perturbations were detected for the N-terminus and the myristate pocket of myrPKA-C, while both perturbations and line broadening was detected in remote regions far away from the myristoyl binding pocket. Such changes were not observed for the non-myristoylated form, suggesting that myristoylation drives the enzyme towards the membrane and may act allosterically on the enzyme. Membrane localization was further supported by intrinsic tryptophan fluorescence measurements which showed drastically different patterns between myrPKA-C and PKA-C. Finally, the insertion of the myristate group into lipid membranes was confirmed using 2H solid-state NMR spectroscopy on myrPKA-C with a deuterated myristoyl group. We propose that the myristoyl group of myrPKA-C steers the enzyme towards the membrane, even in the absence of its regulatory subunit or A-kinase anchoring proteins (AKAPs). These results provide an additional mechanism for PKA localization and recognition of membrane bound substrates. Preliminary data on PKA recognition of the integral membrane protein, phospholamban, are also presented.

The Degree of Response of Philadelphia Chromosome Positive Acute Lymphatic Leukemia to Allosteric Inhibition by GNF-2 Depends on the Presence of Either p185-BCR/ABL or p210-BCR/ABL

AbstractAbstract 598The t(9;22) translocation (Ph+) leads to the formation of the chimeric bcr/abl fusion gene, which encodes the BCR/ABL fusion protein. Depending on the minor and major breakpoint (m-BCR or M-BCR, respectively) on chromosome 22, either the p185-BCR/ABL (p185) or the p210-BCR/ABL (p210) fusion protein is expressed. In contrast to p210, p185 lacks the putatively oncogenic dbl- and pleckstrin homology domains. In the majority of the cases Ph+ ALL patients harbor the p185, but with increasing age the number Ph+ ALL patients with the p210 increases up to an 40%. Only few functional and biological differences between p185 and p210 are known. Both exhibit a constitutively activated kinase activity responsible for the induction of the leukemic phenotype in contrast to their physiological counterpart, c-ABL, whose kinase activity is finely regulated. One reason is that BCR/ABL “escapes” auto-inhibition mechanisms of c-ABL, such as the allosteric inhibition by “capping”. “Capping” is the process by which myristoylation of the N-terminal cap region of c-ABL leads to an auto-inhibited conformation through its binding to the hydrophobic myristoyl binding pocket (MBP) in the kinase domain. The cap region of c-ABL is encoded by the exon I, which is replaced by the N-terminus of BCR in the fusion protein. Myristate “mimicks” and MBP binders, such as GNF-2, aim to restore the allosteric inhibition through the binding to the MBP in BCR/ABL. The aim of our study was to further develop allosteric inhibition in Ph+ ALL. Therefore we investigated the anti-proliferative activity of GNF-2 in different Ph+ leukemia models such as patient derived Ph+ lymphatic cell lines, long term cultures of Ph+ ALL patient derived bone marrow cells (LTC) and factor dependent lymphatic Ba/F3 cells retrovirally transduced with either p185 or p210. Here we report that i.) the IC50 of GNF-2 for p210-positive ALL cells were much lower (cell lines =125nM; LTC from two different patients 100–300nM) than that for p185-positive ALL cells (cell lines=500 nM; LTC from two different patients 500–1000nM); ii.) factor independent growth of Ba/F3 cells expressing p185 was inhibited with an IC50 of 750nM whereas that of Ba/F3 cells expressing p210 was abolished already at 125nM; iii.) 1000nM GNF-2 was able to abolish colony formation in soft agar of p210-positive but not of p185-positive LTC; iv.) increasing concentrations of GNF-2 abolished in p210-positive ALL cells the BCR/ABL kinase activity, whereas in p185-positive cells it only reduced it as shown by the effects of GNF-2 on the autophosphorylation (Y245) and substrate phosphorylation (Crkl) activity as well as on down stream signaling (Stat5) of BCR/ABL.Our here presented data provide first evidence of a differential response of p185- and p210-positive Ph+ ALL cells to the allosteric inhibition by GNF-2, as a expression of functional and biological differences between the two fusion proteins within the same disease entity. It remains to clarify not only the mechanisms of this differential response, such as three dimensional conformation, different kinase activity and related signal transdcution, but also its prognostic significance for patients with p210-positive Ph+ ALL. Disclosures:No relevant conflicts of interest to declare.

GNF-2, An Allosteric BCR-ABL Inhibitor, Identifies a Novel Myristoylation-Mediated Mechanism Regulating the Ability of BCR-ABL to Activate HCK and IGF-1 Signaling.

