scholarly journals Mutation in Abl kinase with altered drug binding kinetics indicates a novel mechanism of imatinib resistance

2021 ◽  
Author(s):  
Agatha Lyczek ◽  
Benedict-Tilman Berger ◽  
Aziz M Rangwala ◽  
YiTing Paung ◽  
Jessica Tom ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Domain ◽  
Protein Kinase Inhibitors ◽  
Drug Binding ◽  
The Body ◽  
Binding Kinetics ◽  
Wild Type ◽  
Resistance Mutations ◽  
Imatinib Resistance ◽  
Abl Kinase

Protein kinase inhibitors are potent anti-cancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for Chronic Myeloid Leukemia (CML) by 80%, but 22-41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, in which more than a hundred different mutations have been identified. Some mutations are located near the imatinib binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug binding site and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies (10). Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease-relevant or resistance-causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than two-fold compared to Abl wild type. Surprisingly, one-third of mutations in Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified a clinical Abl mutation that binds imatinib with wild type-like affinity but dissociates from imatinib three times faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the non-equilibrium environment of the body where drug export and clearance play critical roles.

scholarly journals Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance

2021 ◽  
Vol 118 (46) ◽  
pp. e2111451118
Author(s):  
Agatha Lyczek ◽  
Benedict-Tilman Berger ◽  
Aziz M. Rangwala ◽  
YiTing Paung ◽  
Jessica Tom ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Domain ◽  
Protein Kinase Inhibitors ◽  
Drug Binding ◽  
The Body ◽  
Binding Kinetics ◽  
Wild Type ◽  
Resistance Mutations ◽  
Imatinib Resistance ◽  
Abl Kinase

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type–like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

BCR-ABL Kinase Dynamics and Drug Resistance.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1996-1996 ◽  
Author(s):  
Mohammad Azam ◽  
Valentina Nardi ◽  
William C. Shakespear ◽  
Robert R. Latek ◽  
Darren Veach ◽  
...  
Keyword(s):  
Kinase Domain ◽  
Drug Binding ◽  
Hinge Region ◽  
Activation Loop ◽  
In Vitro Mutagenesis ◽  
Resistance Mutations ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Clinical Resistance

Abstract The aberrant signaling behavior caused by the expression of BCR-ABL is necessary and sufficient to cause chronic myeloid leukemia (CML), an observation which paved the way for the development of imatinib (GleevecTM), a small molecule inhibitor of the BCR-ABL kinase. Enthusiasm for the remarkable efficacy of imatinib has been tempered by the development of clinical resistance. The most common mechanisms for resistance are the development of kinase domain mutations and/or overexpression of the BCR-ABL gene, with mutations in the kinase accounting for ~90 % of all cases. The resistance-conferring lesions are found in regions of the kinase that are critical to its autoregulation, such as P-loop, C-helix, gatekeeper area, activation loop and the SH2-C-lobe interface. Mechanistically, these mutations effect either a steric blockade or a change in the dynamic equilibrium that favors the active kinase conformation that precludes imatinib binding. We have analyzed two dual Src-Abl kinase inhibitors, AP23464 and PD166326, against 58 BCR-ABL kinase variants conferring imatinib resistance. PD166326 binds to the Abl kinase domain in the open although enzymatically inactive conformation, while AP23464 targets the active conformation. Both of these compounds have effectively suppressed the cell growth of imatinib resistance variants, except for a recurrent mutation in the gatekeeper residue (T315I). The P-loop variants are more sensitive to AP23464 than PD166326. Interestingly, the imatinib resistant variants from the C-helix, hinge region, activation loop and SH2-C-lobe region, are hypersensitive to both compounds, as compared to native BCR-ABL. The BCR-ABL variants in the C-helix, gatekeeper area, and the activation loop are more sensitive to AP23464 than PD166326, while variants from the hinge region and the SH2-C-lobe interface are hypersensitive to PD166326. Altogether, these results define a differential requirement for a specific ABL conformation for drug binding of AP23464 and PD166326. In order to better understand their structure activity relationships and the patterns of resistance, we carried out an in-vitro mutagenesis-screen using different concentration of the drug either alone or in combination with imatinib. AP23464 mediates 2–3 time less resistance than PD166326. A higher concentration of all three compounds suppresses all resistance mutations, save for the notable exceptions, T315I and F317L/VandC. Resistance conferring mutations selected at 10–20 fold higher IC50 values are different. AP23464 efficiently suppresses the mutations from the P-loop (except E255K) and two mutations from the activation loop, while PD166326 remains refractory to the mutations in the C-helix and SH2-C-lobe interface. In combination with imatinib, AP23464 and PD166326 suppressed the emergence of most resistance mutations, with the notable exception of T315I. These in-vitro studies demonstrate that the combination of two or three different conformation specific inhibitors is needed to suppress the emergence of resistance. We are characterizing variants of AP23464 that we predict will show activity against the most challenging imatinib resistance mutant T315I.

