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Structural biology contributions to the discovery of drugs to treat chronic myelogenous leukaemia.

Cowan-Jacob SW, Fendrich G, Floersheimer A, Furet P, Liebetanz J, Rummel G, Rheinberger P, Centeleghe M, Fabbro D, Manley PW - Acta Crystallogr. D Biol. Crystallogr. (2006)

Bottom Line: More than 40 such point mutations have been observed in imatinib-resistant patients.The crystal structures of wild-type and mutant Abl kinase in complex with imatinib and other small-molecule Abl inhibitors were determined, with the aim of understanding the molecular basis of resistance and to aid in the design and optimization of inhibitors active against the resistance mutants.These results are presented in a way which illustrates the approaches used to generate multiple structures, the type of information that can be gained and the way that this information is used to support drug discovery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Basel, Switzerland. sandra.jacob@novartis.com

ABSTRACT
Chronic myelogenous leukaemia (CML) results from the Bcr-Abl oncoprotein, which has a constitutively activated Abl tyrosine kinase domain. Although most chronic phase CML patients treated with imatinib as first-line therapy maintain excellent durable responses, patients who have progressed to advanced-stage CML frequently fail to respond or lose their response to therapy owing to the emergence of drug-resistant mutants of the protein. More than 40 such point mutations have been observed in imatinib-resistant patients. The crystal structures of wild-type and mutant Abl kinase in complex with imatinib and other small-molecule Abl inhibitors were determined, with the aim of understanding the molecular basis of resistance and to aid in the design and optimization of inhibitors active against the resistance mutants. These results are presented in a way which illustrates the approaches used to generate multiple structures, the type of information that can be gained and the way that this information is used to support drug discovery.

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(a) Superposition of the four main DFG conformations observed in Abl kinase structures, with the active conformation in cyan, the DFG-out conformation in yellow, the DFG-flip conformation in grey and the Src-like inactive conformation in green. (b) Superposition of all structures reported here plus PDB entry 2g1t. The P-loop is shown in red, the C-helix is cyan, the A-loop is blue and all the ligands are shown in green. The superposition is based on an alignment of the C-terminal lobes to emphasize the relative differences in angles between the N- and C-terminal lobes of the kinase. (c) A stereoview of all the ligands superimposed (imatinib, magenta C atoms; NVP-AFN941, cyan C atoms; NVP-AFG210, yellow C atoms; NVP-AEG082, green C atoms; PD180970, grey C atoms).
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fig4: (a) Superposition of the four main DFG conformations observed in Abl kinase structures, with the active conformation in cyan, the DFG-out conformation in yellow, the DFG-flip conformation in grey and the Src-like inactive conformation in green. (b) Superposition of all structures reported here plus PDB entry 2g1t. The P-loop is shown in red, the C-helix is cyan, the A-loop is blue and all the ligands are shown in green. The superposition is based on an alignment of the C-terminal lobes to emphasize the relative differences in angles between the N- and C-terminal lobes of the kinase. (c) A stereoview of all the ligands superimposed (imatinib, magenta C atoms; NVP-AFN941, cyan C atoms; NVP-AFG210, yellow C atoms; NVP-AEG082, green C atoms; PD180970, grey C atoms).

