Kinase Conformation Research Articles

Ligand discrimination between active and inactive activation loop conformations of Aurora-A kinase is unmodified by phosphorylation.

Structure-based drug design is commonly used to guide the development of potent and specific enzyme inhibitors. Many enzymes - such as protein kinases - adopt multiple conformations, and conformational interconversion is expected to impact on the design of small molecule inhibitors. We measured the dynamic equilibrium between DFG-in-like active and DFG-out-like inactive conformations of the activation loop of unphosphorylated Aurora-A alone, in the presence of the activator TPX2, and in the presence of kinase inhibitors. The unphosphorylated kinase had a shorter residence time of the activation loop in the active conformation and a shift in the position of equilibrium towards the inactive conformation compared with phosphorylated kinase for all conditions measured. Ligand binding was associated with a change in the position of conformational equilibrium which was specific to each ligand and independent of the kinase phosphorylation state. As a consequence of this, the ability of a ligand to discriminate between active and inactive activation loop conformations was also independent of phosphorylation. Importantly, we discovered that the presence of multiple enzyme conformations can lead to a plateau in the overall ligand K d, despite increasing affinity for the chosen target conformation, and modelled the conformational discrimination necessary for a conformation-promoting ligand.

TRK xDFG Mutations Trigger a Sensitivity Switch from Type I to II Kinase Inhibitors

On-target resistance to next-generation kinase inhibitors, drugs specifically designed to maintain potency in the setting of kinase domain mutations, remains poorly characterized. Having identified that xDFG (TRKA G667) mutations confer second-generation TRK inhibitor resistance in patients with TRK fusion-positive cancers, we used computational modeling and biochemical assays to study how these affect drug binding. We found that xDFG mutations stabilize the inactive kinase conformation, hampering the binding of type I second-generation TRK inhibitors, while increasing the affinity for type II multikinase inhibitors. Type II drugs consistently inhibit the growth and TRK-mediated signaling of isogenic and patient-derived models harboring xDFG mutations. Collectively these data demonstrate that TRK xDFG substitutions trigger conformational changes that favor type II binding mechanisms. Given prior identification of paralogous xDFG resistance mutations in other oncogene addicted cancers, these findings provide insight into rational drug design to address these recalcitrant resistant alterations by leveraging inhibitor class affinity switching.

KinaMetrix: a web resource to investigate kinase conformations and inhibitor space

Protein kinases are among the most explored protein drug targets. Visualization of kinase conformations is critical for understanding structure–function relationship in this family and for developing chemically unique, conformation-specific small molecule drugs. We have developed Kinformation, a random forest classifier that annotates the conformation of over 3500 protein kinase structures in the Protein Data Bank. Kinformation was trained on structural descriptors derived from functionally important motifs to automatically categorize kinases into five major conformations with pharmacological relevance. Here we present KinaMetrix (http://KinaMetrix.com), a web resource enabling researchers to investigate the protein kinase conformational space as well as a subset of kinase inhibitors that exhibit conformational specificity. KinaMetrix allows users to classify uploaded kinase structures, as well as to derive structural descriptors of protein kinases. Uploaded structures can then be compared to atomic structures of other kinases, enabling users to identify kinases that occupy a similar conformational space to their uploaded structure. Finally, KinaMetrix also serves as a repository for both small molecule substructures that are significantly associated with each conformation type, and for homology models of kinases in inactive conformations. We expect KinaMetrix to serve as a resource for researchers studying kinase structural biology or developing conformation-specific kinase inhibitors.

Type II Kinase Inhibitors Targeting Cys-Gatekeeper Kinases Display Orthogonality with Wild Type and Ala/Gly-Gatekeeper Kinases.

Analogue-sensitive (AS) kinases contain large to small mutations in the gatekeeper position rendering them susceptible to inhibition with bulky analogues of pyrazolopyrimidine-based Src kinase inhibitors (e.g., PP1). This "bump-hole" method has been utilized for at least 85 of ∼520 kinases, but many kinases are intolerant to this approach. To expand the scope of AS kinase technology, we designed type II kinase inhibitors, ASDO2/6 (analogue-sensitive "DFG-out" kinase inhibitors 2 and 6), that target the "DFG-out" conformation of Cys-gatekeeper kinases with submicromolar potency. We validated this system in vitro against Greatwall kinase (GWL), Aurora-A kinase, and cyclin-dependent kinase-1 and in cells using M110C-GWL-expressing mouse embryonic fibroblasts. These Cys-gatekeeper kinases were sensitive to ASDO2/6 inhibition but not AS kinase inhibitor 3MB-PP1 and vice versa. These compounds, with AS kinase inhibitors, have the potential to inhibit multiple AS kinases independently with applications in systems level and translational kinase research as well as the rational design of type II kinase inhibitors targeting endogenous kinases.

