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Introduction

Potatinib was developed as a treatment option for chronic myeloid leukemia (CML) as other inhibitors in treatment have become ineffective. Further mutations in BCR-ABL, a kinase with a cancerous genetic mutation in chromosome 22 that leaves it always active, has left earlier versions of tyrosine kinases unable to bind in almost 30% of cases over a course of 5 years of treatment. The mutant BCR-ABL kinase’s ability to develop new mutations has pushed for newer developments in inhibitiors like Potatinib [7].

Fibroblast growth factor (FGFR) signaling, the factor that normally activates the BCR-ABL kinase, is the protein behind both tissue development and repair. The activation happens through a series of cascading signals that induce proliferation and migration in cells. Though mutations in the regulation of the FGFR tyrosine kinase family can result in malignant tumor growth [1]. The tyrosine kinase inhibitor Ponatinib has been used to bind to the mutant version of kinase BCR-ABL by the kinase's specific "DFG-out" conformation. The "DFG-out" conformation has the phenylalanine group of BCR-ABL flipped out of its hydrophobic binding site. Ponatinib is the first of its kind to be able to inhibit this specific mutation in BCR-ABL of the "DGF-out" combination [1].

The side effects of have caused Ponatinib to fall under scrutiny from the U.S. Food and Drug Administration (FDA). Ponatinib has shown to increase chances of deadly blood clotting and restenosis in both arteries and veins with a rate of about 1 in 5 patients. The drug has also shown to increase risk of heart attack and overall worsening of heart disease in patients.

Fibrolast Growth Factor Receptor (FGFR) plays a role in causing a variety of cancers. FGFR plays a role in the signaling system with FGF by initiating signaling cascades that control development and tissue repair. The disruption of this signaling system is involved in tumor growth^[2]

Ponatinib is a relatively new anti-cancer drug that inhibits FGFR. While most inhibitors are selective for FGFR1-3 and show a reduced binding to FGFR4, Ponatinib binds to FGFR4^[3]. Further, the drug is a Bcr-Abl tyrosine kinase inhibitor (TKI)^[4]. Ponatinib is specifically prescribed to patients with chronic myeloid leukemia and Philadelphia-chromosome positive active lymphoblastic leukemia. Although, it has recently shown side effects causing blood clots and narrowing blood vessels.

•Plays a role in a variety of cancers

•Ponatinib is a drug prescribed to patients with chronic myeloid leukemia and Philadelphia-chromosome positive active lymphoblastic leukemia. Although, it has recently shown side effects causing blood clots and narrowing blood vessels.

•FGFR plays a role in the signaling system with FGF by initiating signaling cascades that control development and tissue repair. The disruption of this signaling system is involved in tumor growth^[5]

•While most inhibitors are selective for FGFR1-3 and show a reduced binding to FGFR4, Ponatinib binds to FGFR4^[6]

•Ponatinib is a Bcr-Abl tyrosine kinase inhibitor (TKI)^[7]

Overall Structure

•Ligands: SO4, 0LI [C29 H27 F3 N6 O]

•Identical amino acid sequence to cI44 with the exception of 1 residue.^[8]

secondary structure with two key residues in the activation loop (highlighted in light blue) for Ponatinab binding

Binding Interactions

•Ponatinib binds to the "DFG-out" conformation of Bcr-Abl, where the Phe (F) is out of its hydrophobic pocket^[9]

•To bind there must be a a conformational rearrangement of the conserved Asp630-Phe631-Gly632 (DFG) tripeptide motif at the proximal end of the activation loop^[10]

FGFR4 active site for ponatinib binding with involved residues labeled

Kinases are the largest drug targets currently being tested in clinical trials. All kinases possess a biolobal fold that is a smaller N-terminal and a larger C-terminal lobe joined together by a “hinge.” The cofactor ATP binds deeply into a pocket between the lobes and binds to the hinge region. The imposition of any other residue in this ATP-binding pocket controls access to the hydrophobic pocket by separating the adenine binding site from an adjacent hydrophobic pocket. Such residues are termed “gatekeepers,” and are critical considerations in the development of drugs to treat CML because of the mutations that these residues can ensue. Gatekeeper mutations that convert a small hydrophilic residue into a large hydrophobic residue are one example of what has been shown to result in drug resistance, specifically to the most well-known ABL inhibitors like imatinib (Gleevec) [4]. Ponatinib is a third generation type II pan-BCR-ABL kinase inhibitor, which allows it to bind even with the presence of gatekeeper mutations [5]. Type II inhibitors are classified by binding to the hydrophobic and allosteric pocket that is only accessible in the DFG-out conformation and that is next to the ATP binding pocket. Additionally, type II inhibitors extend deep into the adenine pocket and hydrogen bond with the hinge region [4]. This unique ability is caused by ponatinib’s ability to overcome resistances of the BCR-ABL gatekeeper mutant T315I at low concentrations (low IC50s ranging from 0.5 nM to 36 nM) by an ethynyl linker in the conformation [5, 6]. The T315I mutation accounts for 15-20% of all clinically observed mutations and it is resistant to all previous generation drugs (imatinib, nilotinib, dasatinib). Additionally, ponatinib has a very high potency against native ABL which allows the binding energy to be distributed over many protein residues [5].