Inhibition of BCR/ABL-T315I by Dismantling the Hydrophobic Spine.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2129-2129
Author(s):  
Mohammad Azam ◽  
Markus Seeliger ◽  
John T Powers ◽  
Nathanael Gray ◽  
John Kuriyan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Resistance Mutations ◽  
Imatinib Resistance ◽  
Principal Mechanism ◽  
Abl Kinase ◽  
Molecule Inhibitor ◽  
Mutant Forms

Abstract Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance (IMR) in patients with chronic myelogenous leukemia (CML). The second generation BCR/ABL inhibitors, Nilotinib and Dasatinib, are effective in inhibiting essentially all IMR variants, but not the gatekeeper mutant T315I. Substitution of a bulky hydrophobic residue for the gatekeeper threonine not only causes steric blockade to the inhibitor but also stabilizes the active kinase conformation through a network of hydrophobic connections dubbed the hydrophobic-spine. In this study we describe the molecular mechanisms employed by the gatekeeper mutation to stabilize the active conformation, and demonstrate that these structural components can be targeted by the small molecule inhibitor compound #14, which efficiently inhibits native and gatekeeper mutant forms of the BCR/ABL kinase. Structural modeling and mutagenesis of residues constituting the spine suggests that compound #14 inhibits the kinase by disrupting the hydrophobic spine. Screening for drug resistance in vitro selected for clones having compound mutations involving both the P-loop and gatekeeper residues. Our studies provide structural guidance for the design of inhibitors against the gatekeeper mutant, and suggest that combination therapy may be required to prevent the emergence of compound resistance mutations.

Dynamics of BCR-ABL1 Transcripts during Nilotinib Therapy in Patients with Chronic Myeloid Leukemia (CML).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2961-2961
Author(s):  
Alfonso Quintás-Cardama ◽  
Hagop Kantarjian ◽  
Dan Jones ◽  
Susan O’Brien ◽  
Mary Beth Rios ◽  
...  
Keyword(s):  
Transcript Level ◽  
Kinase Domain ◽  
Pcr Analysis ◽  
Common Mechanism ◽  
Molecular Response ◽  
Wild Type ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Frontline Therapy