Mentions: An attractive strategy to overcome or avoid most cases of resistance would be to administer two drugs in combination which utilize different binding interactions to inhibit the Abl kinase. In particular, a useful combination could be a compound which binds to the inactive conformation, such as imatinib, with a compound which binds to an active conformation. Examples of chemotypes used as leads for targeting the active conformation are NVP-AFN941 (tetrahydrostaurosporin) and PD180970 (Fig. 3 ▶). The structures of these complexes show that both inhibitors bind in the ATP site and form hydrogen bonds with the hinge region and that they are both within van der Waals distance of the Thr315 gatekeeper residue, although the contacts between PD180970 and Thr315 are much more extensive. The NVP-AFN941 complex structure essentially resembles that of an active kinase, despite the lack of phosphorylation on the A-loop, although some parts of the A-loop and the P-loop are disordered in the crystals. This structure is very similar to other structures of tyrosine kinases in complex with staurosporin, such as Lck, Zap-70, Syk and Fyn (Zhu et al., 1999 ▶; Jin et al., 2004 ▶; Atwell et al., 2004 ▶; Kinoshita et al., 2006 ▶). There are only minor differences in distant loops and the A-loop near the phosphorylation site, because some of these structures are phosphorylated and the Abl–NVP-AFN941 complex is not. The PD180970 structure, which is similar to that of a complex with a related compound published by Nagar et al. (2002 ▶), shows an inactive conformation of the P-loop and an unusual conformation of the DFG motif (Fig. 3 ▶), but otherwise resembles an active kinase conformation with respect to the position of the C-helix and the path of the rest of the A-loop. Tyr393 is sitting in the same position as the phosphorylated tyrosine in active Lck and there is room in this Abl structure for a phosphate group which could interact with Arg363 and His396. The conformation of the P-loop resembles that of the complex with imatinib, which shows that this conformation can be adopted with different chemotypes and it is not specifically stabilized by imatinib only. The conformation of the DFG motif involves the flipping over of Asp381 to make a strong hydrogen bond with the main-chain carbonyl of Val299 (Fig. 4 ▶). This results in the Asp381 side chain occupying what would be the position of the Phe382 side chain in the active conformation and the Phe382 side chain flipping over to occupy the site of the Asp381 side chain. This ‘DFG-flip’ conformation puts the Phe382 side chain in van der Waals contact distance of the inhibitor. The buffer used to grow the crystals of the Abl–PD180970 complex crystals has a pH of 7.0 (measured as pH 6.8), so it is unlikely that it is the crystal buffer that favours protonation of the Asp381 side chain, although it cannot be ruled out that this conformation is an artefact of the crystallization conditions. A similar conformation of the DFG motif is seen in other Abl structures (Nagar et al., 2002 ▶, 2003 ▶) and it may represent another natural inactive conformation of the kinase. This ‘DFG-flip’ conformation does not expose the pocket beyond the gatekeeper residue that becomes available for inhibitor binding in the ‘DFG-out’ conformation.


Structural biology contributions to the discovery of drugs to treat chronic myelogenous leukaemia.

Cowan-Jacob SW, Fendrich G, Floersheimer A, Furet P, Liebetanz J, Rummel G, Rheinberger P, Centeleghe M, Fabbro D, Manley PW - Acta Crystallogr. D Biol. Crystallogr. (2006)

(a) Superposition of the four main DFG conformations observed in Abl kinase structures, with the active conformation in cyan, the DFG-out conformation in yellow, the DFG-flip conformation in grey and the Src-like inactive conformation in green. (b) Superposition of all structures reported here plus PDB entry 2g1t. The P-loop is shown in red, the C-helix is cyan, the A-loop is blue and all the ligands are shown in green. The superposition is based on an alignment of the C-terminal lobes to emphasize the relative differences in angles between the N- and C-terminal lobes of the kinase. (c) A stereoview of all the ligands superimposed (imatinib, magenta C atoms; NVP-AFN941, cyan C atoms; NVP-AFG210, yellow C atoms; NVP-AEG082, green C atoms; PD180970, grey C atoms).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483489&req=5