The specific binding of ponatinib can be categorized into and explained by five major chemical components, they are: (1) the template that interacts with the hinge region; (2) the A ring that occupies the hydrophobic pocket behind the gatekeeper residue; (3) the key ethynyl linker that joins the template and A ring, and that interacts with the gatekeeper residue (linker 1); (4) the A-B ring linker (linker 2); and (5) the B ring that binds to the DFG-out pocket [5].

Ponatinib binds into the ATP binding pocket between the N and C lobes to induce a shift from the DFG-in to the DFG-out conformation. It covers an immense region that spans from the kinase hinge region (back of kinase) to the catalytic pocket (front of kinase). Three sites are engaged in the ATP binding cleft by ponatinib’s aromatic rings. In the first site, the imidazo[1,2b]pyridazine scaffold takes up the same space as the adenine ring of ATP and it is able to form one hydrogen bone with the backbone amide nitrogen atom of Ala553 in the hinge [7]. Both of its rings form several van der Waals contacts with residues in the N and C lobes of the adenine binding site as well [5]. Rigid acetylene linkage drives the rest of the drug into the back of the ATP binding pocket. In the second site, the methylphenyl group displaces the side chain of the catalytic Lys503 and its aromatic ring binds to the hydrophobic pocket that is formed by Val550, the gatekeeper residue, and Met524 [7]. Val550 is stabilized by the benzimide group [6]. This displacement allows Glu520 in the αC helix to hydrogen bond with the amide linkage between the aromatic rings in ponatinib. In the third site, Phe631 of DFG is expelled out of the cleft by ponatinib’s 3-trifluoromethylphenyl moiety, which takes the place of Phe631. Phe631’s new position enables it to make hydrophobic contact with the drug’s scaffold and acetylene linker. Also, Asp630 of DFG becomes available for hydrogen bonding with the amide linkage between the aromatic rings in ponatinib. This also puts the piperazine ring in the position to engage in hydrogen bonding with the catalytic loop. This is shift forms the DFG-out conformation [7].

Other inhibitors are not as potent as ponatinib against FGFR kinases because they are unable to penetrate far enough to access the third site and cannot assume the DFG-out conformation [7].

Additional Features

Ponatinib is an orally ingested tyrosine kinase inhibitor that has shown promising avenues of treatment for counteracting the effects of angiogenesis in tumor growth. Besides the inhibition of FGFRs, this agent inhibits tyrosine kinases involved in vascular endothelial growth factor receptors. Ponatinib is considered a third generation TKI that can treat even the most drug-therapy resistant mutations that previous TKIs were incapable of treating[8].

The brand name for ponatinib is Iclusig. Iclusig received an accelerated approval grant through the Food and Drug Administration. It was mainly prescribed to patients suffering from CML or ALL who did not make any progress with the first and second generation TKIs. However, the clinical trials data revealed a spike in adverse effects. These consequences include heart failure, stroke, coronary artery disease, loss of blood flow to body parts leading to amputation amongst other narrowing of blood vessels[9].

• FGFR-4 is abundantly present in human prostate cancer

•Variant of FGFR-4 with (Arg(388)) replacing (Gly(388)) is associated with increased human prostate cancer. This causes increased receptor stability and activation. [2]

Quiz Question 1

What allows ponatinib to have increased inhibitory effects compared to other BCR-ABL inhibitors?

