Modular synthesis and modification of novel bifunctional dendrons.

Herein, we report the design and synthesis of two novel bifunctional dendrons bearing multiple amine termini at the periphery and an azide at the focal point. Copper-catalyzed alkyne-azide cycloaddition enabled modular dendritic scaffold assembly resulting in a first generation dendron carrying six amines and a second generation dendron carrying eighteen amines. Peripheral amines were labeled with multiple copies of a metal isotope, whereas the azide functionality at the focal point was employed in conjugation to a single anti-human CD4 antibody. We demonstrated that the highly monomeric first generation dendron-antibody conjugate selectively detected CD4+ T cells in the PMBC culture.

Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site.

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr-Abl, fail to effectively suppress the Bcr-Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr-Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.

Binding or bending: distinction of allosteric Abl kinase agonists from antagonists by an NMR-based conformational assay.

Allosteric inhibitors of Bcr-Abl have emerged as a novel therapeutic option for the treatment of CML. Using fragment-based screening, a search for novel Abl inhibitors that bind to the myristate pocket was carried out. Here we show that not all myristate ligands are functional inhibitors, but that the conformational state of C-terminal helix_I is a structural determinant for functional activity. We present an NMR-based conformational assay to monitor the conformation of this crucial helix_I and show that myristate ligands that bend helix_I are functional antagonists, whereas ligands that bind to the myristate pocket but do not induce this conformational change are kinase agonists. Activation of c-Abl by allosteric agonists has been confirmed in a biochemical assay.

Arylcarboxyamino-substituted diaryl ureas as potent and selective FLT3 inhibitors.

A series of diaryl ureas with an amide substitution at the 4-position was prepared and found to be potent and selective FLT3 inhibitors with good oral bioavailability and efficacy in a tumor xenograft model.

Discovery of Potent and Selective Antibody-Drug Conjugates with Eg5 Inhibitors through Linker and Payload Optimization.

Targeted antimitotic agents are a promising class of anticancer therapies. Herein, we describe the development of a potent and selective antimitotic Eg5 inhibitor based antibody-drug conjugate (ADC). Preliminary studies were performed using proprietary Eg5 inhibitors which were conjugated onto a HER2-targeting antibody using maleimido caproyl valine-citrulline para-amino benzocarbamate, or MC-VC-PABC cleavable linker. However, the resulting ADCs lacked antigen-specificity in vivo, probably from premature release of the payload. Second-generation ADCs were then developed, using noncleavable linkers, and the resulting conjugates (ADC-4 and ADC-10) led to in vivo efficacy in an HER-2 expressing (SK-OV-3ip) mouse xenograft model while ADC-11 led to in vivo efficacy in an anti-c-KIT (NCI-H526) mouse xenograft model in a target-dependent manner.

A small molecule-kinase interaction map for clinical kinase inhibitors.

Kinase inhibitors show great promise as a new class of therapeutics. Here we describe an efficient way to determine kinase inhibitor specificity by measuring binding of small molecules to the ATP site of kinases. We have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, against a panel of 119 protein kinases. We find that specificity varies widely and is not strongly correlated with chemical structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. We also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small molecule-protein interaction map for clinical compounds across a large number of related proteins.

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.

Identification of N-(5-tert-butyl-isoxazol-3-yl)-N'-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride (AC220), a uniquely potent, selective, and efficacious FMS-like tyrosine kinase-3 (FLT3) inhibitor.

Treatment of AML patients with small molecule inhibitors of FLT3 kinase has been explored as a viable therapy. However, these agents are found to be less than optimal for the treatment of AML because of lack of sufficient potency or suboptimal oral pharmacokinetics (PK) or lack of adequate tolerability at efficacious doses. We have developed a series of extremely potent and highly selective FLT3 inhibitors with good oral PK properties. The first series of compounds represented by 1 (AB530) was found to be a potent and selective FLT3 kinase inhibitor with good PK properties. The aqueous solubility and oral PK properties at higher doses in rodents were found to be less than optimal for clinical development. A novel series of compounds were designed lacking the carboxamide group of 1 with an added water solubilizing group. Compound 7 (AC220) was identified from this series to be the most potent and selective FLT3 inhibitor with good pharmaceutical properties, excellent PK profile, and superior efficacy and tolerability in tumor xenograft models. Compound 7 has demonstrated a desirable safety and PK profile in humans and is currently in phase II clinical trials.

Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases.

To realize the full potential of targeted protein kinase inhibitors for the treatment of cancer, it is important to address the emergence of drug resistance in treated patients. Mutant forms of BCR-ABL, KIT, and the EGF receptor (EGFR) have been found that confer resistance to the drugs imatinib, gefitinib, and erlotinib. The mutations weaken or prevent drug binding, and interestingly, one of the most common sites of mutation in all three kinases is a highly conserved "gatekeeper" threonine residue near the kinase active site. We have identified existing clinical compounds that bind and inhibit drug-resistant mutant variants of ABL, KIT, and EGFR. We found that the Aurora kinase inhibitor VX-680 and the p38 inhibitor BIRB-796 inhibit the imatinib- and BMS-354825-resistant ABL(T315I) kinase. The KIT/FLT3 inhibitor SU-11248 potently inhibits the imatinib-resistant KIT(V559D/T670I) kinase, consistent with the clinical efficacy of SU-11248 against imatinib-resistant gastrointestinal tumors, and the EGFR inhibitors EKB-569 and CI-1033, but not GW-572016 and ZD-6474, potently inhibit the gefitinib- and erlotinib-resistant EGFR(L858R/T790M) kinase. EKB-569 and CI-1033 are already in clinical trials, and our results suggest that they should be considered for testing in the treatment of gefitinib/erlotinib-resistant non-small cell lung cancer. The results highlight the strategy of screening existing clinical compounds against newly identified drug-resistant mutant variants to find compounds that may serve as starting points for the development of next-generation drugs, or that could be used directly to treat patients that have acquired resistance to first-generation targeted therapy.