A Chemical Genetic Approach for Covalent Inhibition of Analogue-Sensitive Aurora Kinase

The perturbation of protein kinases with small organic molecules is a powerful approach to dissect kinase function in complex biological systems. Covalent kinase inhibitors that target thiols in the ATP binding pocket of the kinase domain proved to be ideal reagents for the investigation of highly dynamic cellular processes. However, due to the covalent inhibitors' possible off-target reactivities, it is required that the overall shape of the inhibitor as well as the intrinsic reactivity of the electrophile are precisely tuned to favor the reaction with only the desired cysteine. Here we report on the design and biological characterization of covalent anilinoquinazolines as potent inhibitors of genetically engineered Aurora kinase in fission yeast

Selective Detection of Allosteric Phosphatase Inhibitors

Normal cellular function, such as signal transduction, is largely controlled by the reversible phosphorylation of cellular proteins catalyzed by two major classes of enzymes, kinases and phosphatases. A misbalance in this complex and dynamic interplay leads to a variety of severe diseases, such as cancer, inflammation, or autoimmune diseases. This makes kinases as well as phosphatases equally attractive targets for therapeutic manipulation by small molecules. While the development of kinase inhibitors has resulted in several blockbuster drugs, such as imatinib, with remarkable success in the clinic and sales of many billions of U.S. dollars per year, not a single phosphatase inhibitor has yet been approved for clinical use. Similar to the kinase world, substrate-competitive phosphatase inhibitors have been developed but were not suitable for further development into clinical candidates due to their charge and limited selectivity. Research efforts, therefore, have shifted to the exploitation of allosteric sites that can regulate phosphatase activity and may enable the discovery of novel modulators of phosphatase activity with much improved pharmacological properties. However, assay systems, which enable the straightforward discovery of these inhibitor types, are missing. Here, we present a novel binding assay capable of detecting ligands of an allosteric pocket of the protein tyrosine phosphatase 1B. This assay is suitable for high-throughput screening and selectively detects ligands which bind to this unique site with a clear discrimination from substrate-competitive ligands

^<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

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

^<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

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

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

^<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

Direct Binding Assay for the Detection of Type IV Allosteric Inhibitors of Abl

Abelson (Abl) tyrosine kinase is an important cellular enzyme that is rendered constitutively active in the breakpoint cluster region (BCR)-Abl fusion protein, contributing to several forms of leukemia. Although inhibiting BCR-Abl activity with imatinib shows great clinical success, many patients acquire secondary mutations that result in resistance to imatinib. Second-generation inhibitors such as dasatinib and nilotinib can overcome the majority of these mutations but fail to treat patients with an especially prevalent T315I mutation at the gatekeeper position of the kinase domain. However, a combination of nilotinib with an allosteric type IV inhibitor was recently shown to overcome this clinically relevant point mutation. In this study, we present the development of a direct binding assay that enables the straightforward detection of allosteric inhibitors which bind within the myristate pocket of Abl. The assay is amenable to high-throughput screening and exclusively detects the binding of ligands to this unique allosteric site