Abstract 40Pharmacologic inhibition of BCR-ABL, using clinically active ATP-competitive inhibitors imatinib, nilotinib, and dasatinib, has been used to investigate BCR-ABL kinase activated signaling pathways. However, these agents show cross reactivity with other kinases (e.g. Kit, PDGFR, SRC family members), and their multi-targeted nature complicates assigning biological effects to the inhibition of a specific kinase target. Allosteric kinase inhibitors modulate the catalytic activity of protein kinases by binding to a site distant from the active site and inducing a protein conformation that inhibits kinase activity. These agents show promise as clinical agents and may offer advantages over ATP-competitive inhibitors in studying the function of specific kinases because they exploit binding sites and regulatory mechanisms that are unique to a particular kinase. GNF-2, a mono-selective BCR-ABL inhibitor that targets wild-type BCR-ABL and many clinically relevant imatinib resistant mutants, was recently discovered and provided the first demonstration that c-ABL kinase activity could be modulated by an inhibitor that binds outside the ATP or substrate binding sites. GNF-2 binds to a myristoyl-binding pocket in the C-lobe of the c-ABL kinase domain but its mechanism of inhibiting specific BCR-ABL kinase targets remains unclear. We previously reported that BCR-ABL activates an autocrine IGF-1 pro-survival signaling pathway in CML blast crisis cells through HCK-mediated activation of STAT5b. As GNF-2 is known to inhibit STAT5b phosphorylation, and HCK myristolyation is known to regulate its cellular localization, we hypothesized that GNF-2 inhibits BCR-ABL activation of HCK by binding to the ABL myristoyl-binding pocket and blocking access to the HCK myristoyl moiety. In support of this hypothesis, we now show that GNF-2 inhibits HCK phosphorylation and IGF-1 activation, but not HCK binding to BCR-ABL. To confirm the importance of the HCK myristoyl moiety in HCK activation, we mutated the myristoyl attachment site at position 2 in HCK from glycine to alanine. The mutant G2A HCK still interacted with BCR-ABL in co-immunoprecipitation assays but showed significantly lower levels of phosphorylation compared to wild-type HCK. To confirm that the decrease in phosphorylation was not due to mislocalization of G2A HCK, we mutated the myristoylation binding pocket of BCR-ABL by changing glutamic acid at position 505 to lysine. Similar to G2A HCK, E505K BCR-ABL still interacted with HCK, but the phosphorylation levels of HCK were dramatically reduced. To confirm that the HCK myristoyl moiety directly interacted with the ABL myristoyl-binding pocket, we used fluorescent spectroscopy to measure the ability of a myristoylated peptide corresponding to the six N-terminal amino acids of HCK to displace GNF-2. The fluorescence of GNF-2 is enhanced when it associates with the myristoyl-binding pocket of ABL. Using this assay, we calculated the Kd of GNF-2 to be 180 nM. We then assayed the ability of myristolyated HCK peptide to displace GNF-2 from ABL. We calculated the IC50 of the myristolyated HCK peptide to be 25 μM when ABL was saturated with 300 nM GNF-2. Myristate showed an IC50 of 213 μM, which is ∼ 10-fold higher than the myristoylated peptide. No binding was detected between the non-myristoylated peptide and ABL. Together, our study highlights a novel acquired function resulting from the fusion of BCR to the N-terminus of ABL, which converts the myristoyl-binding pocket in ABL from a negative regulator of kinase activity to an HCK activation motif that activates downstream IGF-1 signaling. These results also reveal the mechanism of action of the mono-selective BCR-ABL inhibitor GNF-2 and highlight the ABL myristoyl-binding pocket as a therapeutic target for inhibiting BCR-ABL activity. Disclosures:No relevant conflicts of interest to declare.

Oligomerization Inhibition Combined with Allosteric Inhibition Abrogates the Transformation Potential of T315I-Positive BCR/ABL.