Abstract Background: Mutations in the BCR-ABL kinase domain are a common mechanism (40%) of imatinib-resistance. Nilotinib is approximately 30-fold more potent than imatinib against ABL kinase and has activity against most BCR-ABL kinase mutants, except T315I. We investigated the molecular response to nilotinib in patients (pts) receiving this agent both as frontline therapy as well as after intolerance or resistance to imatinib. Methods: 144 pts in chronic (CP) (n=63), accelerated (AP) (n=44) and blast (BP) (n=37) phase CML received nilotinib therapy. Twenty-seven pts in CP received nilotinib as frontline therapy. Quantitative reverse transcription PCR in peripheral blood samples was performed prior to nilotinib and every 3 mo thereafter. Results: The median BCR-ABL1/ABL1 ratio (%) at nilotinib start was 64.50 (range, 0.67-100), including 67.09 (range, 0.01–100) for pts in CP (100 [range, 6.87–100] for pts receiving frontline nilotinib therapy), 46.44 (range, 0.01–100) for AP, and 73.31 (range, 0.16–100) for BP. Fifty-two (45%) of 116 assessed pts (19/43 in CP, 23/40 in AP, and 10/33 in BP) harbored 26 different BCR-ABL1 mutants. The most common mutations were G250E (n=8), E355G (n=5), M351T (n=4), and T315I (n=4). The lowest PCR values for pts in CP, AP, and BP were achieved after 18 (0.20), 18 (0.88), and 18 (0.18) mos, respectively (0.1 at 12 mos for pts receiving frontline nilotinib therapy). After 24 mos of therapy, the median BCR-ABL1/ABL1 ratios for pts in CP, AP, and BP were 0.21, 6.99, and 0.05, respectively. BCR-ABL1/ABL1 ratio reductions occurred in 80 (72%) of 112 pts who had at least 2 PCR analyses during nilotinib therapy: <1-log in 22 (20%) pts (6 CP, 8 AP, 8 BP) after a median of 18 wks (range, 12 to 96); >1-log in 19 (17%) pts (10 CP, 6 AP, 3 BP) after a median of 36 wks (range, 11 to 96); >2-logs in 20 (18%) pts (12 CP, 6 AP, 2 BP) after a median of 48 wks (range, 12 to 96) and >3-logs in 19 (17%) pts (13 CP, 4 AP, 2 BP) after a median of 36 wks (range, 12 to 116). A major molecular response (MMR) was seen in 21 (19%) pts (12 CP, 7 AP, 2 BP). Nine (8%) pts achieved a complete molecular response (CMR; undetectable BCR-ABL1 transcripts), including 5 CP, 2 AP, and 2 BP. Among pts treated in CP followed for at least 6 months, MMR occurred in 7 (22%) of 32 pts treated after imatinib failure (median follow-up 23 mos) and 6 (55%) of 11 pts treated as frontline therapy (median follow-up 9 mos). CMR occurred in 2 (6%) and 3 (27%) pts, respectively. Fifty-three (47%) pts (21 CP, 22 AP, 10 BP) had at least 1 follow-up PCR analysis after their lowest transcript level, and in 7 pts the BCR-ABL1/ABL1 ratio increased >1 log (baseline mutations: M244V, G250E, wild-type, wild-type, A433T, E355G, Q252H), in 6 pts >2 logs (baseline mutation: F359V, E453K, wild-type, E355G, G250E, wild-type), and in 1 AP >3 logs (wild-type). Conclusion: Nilotinib therapy induces molecular responses in a significant number of pts both as frontline therapy and after imatinib failure. These responses can be observed across a wide variety of BCR-ABL kinase mutations. Longer follow-up is needed to define the stability and durability of MMR and CMR in these pts.

Comparative Analysis of Two BCR-ABL Small Molecule Inhibitors Reveals Overlapping but Distinct Mechanisms of Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 552-552 ◽  
Author(s):  
Michael R. Burgess ◽  
Neil P. Shah ◽  
Brian J. Skaggs ◽  
Francis Y. Lee ◽  
Charles L. Sawyers
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Point Mutations ◽  
Kinase Domain ◽  
Drug Binding ◽  
Saturation Mutagenesis ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract A novel dual SRC/ABL kinase inhibitor, BMS-354825, is showing promise for the treatment of imatinib-resistant chronic myeloid leukemia not only in vitro (Shah NP, et al., Science 305:399), but also in a phase I clinical trial (ASH abstract: Sawyers CL, et al.) Resistance to imatinib is increasingly found in patients due to point mutations in the BCR-ABL kinase domain that do not impair kinase activity but prevent drug binding. BMS-354825 is more potent than imatinib and retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants in vitro. The compound’s ability to inhibit imatinib-resistant forms of BCR-ABL is presumed to be due to its relaxed binding requirements, whereas imatinib requires the adoption of a closed conformation of the kinase to bind. We addressed the hypothesis that the relaxed binding requirements of BMS-354825 would limit the range of BCR-ABL mutations that confer drug resistance. To address this question, we employed a saturation mutagenesis experiment as described by others (Azam M, et al., Cell 112:831) and found that the spectrum of BMS-354825-resistant mutants was reduced compared to that of imatinib. In a series of such screens, mutations at only four amino acids have been isolated, two of which account for the vast majority of resistant clones. In contrast, Azam et al. isolated over 20 mutations in a screen for imatinib resistance, a finding which has been generally reproduced in our lab. All four BMS-354825-resistant mutations map to known BMS-354825 contact residues as shown by co-crystallographic studies (ASH abstract: Tokarski JS et al., Bristol-Myers Squibb). Mutations at L248, T315, and F317 show BMS-354825 resistance and have been previously reported to confer imatinib resistance. Mutation at V299 represents a novel mode of resistance. Interestingly, some point mutations conferring BMS-354825 resistance were at positions known to be mutated in cases of imatinib resistance, but the mutated residues differed. Furthermore, the identity of the mutated residue was crucial in conferring sensitivity or resistance to an individual drug as shown by comparison of cellular IC50’s (see table). For example, F317L was shown previously to confer imatinib resistance. F317V, on the other hand, demonstrates relative BMS-354825-resistance but is still exquisitely sensitive to imatinib. In a screen for mutants simultaneously resistant to both drugs, we consistently recover 30–50 fold fewer mutant clones compared to single drug treatment. All such clones isolated to date encode for T315I. Kinase domain point mutation is becoming an increasingly encountered clinical problem in diseases treated with small molecule inhibitors. Our findings suggest that combination therapy with imatinib and BMS-354825 may be of clinical utility in CML, particularly by delaying the development of resistance. IC50 for growth (nM) Baf3 Clone imatinib BMS-354825 p210 wt < 1,000 < 5 T315I > 10,000 > 500 T315A 1,000 100 F317L 2,000 10 F317V < 1,000 60 V299L 1,000 20 L248R > 10,000 20

