Xanthine Derivatives Reveal an Allosteric Binding Site in Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2).

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays an important role in one-carbon metabolism. The MTHFD2 gene is upregulated in various cancers but very low or undetectable in normal proliferating cells, and therefore a potential target for cancer treatment. In this study, we present the structure of MTHFD2 in complex with xanthine derivative 15, which allosterically binds to MTHFD2 and coexists with the substrate analogue. A kinetic study demonstrated the uncompetitive inhibition of MTHFD2 by 15. Allosteric inhibitors often provide good selectivity and, indeed, xanthine derivatives are highly selective for MTHFD2. Moreover, several conformational changes were observed upon the binding of 15, which impeded the binding of the cofactor and phosphate to MTHFD2. To the best of our knowledge, this is the first study to identify allosteric inhibitors targeting the MTHFD family and our results would provide insights on the inhibition mechanism of MTHFD proteins and the development of novel inhibitors.

Potent and orally active purine-based fetal hemoglobin inducers for treating ß-thalassemia and sickle cell disease.

Reactivation of fetal hemoglobin (HbF) expression by therapeutic agents has been suggested as an alternative treatment to modulate anemia and the related symptoms of severe ß-thalassemia and sickle cell disease (SCD). Hydroxyurea (HU) is the first US FDA-approved HbF inducer for treating SCD. However, approximately 25% of the patients with SCD do not respond to HU. A previous study identified TN1 (1) as a small-molecule HbF inducer. However, this study found that the poor potency and oral bioavailability of compound 1 limits the development of this inducer for clinical use. To develop drug-like compounds, further structure-activity relationship studies on the purine-based structure of 1 were conducted. Herein, we report our discovery of a more potent inducer, compound 13a, that can efficiently induce γ-globin gene expression at non-cytotoxic concentrations. The molecular mechanism of 13a, for the regulation HbF expression, was also investigated. In addition, we demonstrated that oral administration of 13a can ameliorate anemia and the related symptoms in SCD mice. The results of this study suggest that 13a can be further developed as a novel agent for treating hemoglobinopathies, such as ß-thalassemia and SCD.

Identification of a Multitargeted Tyrosine Kinase Inhibitor for the Treatment of Gastrointestinal Stromal Tumors and Acute Myeloid Leukemia.

Gastrointestinal stromal tumors (GISTs) are prototypes of stem cell factor receptor (c-KIT)-driven cancer. Two receptor tyrosine kinases, c-KIT and fms-tyrosine kinase (FLT3), are frequently mutated in acute myeloid leukemia (AML) patients, and these mutations are associated with poor prognosis. In this study, we discovered a multitargeted tyrosine kinase inhibitor, compound 15a, with potent inhibition against single or double mutations of c-KIT developed in GISTs. Moreover, crystal structure analysis revealed the unique binding mode of 15a with c-KIT and may elucidate its high potency in inhibiting c-KIT kinase activity. Compound 15a inhibited cell proliferation and induced apoptosis by targeting c-KIT in c-KIT-mutant GIST cell lines. The antitumor effects of 15a were also demonstrated in GIST430 and GIST patient-derived xenograft models. Further studies demonstrated that 15a inhibited the proliferation of c-KIT- and FLT3-driven AML cells in vitro and in vivo. The results of this study suggest that 15a may be a potential anticancer drug for the treatment of GISTs and AML.

Discovery of Conformational Control Inhibitors Switching off the Activated c-KIT and Targeting a Broad Range of Clinically Relevant c-KIT Mutants.

Drug resistance due to acquired mutations that constitutively activate c-KIT is a significant challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). Herein, we identified 1-(5-ethyl-isoxazol-3-yl)-3-(4-{2-[6-(4-ethylpiperazin-1-yl)pyrimidin-4-ylamino]-thiazol-5-yl}phenyl)urea (10a) as a potent inhibitor against unactivated and activated c-KIT. The binding of 10a induced rearrangements of the DFG motif, αC-helix, juxtamembrane domain, and the activation loop to switch the activated c-KIT back to its structurally inactive state. To the best of our knowledge, it is the first structural evidence demonstrating how a compound can inhibit the activated c-KIT by switching back to its inactive state through a sequence of conformational changes. Moreover, 10a can effectively inhibit various c-KIT mutants and the proliferation of several GIST cell lines. The distinct binding features and superior inhibitory potency of 10a, together with its excellent efficacy in the xenograft model, establish 10a as worthy of further clinical evaluation in the advanced GISTs.

Pyrazolylamine Derivatives Reveal the Conformational Switching between Type I and Type II Binding Modes of Anaplastic Lymphoma Kinase (ALK).