Abstract 2964Poster Board II-940The t(9;22)-related translocation products are the BCR/ABL fusion proteins. Fusion of BCR to ABL leads to constitutive activation of ABL tyrosine kinase (ATK) activity. c-ABL is finely regulated by a variety of stimuli, whereas constitutively activated ABL induces aberrant proliferation and neoplastic transformation by constitutive activation of RAS, PI3 kinase, and JAK/STAT. Constitutively activated ATK is indispensable for the transformation potential of BCR/ABL.Inhibition of BCR/ABL kinase activity by selective ATP-competitors, such as Imatinib, Dasatinib or Nilotinib, is a valid concept for the causal therapy of Ph+ leukemia. Unfortunately, in advanced Ph+ leukemia, CML-blast crisis, and Ph+ ALL, these compounds select for resistant clones mostly due to the acquisition of point mutations in BCR/ABL that change the affinity for these ATP competitors. Tetramerization of ABL through the N-terminal coiled-coil region (CC) of BCR is essential for aberrant ABL-kinase activation. We recently showed that targeting the CC-domain forces BCR/ABL into a monomeric conformation that abolishes its transformation potential by interfering with its kinase activity. This also increases the sensitivity of the leukemic cells to Imatinib and overcomes the Imatinib resistance of BCR/ABL harboring the Y253F and E255K mutations. Another mechanism to target BCR/ABL consists in the allosteric inhibition. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl binding pocket in a process called “capping”, which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, BCR/ABL “escapes” this auto-inhibition. Allosteric inhibition by myristate “mimics”, such as GNF-2, is able to inhibit BCR/ABL. The the “gatekeeper” mutation T315I confers pluri-resistance against ATP competitor, oligomerization as well as allosteric inhibitors Based on data showing that the response to GNF-2 varies with the oligomerization in ABL-chimeras we investigated the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. Therefore we combined GNF-2 with the competitive peptide helix-2, for which we have previously shown the capacity to target BCR/ABL and its Imatinib-resistant mutants. Here we demonstrate that i.) the helix-2 strongly increased the inhibitory effects of 2μM GNF-2 on the factor independent growth of BCR/ABL expressing Ba/F3 progenitors; ii.) both helix-2 and GNF-2 were able to decrease the transformation potential of BCR/ABL in Rat-l fibroblasts; iii.) the combination of helix-2 and GNF-2 completely abolished the colony formation of BCR/ABL-positive Rat-1 cells in semi-solid medium; iv.) in combination helix-2 and GNF-2 were able to abrogate factor independent growth of Ba/F3 cells expressing BCR/ABL-T315I, whereas no effect was seen by the single agents; v.) the helix-2/GNF-2 combination nearly completely abolished the transformation potential of BCR/ABL-T315I in Rat-1 cells; vi.) the inhibitory effects of helix-2/GNF-2 were due to an inhibition of the BCR/ABL dependent signaling as shown by a reduced activation of STAT5 and a block of substrate phosphorylation (Crkl). The fact that the helix-2 is able to overcome the resistance of BCR/ABL-T315I against GNF-2 strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL.In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors. Disclosures:No relevant conflicts of interest to declare.

Oligomerization inhibition, combined with allosteric inhibition, abrogates the transformation potential of T315I-positive BCR/ABL

The t(9;22) translocation leads to the formation of the chimeric bcr/abl fusion gene, which encodes the BCR/ABL fusion protein. In contrast to its physiological counterpart c-ABL, the BCR/ABL kinase is constitutively activated, inducing the leukemic phenotype. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl-binding pocket in a process called 'capping', which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, the BCR/ABL 'escapes' this auto-inhibition. Allosteric inhibition by myristate 'mimics', such as GNF-2, is able to inhibit unmutated BCR/ABL, but not the BCR/ABL that harbors the 'gatekeeper' mutation T315I. In this study, we analyzed the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. We showed that inhibition of oligomerization was able to not only increase the efficacy of GNF-2 on unmutated BCR/ABL, but also overcome the resistance of BCR/ABL-T315I to allosteric inhibition. These results strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors.

Core mutations that promote the calcium-induced allosteric transition of bovine recoverin.

Recoverin is a small calcium binding protein involved in regulation of the phototransduction cascade in retinal rod cells. It functions as a calcium sensor by undergoing a cooperative, ligand-dependent conformational change, resulting in the extrusion of the N-terminal myristoyl group from a hydrophobic pocket. To test the role of certain core residues in tuning this allosteric switch, we have made and characterized two mutants: W31K, which replaces Trp31 with Lys; and a double mutant, I52A/Y53A, in which Ile52 and Tyr53 are both replaced by Ala. These mutations decrease the hydrophobicity of the myristoyl binding pocket. They are thus expected to make sequestering of the myristoyl group less favorable and destabilize the Ca2+-free state. As predicted, the myristoylated forms of the mutants exhibit increased affinity for Ca2+, whether monitored by equilibrium binding of 45Ca2+ (Kd = 17.2, 7.9, and 8.1 microM for wild type, W31K, and I52A/Y53A, respectively) or by the change in tryptophan fluorescence associated with the conformational change (Kd = 17.9, 3.6, and 4.4 microM for wild type, W31K, and I52A/Y53A, respectively). The mutants also exhibit decreased cooperativity of binding (Hill coefficient = 1.2 and 1.0 for W31K and I52A/Y53A vs 1. 4 for wild type). Binding of the mutant proteins to rod outer segment membranes occurs at lower Ca2+ concentrations compared to wild-type protein (K1/2 = 5.6, 2.2, and 1.0 microM for wild type, W31K, and I52A/Y53A, respectively). The unmyristoylated forms of the mutants exhibit biphasic Ca2+ binding curves, nearly identical to that observed for wild type. The binding data for the two mutants can be explained by a concerted allosteric model in which the mutations affect only the equilibrium constant L between the two allosteric forms, T (the Ca2+-free form) and R (the Ca2+-bound form), without affecting the intrinsic binding constants for the two Ca2+ sites. Two-dimensional NMR spectra of the Ca2+-free forms of the mutants have been compared to the wild-type spectrum, whose peaks have been assigned to specific residues (1). Many resonances assigned to residues in the C-terminal domain (residues 100-202) in the wild-type spectrum are identical in the mutant spectra, suggesting that the backbone structure of the C-terminal domain is probably unchanged in both mutants. The N-terminal domain, in which both mutations are located, reveals in each case numerous changes of undetermined spatial extent.