Electrostatic mechanism of V600E mutation-induced B-Raf constitutive activation in colorectal cancer: molecular implications for the selectivity difference between type-I and type-II inhibitors.

The oncogenic mutation V600E in B-Raf activation loop (A-loop) has been frequently observed to cause drug resistance in colorectal cancer chemotherapy. Here, the molecular mechanism of V600E-induced conformational flipping of B-Raf activation loop (A-loop) is investigated systematically via continuum electrostatic analysis. It is found that substitution of the electroneutral Val600 residue with negatively charged glutamic acid Glu600 electrostatically destabilizes the inactive DFG-out conformation of B-Raf kinase and promotes its shifting to active DFG-in conformation. This is analogous with natural phosphorylation of Thr598 and/or Ser601 residues in A-loop to activate the kinase, that is, both the mutation and phosphorylation can introduce negative charge to B-Raf A-loop and then trigger the loop flipping. Energetic analysis reveals that the V600E mutation can affect inhibitor binding indirectly via regulation of kinase conformation. Type-I and type-II inhibitors respond distinctly to V600E mutation; the former is sensitized by the mutation, while the latter generally shows a low sensitivity to the mutation. Based on this guideline, the sophisticated type-I pan-kinase inhibitor Staurosporine as well as its analogs Midostaurin and Lestaurtinib are identified as potent mutant-selective inhibitors by modeling analysis and kinase assay, which exhibit a moderate or high selectivity for B-RafV600E over B-RafWT (3.7-fold, 6.1-fold and > 3.1-fold, respectively).

Identification of inactive conformation-selective interleukin-2-inducible T-cell kinase (ITK) inhibitors based on second-harmonic generation.

Many clinically approved protein kinase inhibitors stabilize an inactive conformation of their kinase target. Such inhibitors are generally highly selective compared to active conformation inhibitors, and consequently, general methods to identify inhibitors that stabilize an inactive conformation are much sought after. Here, we have applied a high‐throughput, second‐harmonic generation (SHG)‐based conformational approach to identify small molecule stabilizers of the inactive conformation of interleukin‐2‐inducible T‐cell kinase (ITK). A single‐site cysteine mutant of the ITK kinase domain was created, labeled with an SHG‐active dye, and tethered to a supported lipid bilayer membrane. Fourteen tool compounds, including stabilizers of the inactive and active conformations as well as nonbinders, were first examined for their effect on the conformation of the labeled ITK protein in the SHG assay. As a result, inactive conformation inhibitors were clearly distinguished from active conformation inhibitors by the intensity of SHG signal. Utilizing the SHG assay developed with the tool compounds described above, we identified the mechanism of action of 22 highly selective, inactive conformation inhibitors within a group of 105 small molecule inhibitors previously identified in a high‐throughput biochemical screen. We describe here the first use of SHG for identifying and classifying inhibitors that stabilize an inactive vs. an active conformation of a protein kinase, without the need to determine costructures by X‐ray crystallography. Our results suggest broad applicability to other proteins, particularly with single‐site labels reporting on specific protein movements associated with selectivity.

Structural and clinical consequences of activation loop mutations in class III receptor tyrosine kinases.

Mutations within the activation loop of members of the class III receptor tyrosine kinase (RTK) subfamily, which includes KIT, PDGFRA, and FLT3, have been observed in multiple human tumor types. These mutations confer constitutive activation as well as resistance to the type II tyrosine kinase inhibitors (TKI) that are currently clinically available, such as imatinib and sunitinib. It is now understood that activation loop mutations in class III RTKs shift the activation state equilibrium away from inactive states, to which type II TKIs bind, to the active state by destabilizing the inactive conformation. Recently, type I TKIs, which can bind to active kinase conformations, have been developed with specificity for class III RTK members. Some type I TKIs, such as crenolanib and avapritinib (BLU-285), have entered clinical studies for patients with activation loop mutations in KIT, PDGFRA, or FLT3. Preliminary results suggest that these type I TKIs show activity in these patient populations that previously lacked effective treatments. This article reviews the inactive and active structures of KIT, PDGFRA, and FLT3, how the mutations seen in human cancers affect kinase structure, and the clinical implications of these mutations in terms of type I vs. type II TKI binding.

