Correspondence on "Synergy and Antagonism between Allosteric and Active-Site Inhibitors of Abl Tyrosine Kinase".

Soellner published on the interplay between allosteric and adenosine triphosphate (ATP)-competitive inhibitors of ABL kinase, showing that the latter preferably binds to different conformational states of ABL compared to allosteric agents that specifically target the ABL myristate pocket (STAMP) and deducing that asciminib cannot bind to ABL simultaneously with ATP-competitive drugs. These results are to some extent in line with ours, although our analyses of dose-response matrices from combinations of asciminib with imatinib, nilotinib or dasatinib, show neither synergy nor antagonism, but suggest additive antiproliferative effects on BCR-ABL-dependent KCL22 cells. Furthermore, our X-ray crystallographic, solution nuclear magnetic resonance (NMR), and isothermal titration calorimetry studies show that asciminib can bind ABL concomitantly with type-1 or -2 ATP-competitive inhibitors to form ternary complexes. Concomitant binding of asciminib with imatinib, nilotinib, or dasatinib might translate to benefit some chronic myeloid leukaemia patients.

A kinase inhibitor which specifically targets the ABL myristate pocket (STAMP), but unlike asciminib crosses the blood-brain barrier.

The ubiquitously expressed ABL1 and ABL2 protein kinases play many important roles in cell function. Although they have been implicated in neuron development, maintenance and signaling, there are no good tool compounds to evaluate the effects of ABL kinase inhibition in the brain. Asciminib is a recently approved drug that specifically and potently inhibits the tyrosine kinase activity of ABL1, ABL2 and that of the chimeric BCR-ABL1 oncoprotein which causes chronic myeloid leukemia. Herein we show that asciminib does not penetrate the intact blood-brain barrier (BBB) following administration to rats, which curtails its utility for assessing the in vivo effects of ABL kinase inhibition in the brain. However, we describe another specific ABL kinase inhibitor, possessing physicochemical characteristics suitable for BBB penetration, and which after administration (either i.v., i.p. or p.o.) to mice achieves substantial, pharmacologically relevant brain concentrations. This bipyridine compound (4) therefore has potential for elucidating the role of ABL kinases in the brain in non-clinical studies.

BET bromodomain inhibitors regulate keratinocyte plasticity.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

Discovery of Potent, Highly Selective, and In Vivo Efficacious, Allosteric MALT1 Inhibitors by Iterative Scaffold Morphing.

MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.

The specificity of asciminib, a potential treatment for chronic myeloid leukemia, as a myristate-pocket binding ABL inhibitor and analysis of its interactions with mutant forms of BCR-ABL1 kinase.

Asciminib is a potent, orally bioavailable, investigational drug that specifically and potently inhibits the tyrosine kinase activity of native ABL1, together with that of the chimeric BCR-ABL1 oncoprotein which causes chronic myeloid leukemia (CML). In contrast to ATP-competitive BCR-ABL1 kinase inhibitors employed to treat CML that target multiple kinases, asciminib binds to the myristate binding pocket on the kinase domains of ABL1 and BCR-ABL1. Hitherto no drugs have been developed whose mechanism of action involves interacting with myristate binding pockets on proteins, and analysis of the structures of such binding sites in proteins other than ABL1/ABL2/BCR-ABL1 strongly suggest that asciminib will not bind to these with high affinity. Accordingly, the drug has no known safety liabilities resulting from any off-target activity, as illustrated by its specificity towards cells expressing BCR-ABL1 and lack of effects on non-kinase targets in biochemical screens. Because asciminib does not bind to the ATP-binding site it maintains substantial activity against kinase domain mutations that impart acquired drug resistance to ATP-competitive drugs. However, in vitro studies in cells have identified BCR-ABL1 mutations that reduce the anti-proliferative activity of asciminib, some of which are associated with clinical resistance towards the drug in patients. Here we review effects of asciminib on mutant forms of BCR-ABL1, analyse their sensitivity towards the drug from a structural perspective and affirm support for employing combinations with ATP-competitive inhibitors to impede the reactivation of BCR-ABL1 kinase activity in patients receiving monotherapy.

Investigations into the Potential Role of Metabolites on the Anti-Leukemic Activity of Imatinib, Nilotinib and Midostaurin.

The efficacy and side-effects of drugs do not just reflect the biochemical and pharmacodynamic properties of the parent compound, but often comprise of cooperative effects between the properties of the parent and active metabolites. Metabolites of imatinib, nilotinib and midostaurin have been synthesised and evaluated in assays to compare their properties as protein kinase inhibitors with the parent drugs. The N-desmethyl-metabolite of imatinib is substantially less active than imatinib as a BCR-ABL1 kinase inhibitor, thus providing an explanation as to why patients producing high levels of this metabolite show a relatively low response rate in chronic myeloid leukaemia (CML) treatment. The hydroxymethylphenyl and N-oxide metabolites of imatinib and nilotinib are only weakly active as BCR-ABL1 inhibitors and are unlikely to play a role in the efficacy of either drug in CML. The 3-(R)-HO-metabolite of midostaurin shows appreciable accumulation following chronic drug administration and, in addition to mutant forms of FLT3, potently inhibits the PDPK1 and VEGFR2 kinases (IC50 values <100 nM), suggesting that it might contribute to drug efficacy in acute myeloid leukaemia patients. The case studies discussed here provide further examples of how the synthesis and characterisation of metabolites can make important contributions to understanding the clinical efficacy of drugs.