Molecular Interactions between the Highly Selective pan-Bcr-Abl Inhibitor, AMN107, and the Tyrosine Kinase Domain of Abl.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3365-3365 ◽  
Author(s):  
Paul W. Manley ◽  
Sandra W. Cowan-Jacob ◽  
Gabriele Fendrich ◽  
Jürgen Mestan
Keyword(s):  
Hydrogen Bond ◽  
Catalytic Activity ◽  
Binding Site ◽  
Kinase Domain ◽  
Side Chain ◽  
Wild Type ◽  
Inactive Conformation ◽  
Abl Kinase ◽  
Hydrogen Bond Interactions ◽  
Imatinib Resistant

Abstract AMN107 (Novartis Pharma AG), is a new, highly potent and selective inhibitor of the Abl tyrosine kinase activity of the Bcr-Abl oncoprotein, which causes chronic myelogenous leukemia (CML). In addition to inhibiting wild-type Bcr-Abl, AMN107 inhibits the activity of 32/33 mutant forms of this protein which occur in imatinib-resistant patients. Following promising results from Phase I clinical studies in imatinib-resistant CML patients, AMN107 has now entered Phase II clinical trials. In order to better understand the molecular basis for the activity of this compound we have examined the x-ray crystallographic structures of complexes between AMN107 and the Abl kinase domain of wild-type and mutant Bcr-Abl. For these studies, recombinant proteins (residues 229–500) corresponding to the wild type and M351T mutant of the human Abl kinase domain were expressed in baculovirus infected insect cells and purified in the presence of AMN107. In each case the crystals obtained contained four independent copies of the complex in the unit cell and they diffracted to a resolution of 2.2 and 2.7 Å, respectively. As has been reported for the prototype Bcr-Abl inhibitor, imatinib (Nagar et al., Cancer Research2002;62:4236), AMN107 binds to an inactive conformation of Abl, in which the glycine-rich, P-loop folds over the ATP binding site and the activation-loop adopts a conformation in which it occludes the substrate binding site and disrupts the ATP-phosphate binding site to block the catalytic activity of the enzyme. In order to induce the inactive conformation of the protein, AMN107 participates in hydrogen-bond interactions between (i) the pyridine-N and the backbone-C=O of Met318, (ii) the anilino-NH and the side-chain hydroxyl of Thr315, (iii) the carboxamido-C=O and the backbone-NH of Asp381, and (iv) the carboxamido-NH and the side-chain carboxylate of Glu286. These hydrogen-bond interactions are complemented by a large number of lipophilic interactions, surrounding the pyridine and 4-methylimidazole moieties in particular. Furthermore, careful analysis has revealed the presence of interactions between the protein and the trifluoromethyl group of AMN107, in which a fluorine atom is in close contact with the backbone-C=O of Asp381, with a mean F-C distance of 3.02 Å (4 mols/unit cell: 2.95, 2.97, 3.08 and 3.09). This compares with a value of 3.30 Å for the sum of the van der Waals radii of fluorine and carbon. Similar F-C=O interactions (mean F-C distance 3.02 Å) are observed in a complex between AMN107 and M351TAbl. Such interactions have been reported in the literature (Olsen et al., ChemBioChem2004;5:666) and are the result of dipolar interactions between the electronegative fluorine and the positively polarised carbon of the carbonyl group. In order to evaluate the contribution of the fluorine interactions to the binding of AMN107 to Abl we compared the effects of AMN107 on Bcr-Abl autophosphorylation in Ba/F3 cells to those of the corresponding analogue of AMN107, possessing a methyl group in place of trifluoromethyl. Whereas AMN107 displayed a mean IC50 value of 17 ± 0.5 nM, the methyl analogue had a mean IC50 of 83 ± 13 nM, which translates into a difference in binding energy in the region of 0.7 kcal/mole. Although Asp381 is therefore important for the binding of AMN107 to Abl through a hydrogen bond and through F-C interactions, resistance-mutations of this residue are unlikely the arise, since it plays a key role in the catalytic activity of the enzyme by interacting with a Mg ion which coordinates the phosphate groups of ATP.

