Abstract Philadelphia chromosome or Philadelphia translocation, a chromosomal abnormality associated with Chronic Myelogenous Leukemia (CML) by a reciprocal translocation between chromosome 9 and 22, results in abnormal protein translation by newly formed oncogene Abl-Bcr. Drug Imatinib (Gleevec) has helped in inhibiting BCR-ABL expression and has dampened tumorogensis in many patients with CML. But resistance attributed to kinase domain mutations can lead to relapse, for which second-line therapy with nilotinib or dasatinib has been applied. Inspite of the three approved therapeutic options, the cross- resistant BCR-ABLT315I mutation and compound mutants selected on sequential inhibitor therapy still remain major clinical challenges.
Protein Background For the above reasons, an artificial inhibitor protein, AP24534 (Ponatinib) was designed and first reported by Ariad Scientists, Oregon Health and Science University, Knight Cancer Institute and Howard Hughes Medical Institute. AP24534, an orally available multi-targeted kinase inhibitor was clinically active against T315I and other BCR-ABL mutants. AP24534 also inhibited all tested BCR-ABL mutants in cellular and biochemical assays.
Protein AP24534, a pan-BCR-ABL inhibitor for treatment of CML
Treatment of CML Primary Cells withAP24534 Inhibits Cellular ProliferationMononuclear cells derived from blood orbone marrow of CML patients with BCR-ABL-driven leukemia were exposed tograded concentrations of AP24534 andassayed viable cells after 72 hr.Patients with myeloid blast crisisharboring native BCR-ABL or BCR-ABLT315I andCells from healthy individuals to werealso exposed to similar conditions.Results: AP24534 induced a selectivereduction of viable cell numbers inprimary CML cells, with IC50 valuesapproximately 500-fold lowerthan those observed with normal cells(Figure A).
AP24534 Inhibits the Catalytic Activity ofABLT315I
Chemical structure of AP24534. The structure of AP24534 in complex with ABLT315I was determined by molecular replacement by AMoRe (Navaza, 1994) using the structure of native ABL bound with imatinib (PDB code: 1IEP). There were two ABLT315I molecules in the asymmetric unit.
PDB Code for AP24534: 3IK3 Batch Precipitation, Batch Dialysis, Vapor Diffusion byExtraction Methodology Hanging Drop and Sitting DroppH 8.5Temperature 277.0 0.1 M Tris-HCl (pH 8.5), 30% w/v polyethylene glycol 4000, and 0.2 M sodiumDetails acetate, VAPOR DIFFUSION, HANGING DROP, temperature 277K
Extraction Procedure:•Kinase domain of murine ABLT315I (residues 229–515) were co-expressed with YopH protein tyrosine phosphatase in E. coli (Zhou et al., 2007) and purified in the presence of AP24534 to near homogeneity (> 95%) using metal affinity, Mono Q, and size exclusion chromatography (O’Hare et al. 2009).•The typical yield of purified ABLT315I bound with AP24534 was about 1 mg/L.•Co-crystals of ABLT315I and AP24534 were grown by the hanging drop vapor diffusion method at 4oC by mixing equal volumes of the AP24534:ABLT315I complex (25 mg/mL) and well solution (30% w/v polyethylene 4000, 0.2 M sodium acetate, 0.1 M Tris, pH 8.5).•After 1–2 days, crystals reached a typical size of 50 × 50 × 300 μm3 and were harvested in mother liquor and supplemented with 30% v/v glycerol as cyroprotectant.•X-ray diffraction data were collected at 100K at beamline 19 BM (Advanced Photon Service, Argonne, IL). The data were indexed and scaled in space group P21 by using the HKL2000 package (Otwinowski and Minor,1997).•The structure of AP24534 in complex with ABLT315I was determined by molecular replacement by AMoRe (Navaza, 1994) using the structure of native ABL boun with imatinib (PDB code: 1IEP). There were two ABLT315I molecules in the asymmetric unit.
Crystallographic Analysis of AP24534: ABLT315I Crystal structure of AP24534 in complex with the ABL T315I mutant kinase. And AP24534 (green). The side chain of the mutated gatekeeper residue Ile315 (red). The side chains of Y253 and E255 and locations of point mutations appearing in the resistant outgrowth screen of AP24534 are shown in grey along with the C-helix (αC). Imatinib, nilotinib and dasatinib each form a hydrogen bond with the side chain of T315 in native ABL, ligands were designed to devoid this interaction by introducing vinyl and ethyl linkages into a purine- based inhibitor scaffold.
Crystallographic Analysis of the Bcr-Abl inhibitorsAP24534 in complex with the murine ABL T315I kinasedomain confirmed that AP24534 binds in the DFG-out mode(Figure C) and maintains a network of protein contacts similarto imatinib (Figure D and E).
Blastp of AP24534: Obtain protein sequences to see similarity in different species http://blast.ncbi.nlm.nih.gov/Blast.cgi Identify conserved regions indicating evolutionary relatedness http://www.ebi.ac.uk/Tools/clustalw2/index.html
ClustalW Analysis The “*” refers to identical amino acids The “:” refers to highly conserved areas The “.” refers to somewhat similar areas The gap refers to dissimilar areas Therefore, the first 16 amino acids have the most conserved domain
ClustalW Analysis The Cladogram shows that AP24534 is closely related to the Tyrosine-protein kinase ABL1 polypeptide(L) protein (3KFA) and not so much to the muscle specific tyrosine kinase (1LUF).
Structural Similarity to 3KFA.A3KFA.A: Results from a kinase mutation inpurine templates of DFG-in and DFG-outand is also a dual Src-Abl inhibitors.
Future ScopeARIAD Pharmaceuticals, Inc. AnnouncesInitiation of Ponatinib (AP24534) PivotalTrial in Drug-Resistant or IntolerantChronic Myeloid LeukemiaPonatinib (previously known as AP24534), inpatients with resistant or intolerant chronicmyeloid leukemia (CML) and Philadelphiapositive acute lymphoblastic leukemia (Ph+ALL). The PACE (Ponatinib Ph+ ALLand CML Evaluation) trial is designed toprovide definitive clinical data for regulatory “The start of the pivotal PACE trialapproval of ponatinib in this setting. Ponatinib represents an important step in thehas been granted orphan drug status in both development of our second molecularlythe United States and Europe for the targeted cancer therapy,” statedtreatment of CML and Ph+ ALL. Harvey J. Berger, M.D., chairman and chief executive officer at ARIAD.
Future ScopePhase I trial indicates ponatinib maythwart most resistant CMLDrug also acts against chronic myeloidleukemia with untreatable T135Imutation "Ponatinib seems to be filling the gap we had for patients who right now have no good treatments left," said Jorge Cortes, M.D.
References: Amor, J., Harrison, D., Kahn, R., & Ringe, D. (1994). Structure of the human ADP-ribosylatioin factor 1 complexed with GDP. Letters to Nature, 372, 704-708. Navaza J. A MoRe. Acta Crystallogr A 1994;A50:157–163. Otwinowski Z, Minor W. Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods Zhou T, Parillon L, Li F, Wang Y, Keats J, Lamore S, Xu Q, Shakespeare W, Dalgarno D, Zhu X. Crystal structure of the T315I mutant of Abl kinase. Chem Biol Drug Des 2007;70:171–181. [PubMed:17718712]