Redefining the Protein Kinase Conformational Space with Machine Learning

Protein kinases are dynamic, adopting different conformational states that are critical for their catalytic activity. We assess a range of structural features derived from the conserved αC helix and DFG motif to define the conformational space of the catalytic domain of protein kinases. We then construct Kinformation, a random forest classifier, to annotate the conformation of 3,708 kinase structures in the PDB. Our classification scheme captures known active and inactive kinase conformations and defines an additional conformational state, thereby refining the current understanding of the kinase conformational space. Furthermore, network analysis of the small molecules recognized by each conformation captures chemical substructures that are associated with each conformation type. Our description of the kinase conformational space is expected to improve modeling of protein kinase structures, as well as guide the development of conformation-specific kinase inhibitors with optimal pharmacological profiles.

Hypoxia-selective allosteric destabilization of activin receptor-like kinases: A potential therapeutic avenue for prophylaxis of heterotopic ossification.

Heterotopic ossification (HO), the pathological extraskeletal formation of bone, can arise from blast injuries, severe burns, orthopedic procedures and gain-of-function mutations in a component of the bone morphogenetic protein (BMP) signaling pathway, the ACVR1/ALK2 receptor serine-threonine (protein) kinase, causative of Fibrodysplasia Ossificans Progressiva (FOP). All three ALKs (-2, -3, -6) that play roles in bone morphogenesis contribute to trauma-induced HO, hence are well-validated pharmacological targets. That said, development of inhibitors, typically competitors of ATP binding, is inherently difficult due to the conserved nature of the active site of the 500+ human protein kinases. Since these enzymes are regulated via inherent plasticity, pharmacological chaperone-like drugs binding to another (allosteric) site could hypothetically modulate kinase conformation and activity. To test for such a mechanism, a surface pocket of ALK2 kinase formed largely by a key allosteric substructure was targeted by supercomputer docking of drug-like compounds from a virtual library. Subsequently, the effects of docked hits were further screened in vitro with purified recombinant kinase protein. A family of compounds with terminal hydrogen-bonding acceptor groups was identified that significantly destabilized the protein, inhibiting activity. Destabilization was pH-dependent, putatively mediated by ionization of a histidine within the allosteric substructure with decreasing pH. In vivo, nonnative proteins are degraded by proteolysis in the proteasome complex, or cellular trashcan, allowing for the emergence of therapeutics that inhibit through degradation of over-active proteins implicated in the pathology of diseases and disorders. Because HO is triggered by soft-tissue trauma and ensuing hypoxia, dependency of ALK destabilization on hypoxic pH imparts selective efficacy on the allosteric inhibitors, providing potential for safe prophylactic use.

The PKC inhibitor BimI functions as a bi‐topic ligand that toggles a conserved allosteric switch to drive potent kinase inhibition

ATP‐competitive small‐molecule kinase inhibitors (SMKIs) are viewed primarily from the lens of tight and specific binding to the conserved catalytic domain. The allosteric effects of ATP‐competitive SMKIs are generally ignored, despite evidence that they can influence the potency and selectivity of kinase inhibition. We have recently showed that the ATP‐competitive PKC inhibitor BimI can displace substrate binding leading to potent inhibition of the kinase (Sommese and Sivaramakrishnan, JBC, 2016). In this study, we examine the substrate‐competitive behavior of a range of SMKIs derived from staurosporine and nucleotide analogs. We find that BimI functions like a bi‐topic ligand that acts on both the orthosteric ATP binding pocket and a remote allosteric site that determines overall kinase conformation. Combining novel FRET‐based sensors and multi‐scale MD simulations we identify a novel, conserved structural mechanism that regulates substrate binding in protein kinases. Bi‐topic ligands such as BimI disrupt a conserved ionic‐lock that drives global reorganization of the kinase domain through long‐range allosteric pathways that connect the N‐ and C‐terminal lobes. Together our studies identify a novel allosteric switch that regulates substrate binding in protein kinases that can be leveraged to develop highly selective kinase inhibitors.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An active‐like Src conformation explains its dynamic regulation