Tyrosine kinase inhibitors relax pulmonary arteries in human and murine precision-cut lung slices.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) inhibit the platelet derived growth factor receptor (PDGFR) and gain increasing significance in the therapy of proliferative diseases, e.g. pulmonary arterial hypertension (PAH). Moreover, TKIs relax pulmonary vessels of rats and guinea pigs. So far, it is unknown, whether TKIs exert relaxation in human and murine pulmonary vessels. Thus, we studied the effects of TKIs and the PDGFR-agonist PDGF-BB in precision-cut lung slices (PCLS) from both species. METHODS: The vascular effects of imatinib (mice/human) or nilotinib (human) were studied in Endothelin-1 (ET-1) pre-constricted pulmonary arteries (PAs) or veins (PVs) by videomicroscopy. Baseline initial vessel area (IVA) was defined as 100%. With regard to TKI-induced relaxation, K+-channel activation was studied in human PAs (PCLS) and imatinib/nilotinib-related changes of cAMP and cGMP were analysed in human PAs/PVs (ELISA). Finally, the contractile potency of PDGF-BB was explored in PCLS (mice/human). RESULTS: Murine PCLS: Imatinib (10 µM) relaxed ET-1-pre-constricted PAs to 167% of IVA. Vice versa, 100 nM PDGF-BB contracted PAs to 60% of IVA and pre-treatment with imatinib or amlodipine prevented PDGF-BB-induced contraction. Murine PVs reacted only slightly to imatinib or PDGF-BB. Human PCLS: 100 µM imatinib or nilotinib relaxed ET-1-pre-constricted PAs to 166% or 145% of IVA, respectively, due to the activation of KATP-, BKCa2+- or Kv-channels. In PVs, imatinib exerted only slight relaxation and nilotinib had no effect. Imatinib and nilotinib increased cAMP in human PAs, but not in PVs. In addition, PDGF-BB contracted human PAs/PVs, which was prevented by imatinib. CONCLUSIONS: TKIs relax pre-constricted PAs/PVs from both, mice and humans. In human PAs, the activation of K+-channels and the generation of cAMP are relevant for TKI-induced relaxation. Vice versa, PDGF-BB contracts PAs/PVs (human/mice) due to PDGFR. In murine PAs, PDGF-BB-induced contraction depends on intracellular calcium. So, PDGFR regulates the tone of PAs/PVs. Since TKIs combine relaxant and antiproliferative effects, they may be promising in therapy of PAH.

Discovery of Asciminib (ABL001), an Allosteric Inhibitor of the Tyrosine Kinase Activity of BCR-ABL1.

Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) that target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding. We describe the discovery of asciminib (ABL001), the first allosteric BCR-ABL1 inhibitor to reach the clinic. Asciminib binds to the myristate pocket of BCR-ABL1 and maintains activity against TKI-resistant ATP-site mutations. Although resistance can emerge due to myristate-site mutations, these are sensitive to ATP-competitive inhibitors so that combinations of asciminib with ATP-competitive TKIs suppress the emergence of resistance. Fragment-based screening using NMR and X-ray yielded ligands for the myristate pocket. An NMR-based conformational assay guided the transformation of these inactive ligands into ABL1 inhibitors. Further structure-based optimization for potency, physicochemical, pharmacokinetic, and drug-like properties, culminated in asciminib, which is currently undergoing clinical studies in CML patients.

Comparison of the Kinase Profile of Midostaurin (Rydapt) with That of Its Predominant Metabolites and the Potential Relevance of Some Newly Identified Targets to Leukemia Therapy.

The multitargeted protein kinase inhibitor midostaurin is approved for the treatment of both newly diagnosed FLT3-mutated acute myeloid leukemia (AML) and KIT-driven advanced systemic mastocytosis. AML is a heterogeneous malignancy, and investigational drugs targeting FLT3 have shown disparate effects in patients with FLT3-mutated AML, probably as a result of their inhibiting different targets and pathways at the administered doses. However, the efficacy and side effects of drugs do not just reflect the biochemical and pharmacodynamic properties of the parent compound but are often comprised of complex cooperative effects between the properties of the parent and active metabolites. Following chronic dosing, two midostaurin metabolites attain steady-state plasma trough levels greater than that of the parent drug. In this study, we characterized these metabolites and determined their profiles as kinase inhibitors using radiometric transphosphorylation assays. Like midostaurin, the metabolites potently inhibit mutant forms of FLT3 and KIT and several additional kinases that either are directly involved in the deregulated signaling pathways or have been implicated as playing a role in AML via stromal support, such as IGF1R, LYN, PDPK1, RET, SYK, TRKA, and VEGFR2. Consequently, a complex interplay between the kinase activities of midostaurin and its metabolites is likely to contribute to the efficacy of midostaurin in AML and helps to engender the distinctive effects of the drug compared to those of other FLT3 inhibitors in this malignancy.