The document describes the development of the protein inhibitor AP24534 (Ponatinib) for treatment of chronic myeloid leukemia (CML). AP24534 was shown to inhibit the BCR-ABL protein, including the drug-resistant T315I mutation. Crystallography analysis revealed that AP24534 binds to BCR-ABL in the inactive conformation and maintains similar protein contacts as Imatinib. A phase I clinical trial found that AP24534 was selectively toxic to CML cells and active against T315I.

1. Brad Pamnani
NYU Medical Center

2. 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.

3. 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.

4. Protein AP24534, a pan-BCR-ABL inhibitor
for treatment of CML

5. Treatment of CML Primary Cells with
AP24534 Inhibits Cellular Proliferation
Mononuclear cells derived from blood or
bone marrow of CML patients with BCR-
ABL-driven leukemia were exposed to
graded concentrations of AP24534 and
assayed viable cells after 72 hr.
Patients with myeloid blast crisis
harboring native BCR-ABL or BCR-
ABLT315I and
Cells from healthy individuals to were
also exposed to similar conditions.
Results: AP24534 induced a selective
reduction of viable cell numbers in
primary CML cells, with IC50 values
approximately 500-fold lower
than those observed with normal cells
(Figure A).

6. AP24534 Inhibits the Catalytic Activity of
ABLT315I

7. 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.

8. PDB Code for AP24534: 3IK3
Batch Precipitation, Batch
Dialysis, Vapor Diffusion by
Extraction Methodology
Hanging Drop and Sitting
Drop
pH 8.5
Temperature 277.0
0.1 M Tris-HCl (pH 8.5),
30% w/v polyethylene glycol
4000, and 0.2 M sodium
Details
acetate, VAPOR
DIFFUSION, HANGING
DROP, temperature 277K

9. 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.

10. 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.

11. Crystallographic Analysis of the Bcr-Abl
inhibitors
AP24534 in complex with the murine ABL T315I kinase
domain confirmed that AP24534 binds in the DFG-out mode
(Figure C) and maintains a network of protein contacts similar
to imatinib (Figure D and E).

13. UniProt Analysis:

14. 3IK3 Chain Representation
 Description of Proto-oncogene tyrosine-protein kinase ABL1
Identical chains
 Chain Type polypeptide(L): Length 288 residues
 Dssp secondary structure: 44% helical (15 helices; 128 residues)
15% beta sheet (12 strands; 45 residues)

15. 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

16. Blastp Results for AP24534

17. ClustalW Analysis

18. 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

19. 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).

20. Structural Similarity to 3KFA.A
3KFA.A: Results from a kinase mutation in
purine templates of DFG-in and DFG-out
and is also a dual Src-Abl inhibitors.

21. Structural Similarity to 3KFA.A

22. Future Scope
ARIAD Pharmaceuticals, Inc. Announces
Initiation of Ponatinib (AP24534) Pivotal
Trial in Drug-Resistant or Intolerant
Chronic Myeloid Leukemia
Ponatinib (previously known as AP24534), in
patients with resistant or intolerant chronic
myeloid leukemia (CML) and Philadelphia
positive acute lymphoblastic leukemia (Ph+
ALL). The PACE (Ponatinib Ph+ ALL
and CML Evaluation) trial is designed to
provide definitive clinical data for regulatory “The start of the pivotal PACE trial
approval of ponatinib in this setting. Ponatinib represents an important step in the
has been granted orphan drug status in both development of our second molecularly
the United States and Europe for the targeted cancer therapy,” stated
treatment of CML and Ph+ ALL. Harvey J. Berger, M.D., chairman and
chief executive officer at ARIAD.

23. Future Scope
Phase I trial indicates ponatinib may
thwart most resistant CML
Drug also acts against chronic myeloid
leukemia with untreatable T135I
mutation
"Ponatinib seems to be filling the gap
we had for patients who right now have
no good treatments left," said Jorge
Cortes, M.D.

24. 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]