Biological signaling by protein kinases plays an essential role in cellular function. The switch between inactive and active protein kinase conformations is central to their regulation. The active conformations of protein kinases are triggered and maintained by various post‐translational modifications. The inactive conformation of Src tyrosine kinase has a non‐phosphorylated activation loop and a rolled‐out αC helix. The R‐spine at the Src kinase core is disassembled via the RS3 residue when the αC helix is disengaged. Maneuvering of the C‐spine is crucial for obtaining a pseudokinase conformation of Src that could be crucial for exploring its scaffolding functions. Our Umbrella sampling methods allow for mapping the conformational landscape of the Src kinase domain and characterizing four defining states; the inactive, active conformations and two intermediates. Mutation of the C‐spine V281F allows for the closing of the two kinase lobes in the absence of ATP. An allosteric region at the β3‐αC loop allows for engaging the αC helix and the RS3 residue of the R‐spine via the L297F mutation. The L297F/ V281F double mutant allows for stabilization of a unique catalytically dead kinase conformation in the active‐like state. These studies allow for exploration of active‐like Src kinase states that define both the underlying kinase activation switch and the dynamic allostery in the molecular communication at the kinase core.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel inhibitor stabilizes the inactive conformation of MAPK-interacting kinase 1.

Mitogen-activated protein kinase (MAPK)-interacting kinases 1 (Mnk1) and 2 (Mnk2) modulate translation initiation through the phosphorylation of eukaryotic translation initiation factor 4E, which promotes tumorigenesis. However, Mnk1 and Mnk2 are dispensable in normal cells, suggesting that the inhibition of Mnk1 and Mnk2 could be effective in cancer therapy. To provide a structural basis for Mnk1 inhibition, a novel Mnk1 inhibitor was discovered and the crystal structure of Mnk1 in complex with this inhibitor was determined. The crystal structure revealed that the inhibitor binds to the autoinhibited state of Mnk1, stabilizing the Mnk-specific DFD motif in the DFD-out conformation, thus preventing Mnk1 from switching to the active conformation and thereby inhibiting the kinase activity. These results provide a valuable platform for the structure-guided design of Mnk1 inhibitors.

Abstract PD4-01: The role of FGF/FGFR axis in resistance to SERDs and CDK4/6 inhibitors in ER+ breast cancer

Abstract Approximately 70% of breast cancers express the estrogen receptor (ER), and estrogen signaling drives breast cancer cell growth and progression. ER-directed therapies are commonly used to treat ER+ breast cancer and have improved survival for patients, yet resistance to those therapies inevitably occurs. Mutations in the estrogen receptor itself occur in ∼25-30% of patients with ER+ metastatic breast cancer that has developed resistance to aromatase inhibitors. Beyond these ER mutations, other resistance mechanisms are not well described. Moreover, clinical mechanisms of resistance to another class of ER-targeted agents, selective estrogen receptor degraders (SERDs), such as fulvestrant have not been clearly identified. Here we report two FGFR2 mutations identified in patients with resistant ER+ metastatic breast cancer, N550K and M538I. N550K is a well-known activating FGFR2 mutation; M538I stabilizes the active kinase conformation and it has not previously been described in breast cancer. When expressed in ER+ T47D cells, FGFR2 M538I and N550K led to resistance to fulvestrant, and the CDK4/6 inhibitor palbociclib and the combination of the two agents. FGFR2 M538I induced hyperactivity of p-FRS2, p-ERK and p-AKT, which is higher than wildtype FGFR2 and comparable to other known activating mutations N550K and K660N. In addition, overexpression of M538I mutant reduced sensitivity to FGFR inhibitors PD173074 and dovitinib in T47D cells, suggesting M538I is also functionally activating. Due to the hyperactive downstream signaling elicited by the mutation, cells overexpressing FGFR2 M538I achieved optimal growth in the presence of low dose of FGFR inhibitor. Under such conditions, FGFR2 M538I conferred more potent resistance to fulvestrant as compared to wildtype FGFR2. However, drug resistance resulting from M538I mutant can be fully resensitized to fulvestrant and/or palbociclib with high dose of FGFR inhibitors. In summary, we have identified activating FGFR2 mutations (M538I and N550K) in ER+ breast cancer patients, which may contribute to the development of resistance to SERDs and CDK4/6 inhibitors. Additional FGFR2 mutations have been recently identified in other cohorts of patients with resistant ER+ metastatic breast cancer, suggesting that this may be a clinical mechanism of resistance in some patients. Patients with activating FGFR2 mutations may benefit from the treatment with an FGFR inhibitor in combination with SERDs and CDK4/6 inhibitors. Citation Format: Mao P, Kusiel J, Cohen O, Wagle N. The role of FGF/FGFR axis in resistance to SERDs and CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-01.

