Strukturbiologische Untersuchungen von Kinaseinhibitoren

The main function of protein kinases is widely considered the activation of their substrates by catalytic phosphotransfer from adenosine-5‘-triphosphate (ATP), explaining the central role of kinases in cellular signaling cascades. Recently, more and more functions independent from this catalytic mechanism were described, that are involved in several biological processes, e.g., by regulation or localization of their substrates via protein-protein interactions or the formation of multi-enzyme complexes, respectively. This is usually realized by addressing alternative binding regions distant from the enzyme’s active site. Hence, the discovery of corresponding binding pockets and the development of small molecules capable of modulating scaffolding functions are of great interest in current scientific research. In the present work, the structure-based design and organic synthesis of 2-arylquinazolines, which were identified as ligands of a lipophilic binding pocket (LiPoLis) in p38α mitogen-activated protein kinase (MAPK), is described. Since the function of this pocket is not fully understood yet, the LiPoLis were thought to aid in elucidating its biological role. The generated compounds were characterized applying several biophysical methods to determine their affinity towards the enzyme and the suggested binding mode was validated by means of protein crystallography. Concluding these studies, the ligands were shown to positively address the lipophilic pocket (LP) but exhibiting a very low affinity, making them unsuitable to serve as molecular probes in biological systems. Consequently, electrophile-decorated LiPoLis were designed to target cysteine mutants of p38α MAPK, forming a covalent bond within the LP to maximize the residence time at the enzyme. Employing mass spectrometric analysis, specific ligand-protein pairs were identified and in tandem measurements and via co-crystallization, the introduced cysteines were verified as the actual labeling positions. Therefore, it could be shown that the thus found ligands exhibit a favorable reactivity and accordingly irreversible modifications of the target enzyme, rendering them suitable to be used in future studies to dissect the biological function of the described binding pocket in p38α MAPK. In another part of this thesis, various complex crystal structures of diverse ATP-competitive p38α MAPK inhibitors could be solved and were discussed, significantly contributing insightful results with respect to the underlying scientific questions

Mechanistic insight into RET kinase inhibitors targeting the DFG-out conformation in RET-rearranged cancer

Oncogenic fusion events have been identified in a broad range of tumors. Among them, RET rearrangements represent distinct and potentially druggable targets that are recurrently found in lung adenocarcinomas. Here, we provide further evidence that current anti-RET drugs may not be potent enough to induce durable responses in such tumors. We report that potent inhibitors such as AD80 or ponatinib that stably bind in the DFG-out conformation of RET may overcome these limitations and selectively kill RET-rearranged tumors. Using chemical genomics in conjunction with phosphoproteomic analyses in RET-rearranged cells we identify the CCDC6-RETI788N mutation and drug-induced MAPK pathway reactivation as possible mechanisms, by which tumors may escape the activity of RET inhibitors. Our data provide mechanistic insight into the druggability of RET kinase fusions that may be of help for the development of effective therapies targeting such tumors

Structure-based design, synthesis and crystallization of 2-arylquinazolines as lipid pocket ligands of p38α MAPK.

In protein kinase research, identifying and addressing small molecule binding sites other than the highly conserved ATP-pocket are of intense interest because this line of investigation extends our understanding of kinase function beyond the catalytic phosphotransfer. Such alternative binding sites may be involved in altering the activation state through subtle conformational changes, control cellular enzyme localization, or in mediating and disrupting protein-protein interactions. Small organic molecules that target these less conserved regions might serve as tools for chemical biology research and to probe alternative strategies in targeting protein kinases in disease settings. Here, we present the structure-based design and synthesis of a focused library of 2-arylquinazoline derivatives to target the lipophilic C-terminal binding pocket in p38α MAPK, for which a clear biological function has yet to be identified. The interactions of the ligands with p38α MAPK was analyzed by SPR measurements and validated by protein X-ray crystallography

Binding modes of active site inhibitors and LiPoLis in p38<i>α</i> MAPK.

<p>(A) Superposed kinase domains of p38<i>α</i> MAPK (cyan) in complex with active site inhibitor BIRB-796 (yellow) (PDB: 1KV2) and the quinazoline-based LiPoLi <b>3</b> (green) (PDB: 4DLJ). (B) Detailed binding mode of <b>3</b> (green) in the LP of p38<i>α</i> MAPK (cyan), highlighting key structural elements and main interactions formed between the protein and the ligand. (C) Chemical structure of <b>3</b> with systematic numbering of the quinazoline scaffold and highlighted moieties selected for derivatization.</p

^<i>a</i> Synthesis of amine building block 16.

<p>^<i>a</i> Reagents and conditions: (a) ethane-1,2-diamine, K₂CO₃, I₂, <i>t</i>BuOH, 70°C, 3.5 h, 54%.</p

^<i>a</i> Synthesis and identity of 2-arylquinazolines.

<p>^<i>a</i> Reagents and conditions: (a) method A: benzamidine hydrochloride hydrate, AcOH, 2-methoxyethanol, 130°C, 18 h, 18–32%; method B: benzoic anhydride, formamide, 200°C, 5 min, MW, 31–37%; (b) method C: aldehyde, NaHSO₃, <i>p</i>TSA, DMAc, 155°C, 18 h, 10–34%; (c) method A: 1) SOCl₂, DMF, 80°C, 4 h, 2) amine, DIPEA, DCM/<i>i</i>PrOH (3:2), rt, 18 h, 66–95%; method B: 1) HCCP, DIPEA, MeCN, rt, 1 h, 2) amine, rt, 18 h, 68–87%; (d) method A: 10% Pd/C, ammonium formate, EtOH, 80°C, 1–3 h, 56–96%; method B: Fe, NH₄Cl, MeOH:H₂O (4:1), 80°C, 3–6 h, 81–87%; (e) acyl chloride, DIPEA, DCM, 0°C to rt or 50°C, 1–6 h, 69–80%.</p

^<i>a</i> Synthesis of aldehyde building blocks 17a and 17b.

<p>^<i>a</i> Reagents and conditions: (a) amine, Na₂CO₃, H₂O, reflux, 18 h, 55–58%.</p

Conformational changes of the LP upon LiPoLi binding.

<p>(A) Alignment of the p38<i>α</i>-<b>9c</b> (cyan) and p38<i>α</i>-<b>9h</b> (yellow) complex crystal structures. Displacement of helix 2L14 and minor rearrangement of loop <i>α</i>EF/<i>α</i>F and helix 1L14 (trajectories are shown as red dots); (B) Opening of the LP (red arrows) when <b>9c</b> is bound compared to the closed LP in presence of <b>9h</b>.</p

Compound design.

<p>(A) Design of LiPoLis based on the alignment of the crystal structures of <b>3</b> (green) and <b>1</b> (yellow) in complex with p38<i>α</i> (PDB-codes: 4DLJ and 3HVC). Overlay of <b>1</b> and modeled structures of (B) <b>9c</b> (cyan), (C) <b>9j</b> (white) and (D) <b>9i</b> (magenta) showcasing the proposed binding modes.</p

^<i>a</i> Synthesis of 19.

<p>^<i>a</i> Reagents and conditions: (a) <i>m</i>CPBA, DCM, rt, 3 h, 87%.</p