fig4: (a) Superposition of the four main DFG conformations observed in Abl kinase structures, with the active conformation in cyan, the DFG-out conformation in yellow, the DFG-flip conformation in grey and the Src-like inactive conformation in green. (b) Superposition of all structures reported here plus PDB entry 2g1t. The P-loop is shown in red, the C-helix is cyan, the A-loop is blue and all the ligands are shown in green. The superposition is based on an alignment of the C-terminal lobes to emphasize the relative differences in angles between the N- and C-terminal lobes of the kinase. (c) A stereoview of all the ligands superimposed (imatinib, magenta C atoms; NVP-AFN941, cyan C atoms; NVP-AFG210, yellow C atoms; NVP-AEG082, green C atoms; PD180970, grey C atoms).
Mentions: An attractive strategy to overcome or avoid most cases of resistance would be to administer two drugs in combination which utilize different binding interactions to inhibit the Abl kinase. In particular, a useful combination could be a compound which binds to the inactive conformation, such as imatinib, with a compound which binds to an active conformation. Examples of chemotypes used as leads for targeting the active conformation are NVP-AFN941 (tetrahydrostaurosporin) and PD180970 (Fig. 3 ▶). The structures of these complexes show that both inhibitors bind in the ATP site and form hydrogen bonds with the hinge region and that they are both within van der Waals distance of the Thr315 gatekeeper residue, although the contacts between PD180970 and Thr315 are much more extensive. The NVP-AFN941 complex structure essentially resembles that of an active kinase, despite the lack of phosphorylation on the A-loop, although some parts of the A-loop and the P-loop are disordered in the crystals. This structure is very similar to other structures of tyrosine kinases in complex with staurosporin, such as Lck, Zap-70, Syk and Fyn (Zhu et al., 1999 ▶; Jin et al., 2004 ▶; Atwell et al., 2004 ▶; Kinoshita et al., 2006 ▶). There are only minor differences in distant loops and the A-loop near the phosphorylation site, because some of these structures are phosphorylated and the Abl–NVP-AFN941 complex is not. The PD180970 structure, which is similar to that of a complex with a related compound published by Nagar et al. (2002 ▶), shows an inactive conformation of the P-loop and an unusual conformation of the DFG motif (Fig. 3 ▶), but otherwise resembles an active kinase conformation with respect to the position of the C-helix and the path of the rest of the A-loop. Tyr393 is sitting in the same position as the phosphorylated tyrosine in active Lck and there is room in this Abl structure for a phosphate group which could interact with Arg363 and His396. The conformation of the P-loop resembles that of the complex with imatinib, which shows that this conformation can be adopted with different chemotypes and it is not specifically stabilized by imatinib only. The conformation of the DFG motif involves the flipping over of Asp381 to make a strong hydrogen bond with the main-chain carbonyl of Val299 (Fig. 4 ▶). This results in the Asp381 side chain occupying what would be the position of the Phe382 side chain in the active conformation and the Phe382 side chain flipping over to occupy the site of the Asp381 side chain. This ‘DFG-flip’ conformation puts the Phe382 side chain in van der Waals contact distance of the inhibitor. The buffer used to grow the crystals of the Abl–PD180970 complex crystals has a pH of 7.0 (measured as pH 6.8), so it is unlikely that it is the crystal buffer that favours protonation of the Asp381 side chain, although it cannot be ruled out that this conformation is an artefact of the crystallization conditions. A similar conformation of the DFG motif is seen in other Abl structures (Nagar et al., 2002 ▶, 2003 ▶) and it may represent another natural inactive conformation of the kinase. This ‘DFG-flip’ conformation does not expose the pocket beyond the gatekeeper residue that becomes available for inhibitor binding in the ‘DFG-out’ conformation.

Bottom Line: More than 40 such point mutations have been observed in imatinib-resistant patients.The crystal structures of wild-type and mutant Abl kinase in complex with imatinib and other small-molecule Abl inhibitors were determined, with the aim of understanding the molecular basis of resistance and to aid in the design and optimization of inhibitors active against the resistance mutants.These results are presented in a way which illustrates the approaches used to generate multiple structures, the type of information that can be gained and the way that this information is used to support drug discovery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Basel, Switzerland. sandra.jacob@novartis.com

ABSTRACT
Chronic myelogenous leukaemia (CML) results from the Bcr-Abl oncoprotein, which has a constitutively activated Abl tyrosine kinase domain. Although most chronic phase CML patients treated with imatinib as first-line therapy maintain excellent durable responses, patients who have progressed to advanced-stage CML frequently fail to respond or lose their response to therapy owing to the emergence of drug-resistant mutants of the protein. More than 40 such point mutations have been observed in imatinib-resistant patients. The crystal structures of wild-type and mutant Abl kinase in complex with imatinib and other small-molecule Abl inhibitors were determined, with the aim of understanding the molecular basis of resistance and to aid in the design and optimization of inhibitors active against the resistance mutants. These results are presented in a way which illustrates the approaches used to generate multiple structures, the type of information that can be gained and the way that this information is used to support drug discovery.

Show MeSH
Related in: MedlinePlus