Correction: Pockets as structural descriptors of EGFR kinase conformations

[This corrects the article DOI: 10.1371/journal.pone.0189147.].

An Allosteric Region of Src Tyrosine Kinase Allows for Stabilization of Its Active-Like Conformation

Protein kinases play an essential role as signaling enzymes and their sensitive action in the needs of the cell requires their tight regulation. Their active conformations are exquisitely regulated and their populations controlled by various post-translational modifications. The inactive conformation of Src tyrosine kinase is characterized by its non-phosphorylated activation loop and the rolled-out αC helix in the N-lobe. The inactive conformation has the regulatory kinase spine disassembled as the RS3 residue from the αC helix is displaced. Our umbrella sampling methods to map the energy landscape of these conformations has allowed for defining four states; the inactive, active conformations and two intermediate states. In the present study we extend our umbrella sampling technique to understand the activation allostery in Src tyrosine kinase in three different mutants. The first mutant is the L297F that occupies an allosteric region in the N-terminal of the αC helix and allows for activation of Src tyrosine kinase analogous to RAFs. The second mutant is the V281F mutation in the catalytic spine that allows for the closing of the two kinase lobes in the absence of nucleotide. A double mutant of Src tyrosine kinase V281F/L297F allows for stabilization of a unique catalytically dead kinase conformation in the active-like state. This unique conformation allows for understanding of kinase switching function independent of its catalytic role. A third M314V mutation in the regulatory spine allows us to explore the maneuvering of hydrophobicity in the Src tyrosine kinase core. These studies provide a fresh perspective to understanding conformational switching in Src family of tyrosine kinases and the dynamic allostery that underlies this molecular communication. Our community map analysis of residue networks provides an additional layer of understanding to these observations.

Structure-Based Kinase Profiling To Understand the Polypharmacological Behavior of Therapeutic Molecules.

Several drugs elicit their therapeutic efficacy by modulating multiple cellular targets and possess varied polypharmacological actions. The identification of the molecular targets of a potent bioactive molecule is essential in determining its overall polypharmacological profile. Experimental procedures are expensive and time-consuming. Therefore, computational approaches are actively implemented in rational drug discovery. Here, we demonstrate a computational pipeline, based on reverse virtual screening technique using several consensus scoring strategies, and perform structure-based kinase profiling of 12 FDA-approved drugs. This target prediction showed an overall good performance, with an average AU-ROC greater than 0.85 for most drugs, and identified the true targets even at the top 2% cutoff. In contrast, 10 non-kinase binder drugs exhibited lower binding efficiency and appeared in the bottom of ranking list. Subsequently, we validated this pipeline on a potent therapeutic molecule, mahanine, whose polypharmacological profile related to targeting kinases is unknown. Our target-prediction method identified different kinases. Furthermore, we have experimentally validated that mahanine is able to modulate multiple kinases that are involved in cross-talk with different signaling molecules, which thereby exhibits its polypharmacological action. More importantly, in vitro kinase assay exhibited the inhibitory effect of mahanine on two such predicted kinases' (mTOR and VEGFR2) activity, with IC50 values being ∼12 and ∼22 μM, respectively. Next, we generated a comprehensive drug-protein interaction fingerprint that explained the basis of their target selectivity. We observed that it is controlled by variations in kinase conformations followed by significant differences in crucial hydrogen-bond and van der Waals interactions. Such structure-based kinase profiling could provide useful information in revealing the unknown targets of therapeutic molecules from their polypharmacological behavior and would assist in drug discovery.

Pockets as structural descriptors of EGFR kinase conformations.

Epidermal Growth Factor Receptor (EGFR), a tyrosine kinase receptor, is one of the main tumor markers in different types of cancers. The kinase native state is mainly composed of two populations of conformers: active and inactive. Several sequence variations in EGFR kinase region promote the differential enrichment of conformers with higher activity. Some structural characteristics have been proposed to differentiate kinase conformations, but these considerations could lead to ambiguous classifications. We present a structural characterisation of EGFR kinase conformers, focused on active site pocket comparisons, and the mapping of known pathological sequence variations. A structural based clustering of this pocket accurately discriminates active from inactive, well-characterised conformations. Furthermore, this main pocket contains, or is in close contact with, ≈65% of cancer-related variation positions. Although the relevance of protein dynamics to explain biological function has been extensively recognised, the usage of the ensemble of conformations in dynamic equilibrium to represent the functional state of proteins and the importance of pockets, cavities and/or tunnels was often neglected in previous studies. These functional structures and the equilibrium between them could be structurally analysed in wild type as well as in sequence variants. Our results indicate that biologically important pockets, as well as their shape and dynamics, are central to understanding protein function in wild-type, polymorphic or disease-related variations.