a) Binding site 1

b) Gatekeeper mutant T315I

c) DFG-out conformation

d) piperazine ring

See Also

• Fibroblast growth factor receptor
• 4uxq
• 4v01
• 4v04
• 4v05

Credits

Introduction - Emily & Cory

Overall Structure - Nicole & Connor

Drug Binding Site - Julie & Cory

Additional Features - Emily & Nicole

Quiz Question 1 - Julie & Connor

References

1. ↑ Tucker JA, Klein T, Breed J, Breeze AL, Overman R, Phillips C, Norman RA. Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4. Structure. 2014 Dec 2;22(12):1764-74. doi: 10.1016/j.str.2014.09.019. Epub 2014, Nov 20. PMID:25465127 doi:http://dx.doi.org/10.1016/j.str.2014.09.019
2. ↑ Tucker JA, Klein T, Breed J, Breeze AL, Overman R, Phillips C, Norman RA. Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4. Structure. 2014 Dec 2;22(12):1764-74. doi: 10.1016/j.str.2014.09.019. Epub 2014, Nov 20. PMID:25465127 doi:http://dx.doi.org/10.1016/j.str.2014.09.019
3. ↑ Tucker JA, Klein T, Breed J, Breeze AL, Overman R, Phillips C, Norman RA. Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4. Structure. 2014 Dec 2;22(12):1764-74. doi: 10.1016/j.str.2014.09.019. Epub 2014, Nov 20. PMID:25465127 doi:http://dx.doi.org/10.1016/j.str.2014.09.019
4. ↑ Tucker JA, Klein T, Breed J, Breeze AL, Overman R, Phillips C, Norman RA. Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4. Structure. 2014 Dec 2;22(12):1764-74. doi: 10.1016/j.str.2014.09.019. Epub 2014, Nov 20. PMID:25465127 doi:http://dx.doi.org/10.1016/j.str.2014.09.019
5. ↑ Tucker JA, Klein T, Breed J, Breeze AL, Overman R, Phillips C, Norman RA. Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4. Structure. 2014 Dec 2;22(12):1764-74. doi: 10.1016/j.str.2014.09.019. Epub 2014, Nov 20. PMID:25465127 doi:http://dx.doi.org/10.1016/j.str.2014.09.019
6. ↑ Tucker JA, Klein T, Breed J, Breeze AL, Overman R, Phillips C, Norman RA. Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4. Structure. 2014 Dec 2;22(12):1764-74. doi: 10.1016/j.str.2014.09.019. Epub 2014, Nov 20. PMID:25465127 doi:http://dx.doi.org/10.1016/j.str.2014.09.019
7. ↑ Tucker JA, Klein T, Breed J, Breeze AL, Overman R, Phillips C, Norman RA. Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4. Structure. 2014 Dec 2;22(12):1764-74. doi: 10.1016/j.str.2014.09.019. Epub 2014, Nov 20. PMID:25465127 doi:http://dx.doi.org/10.1016/j.str.2014.09.019
8. ↑ Ron D, Reich R, Chedid M, Lengel C, Cohen OE, Chan AM, Neufeld G, Miki T, Tronick SR. Fibroblast growth factor receptor 4 is a high affinity receptor for both acidic and basic fibroblast growth factor but not for keratinocyte growth factor. J Biol Chem. 1993 Mar 15;268(8):5388-94. PMID:7680645
9. ↑ Tucker JA, Klein T, Breed J, Breeze AL, Overman R, Phillips C, Norman RA. Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4. Structure. 2014 Dec 2;22(12):1764-74. doi: 10.1016/j.str.2014.09.019. Epub 2014, Nov 20. PMID:25465127 doi:http://dx.doi.org/10.1016/j.str.2014.09.019
10. ↑ Tucker JA, Klein T, Breed J, Breeze AL, Overman R, Phillips C, Norman RA. Structural Insights into FGFR Kinase Isoform Selectivity: Diverse Binding Modes of AZD4547 and Ponatinib in Complex with FGFR1 and FGFR4. Structure. 2014 Dec 2;22(12):1764-74. doi: 10.1016/j.str.2014.09.019. Epub 2014, Nov 20. PMID:25465127 doi:http://dx.doi.org/10.1016/j.str.2014.09.019

[1] Ron D, Reich R, Chedid M, Lengel C, Cohen OE, Chan AM, Neufeld G, Miki T, Tronick SR. Fibroblast growth factor receptor 4 is a high affinity receptor for both acidic and basic fibroblast growth factor but not for keratinocyte growth factor. J Biol Chem. 1993 Mar 15;268(8):5388-94. PMID:http://www.ncbi.nlm.nih.gov/pubmed/7680645

[2] Wang J, Yu W, Cai Y, Ren C, Ittmann MM. Altered fibroblast growth factor receptor 4 stability promotes prostate cancer progression. Neoplasia. 2008 Aug;10(8):847-56. PMID: http://www.ncbi.nlm.nih.gov/pubmed/18670643

[3] Tucker, J. A.; Klein, T.; Breed, J.; Breeze, A. L.; Overman, R.; Phillips, C.; Norman, R. A. Structural insights into FGFR kinase isoform selectivity: diverse binding modes of AZD4547 and ponatinib in complex with FGFR1 and FGFR4. Structure 2014, 22, 1764-1774.

[4] Huang, Zhifeng et al. "DFG-Out Mode Of Inhibition By An Irreversible Type-1 Inhibitor Capable Of Overcoming Gate-Keeper Mutations In FGF Receptors". ACS Chem. Biol. 10.1 (2015): 299-309. Web.

[5] Lesca, E. et al. "Structural Analysis Of The Human Fibroblast Growth Factor Receptor 4 Kinase". Journal of Molecular Biology 426.22 (2014): 3744-3756. Web.

[6] Vijayan, R. S. K. et al. "Conformational Analysis Of The DFG-Out Kinase Motif And Biochemical Profiling Of Structurally Validated Type II Inhibitors". J. Med. Chem. 58.1 (2015): 466-479. Web.

[7] Zhou, Tianjun et al. "Structural Mechanism Of The Pan-BCR-ABL Inhibitor Ponatinib (AP24534): Lessons For Overcoming Kinase Inhibitor Resistance". Chemical Biology & Drug Design 77.1 (2010): 1-11. Web.

[8]Price KE, Saleem N, Lee G, Steinberg M. Potential of ponatinib to treat chronic myeloid leukemia and acute lymphoblastic leukemia. OncoTargets and therapy. 2013;6:1111-1118. doi:10.2147/OTT.S36980.

[9]FDA Drug Safety Communication: FDA investigating leukemia drug Iclusig (ponatinib) after increased reports of serious blood clots in arteries and veins; Drug Safety and Availability; United States Food and Drug Administration (2013). Web.