Identification of a Novel Mode of Kinase Inhibitor Resistance: An F604S Exchange in FIP1L1-PDGFRA Modulates FIP1L1-PDGFRA Protein Stability in a SHP-2 and SRC-Dependent Manner

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1739-1739
Author(s):  
Sivahari Prasad Gorantla ◽  
Nikolas von Bubnoff ◽  
Christian Peschel ◽  
Justus Duyster
Keyword(s):  
Protein Stability ◽  
Kinase Domain ◽  
Drug Binding ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Resistance Mutations ◽  
Imatinib Resistance ◽  
Cell Clones ◽  
Imatinib Resistant ◽  
Pdgfr Alpha

Abstract Abstract 1739 FIP1L1-PDGFR alpha is a constitutively activated protein kinase which was reported in chronic eosinophilic leukemia (CEL) and in cases of hypereosinophilic syndrome and mastocytosis with eosinophilia. Imatinib is clinically active against FIP1L1-PDGFRA positive disease. However, clinical resistance to imatinib has been observed in FIP1L1-PDGFRA positive leukemia and was shown to occur due to a secondary mutation (T674I) in the PDGFR alpha kinase domain. Using a screening strategy to identify imatinib resistant mutations, we generated numerous imatinib resistant cell clones. Analysis of the PDGFRA kinase domain in these cell clones revealed a broad spectrum of resistance mutations including the clinically reported exchange T674I. Interestingly, one of the abundant mutations was a Phe to Ser exchange at position 604 (F604S), which occurred alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S did not increase the biochemical or cellular IC50 value to imatinib when compared to wild-type (WT FP). However, F604S and F604S+D842H transformed Ba/F3 and mouse bone marrow more efficiently compared to WT and D842H, respectively. Immunoprecipitation and immunoblotting indicated increased amounts of FIP1L1-PDGFRA protein in F604S versus WT cells. Pulse chase analysis revealed that FIP1L1-PDGFRA/F604S is strongly stabilized compared to WT. SRC coimmunoprecipitated with FIP1L1-PDGFRA in WT, but not F604S cells. Co-expression of SRC in 293T cells augmented degradation of WT, but not F604S FIP1L1-PDGFRA, indicating that SRC is a negative regulator of FIP1L1-PDGFRA protein stability. Importantly both, the SRC inhibitor PD166326 and SRC siRNA mimicked the F604S phenotype and resulted in stabilization of the WT protein. Importantly, phosphatase inhibitor treatment of FIP1L1-PDGFRA/F604S led to destabilization and SRC recruitment indicating that phosphatases might be responsible for the enhanced stability of FIP1L1-PDGFRA/F604S. In fact, coimmunuprecipitaion experiments identified the phosphatase SHP2 as a specific binding partner of F604S and mapping experiments revealed that the phosphatase domain of SHP-2 directly interacted with FIP1L1-PDGFRA/F604S but not with wt- FIP1L1-PDGFRA. Together, these results suggest that stabilization of FIP1L1-PDGFRA/F604S is due to dephosphorylation by SHP-2 leading to lesser activation of the SRC and Cbl mediated ubiquitination machinery. Finally a novel exchange (L629P) identified in imatinib resistance CEL patient also leads to the stabilization of FIP1L1-PDGFRA protein similar to F604S. This indicates that stabilization of FIP1L1-PDGFRA is a common mode of drug resistance in FIP1L1-PDGFRA positive HES or CEL. In summary, imatinib resistance screening identified a novel class of resistance mutations in FIP1L1-PDGFRA, that do not act by impeding drug binding to the target, but increased target protein stability and abundance by interfering with SRC- mediated degradation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An Activating Mutation in the ATP Binding Site of the ABL Kinase Domain