Molecular insight into mutation-induced conformational change in metastasic bowel cancer BRAF kinase domain and its implications for selective inhibitor design

Oncogenic BRAF V600E mutation confers constitutive activation for the kinase and is closely related to the pathogenesis of metastasic bowel cancer (MBC). Here, the V600E-induced conformational change in MBC BRAF kinase domain is characterized systematically at structural, energetic and dynamic levels. The mutation is observed to cause a conformational conversion of the kinase’s activation loop from DFG-out to DFG-in, thus activating the kinase. Electrostatic force is primarily responsible for the conformational conversion and stabilization of DFG-in associated with the mutation. Molecular docking calculations are employed to analyze the binding mode difference of mutant-selective inhibitors between the DFG-out and DFG-in conformations of BRAF kinase. It is revealed that the mutation can reshape inhibitor selectivity profile by altering kinase loop conformation. Several compounds are determined to have a high or moderate selectivity for mutant over wild-type kinase. The selectivity is primarily originated from hydrogen bond interactions of inhibitor ligands with mutant rather than wild type due to the conformational difference in kinase domain.

Molecular Simulations Reveal an Unresolved Conformation of the Type IA Protein Kinase A Regulatory Subunit and Suggest Its Role in the cAMP Regulatory Mechanism.

We identify a previously unresolved, unrecognized, and highly stable conformation of the protein kinase A (PKA) regulatory subunit RIα. This conformation, which we term the "Flipback" structure, bridges conflicting characteristics in crystallographic structures and solution experiments of the PKA RIα heterotetramer. Our simulations reveal a hinge residue, G235, in the B/C helix that is conserved through all isoforms of RI. Brownian dynamics simulations suggest that the Flipback conformation plays a role in cAMP association to the A domain of the R subunit.

Abstract 3168: Overcoming acquired drug resistance by TPX-0005, an ALK, ROS1 and pan-TRK inhibitor

Abstract The inevitable development of clinical drug resistance presents a common challenge for targeted cancer therapy. In non-small cell lung cancer (NSCLC) patients with ALK and ROS1 rearrangements, the emergence of mutations in the targeted oncogenes was identified as one of the mechanisms that confer drug resistance. In particular, a group of mutations known as solvent front mutations, such as ALKG1202R and ROS1G2032R, render common resistance to ALK and ROS1 inhibitors. Similar solvent front mutations, such as TRKAG595R and TRKCG623R, have also been identified in cancer patients with rearrangement of NTRK family genes who developed resistance to TRK-targeted therapies. TPX-0005, a compact three-dimensional macrocyclic molecule, was designed to completely locate at the adenine binding site of ATP in order to efficiently target the active kinase conformation and systematically circumvent the steric interference from various clinical resistant mutations, especially the solvent front mutations. TPX-0005 is an orally available and potent ATP-competitive inhibitor against ALK, ROS1, TRKA, TRKB and TRKC recombinant kinases and their corresponding clinical resistant mutants. TPX-0005 demonstrated potent anti-proliferative activity in the range of sub-nanomolar to low nanomolar in a number of human cancer cell lines and engineered stable cell lines expressing the targeted oncogenes or their solvent front mutants, accompanied by inhibition of target phosphorylation and concomitant inactivation of downstream effectors such as ERK, AKT and STAT3. In patient derived xenograft tumor models, TPX-0005 treatment resulted in significant regression of tumors harboring the oncogenic ALK, ROS1 and TRKC fusions. Moreover, in a series of mouse xenograft tumor models, TPX-0005 exhibited marked anti-tumor activity not only in tumors harboring the wildtype oncogenic targets but also in tumors harboring the oncogenes with the solvent front mutations via inhibition of the target phosphorylation. Taken together, these pre-clinical studies have demonstrated the potent activities of TPX-0005 against not only wild type oncogenic ALK, ROS1, TRK fusions but also their corresponding solvent front mutations, which will bring in a new therapy for cancer patients resistant to currently available ALK, ROS1, and TRK inhibitors in clinic. Citation Format: Wei Deng, John Huang, Dayong Zhai, Evan Rogers, Jean Cui. Overcoming acquired drug resistance by TPX-0005, an ALK, ROS1 and pan-TRK inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3168. doi:10.1158/1538-7445.AM2017-3168