1996 ◽  
Vol 271 (32) ◽  
pp. 19585-19591 ◽  
Author(s):  
Patrick B. Allen ◽  
Leanne M. Wiedemann
Keyword(s):  
Binding Site ◽  
Kinase Domain ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Abl Kinase ◽  
Activating Mutation

scholarly journals Plasmodium falciparum DHODH inhibitor complexes reveal flexible binding site

2014 ◽  
Vol 70 (a1) ◽  
pp. C1793-C1793
Author(s):  
Paul Rowland ◽  
Onkar SINGH ◽  
Leila Ross ◽  
Francisco Gamo ◽  
Maria Lafuente-Monasterio ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Binding Site ◽  
Crystal Structures ◽  
Point Mutations ◽  
Crystal Form ◽  
Drug Binding ◽  
Binding Modes ◽  
Resistance Mutations ◽  
Drug Binding Site

Malaria is a preventable and treatable disease, yet annually there are still hundreds of thousands of malaria-related deaths. The disease is caused by infection with mosquito-borne Plasmodium parasites. With hundreds of millions of cases each year there is a very high potential for drug resistance and this has compromised many existing therapies. One target under investigation is the enzyme dihydroorotate dehydrogenase (DHODH) which catalyses the rate-limiting step of pyrimidine biosynthesis and is an essential enzyme in the malaria parasite. There are currently several Plasmodium-selective DHODH inhibitors under development. To investigate the potential for drug resistance against DHODH inhibitors in vitro resistance selections were carried out using known inhibitors from different structural classes [1]. These studies identified point mutations in the drug binding site which lead to reduced sensitivity to the inhibitors, and in some cases increased sensitivity to a different inhibitor, suggesting a novel combination therapy approach to combat resistance. To help understand the significance of the inhibitor binding site mutations we determined the crystal structures of P. falciparum DHODH in complex with the inhibitors Genz-669178, IDI-6253 and IDI-6273. Co-crystallisation experiments led to a new crystal form in each case. Here we describe the crystal structures, the binding modes of the inhibitors and the great flexibility of the binding site, which is able to adjust to accommodate different inhibitor series. The structural role of the resistance mutations is also discussed.

Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4611-4614 ◽  
Author(s):  
Amie S. Corbin ◽  
Paul La Rosée ◽  
Eric P. Stoffregen ◽  
Brian J. Druker ◽  
Michael W. Deininger
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Cellular Assays ◽  
Kinase Domain Mutations

Abstract Imatinib mesylate is a selective Bcr-Abl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Kinase domain mutations are the most commonly identified mechanism associated with relapse. Many of these mutations decrease the sensitivity of the Abl kinase to imatinib, thus accounting for resistance to imatinib. The role of other mutations in the emergence of resistance has not been established. Using biochemical and cellular assays, we analyzed the sensitivity of several mutants (Met244Val, Phe311Leu, Phe317Leu, Glu355Gly, Phe359Val, Val379Ile, Leu387Met, and His396Pro/Arg) to imatinib mesylate to better understand their role in mediating resistance.While some Abl mutations lead to imatinib resistance, many others are significantly, and some fully, inhibited. This study highlights the need for biochemical and biologic characterization, before a resistant phenotype can be ascribed to a mutant.

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