Most anaplastic lymphoma kinase (ALK) inhibitors adopt a type I binding mode, but only limited type II ALK structural studies are available. Herein, we present the structure of ALK in complex with N1-(3-4-[([5-(tert-butyl)-3-isoxazolyl]aminocarbonyl)amino]-3-methylphenyl-1H-5-pyrazolyl)-4-[(4-methylpiperazino)methyl]benzamide (5a), a novel ALK inhibitor adopting a type II binding mode. It revealed binding of 5a resulted in the conformational change and reposition of the activation loop, αC-helix, and juxtamembrane domain, which are all important domains for the autoinhibition mechanism and downstream signal pathway regulation of ALK. A structure-activity relationship study revealed that modifications to the structure of 5a led to significant differences in the ALK potency and altered the protein structure of ALK. To the best of our knowledge, this is the first structural biology study to directly observe how changes in the structure of a small molecule can regulate the switch between the type I and type II binding modes and induce dramatic conformational changes.

Identification of a potent 5-phenyl-thiazol-2-ylamine-based inhibitor of FLT3 with activity against drug resistance-conferring point mutations.

Numerous FLT3 inhibitors have been explored as a viable therapy for the treatment of acute myeloid leukemia (AML). However, clinical data have been underwhelming due to incomplete inhibition of FLT3 or the emergence of resistant mutations treated with these older agents. We previously developed a series of 3-phenyl-1H-5-pyrazolylamine derivatives as highly potent and selective FLT3 inhibitors with good in vivo efficacy using an intravenous (IV) route. However, the poor bioavailability of these pyrazole compounds limits the development of these promising antileukemic compounds for clinical use. Herein, we describe a novel class of 5-phenyl-thiazol-2-ylamine compounds that are multi-targeted FLT3 inhibitors. From this class of compounds, compound 7h was very potent against AML cell lines and exhibited excellent oral efficacy in AML xenograft models. In addition, further studies demonstrated that compound 7h exhibited potent in vitro and in vivo activities against clinically relevant AC220 (3)-resistant kinase domain mutants of FLT3-ITD.

Optimization of ligand and lipophilic efficiency to identify an in vivo active furano-pyrimidine Aurora kinase inhibitor.

Ligand efficiency (LE) and lipophilic efficiency (LipE) are two important indicators of "drug-likeness", which are dependent on the molecule's activity and physicochemical properties. We recently reported a furano-pyrimidine Aurora kinase inhibitor 4 (LE = 0.25; LipE = 1.75), with potent activity in vitro; however, 4 was inactive in vivo. On the basis of insights obtained from the X-ray co-crystal structure of the lead 4, various solubilizing functional groups were introduced to optimize both the activity and physicochemical properties. Emphasis was placed on identifying potential leads with improved activity as well as better LE and LipE by exercising tight control over the molecular weight and lipophilicity of the molecules. Rational optimization has led to the identification of Aurora kinase inhibitor 27 (IBPR001; LE = 0.26; LipE = 4.78), with improved in vitro potency and physicochemical properties, resulting in an in vivo active (HCT-116 colon cancer xenograft mouse model) anticancer agent.

Discovery of 3-phenyl-1H-5-pyrazolylamine derivatives containing a urea pharmacophore as potent and efficacious inhibitors of FMS-like tyrosine kinase-3 (FLT3).

Preclinical investigations and early clinical trials suggest that FLT3 inhibitors are a viable therapy for acute myeloid leukemia. However, early clinical data have been underwhelming due to incomplete inhibition of FLT3. We have developed 3-phenyl-1H-5-pyrazolylamine as an efficient template for kinase inhibitors. Structure-activity relationships led to the discovery of sulfonamide, carbamate and urea series of FLT3 inhibitors. Previous studies showed that the sulfonamide 4 and carbamate 5 series were potent and selective FLT3 inhibitors with good in vivo efficacy. Herein, we describe the urea series, which we found to be potent inhibitors of FLT3 and VEGFR2. Some inhibited growth of FLT3-mutated MOLM-13 cells more strongly than the FLT3 inhibitors sorafenib (2) and ABT-869 (3). In preliminary in vivo toxicity studies of the four most active compounds, 10f was found to be the least toxic. A further in vivo efficacy study demonstrated that 10f achieved complete tumor regression in a higher proportion of MOLM-13 xenograft mice than 4 and 5 (70% vs 10% and 40%). These results show that compound 10f possesses improved pharmacologic and selectivity profiles and could be more effective than previously disclosed FLT3 inhibitors in the treatment of acute myeloid leukemia.

3-Phenyl-1H-5-pyrazolylamine-based derivatives as potent and efficacious inhibitors of FMS-like tyrosine kinase-3 (FLT3).

A new class of FLT3 inhibitors has been identified based on the 3-phenyl-1H-5-pyrazolylamine scaffold. The structure-activity relationships led to the discovery of two carbamate series, and some potent compounds within these two series exhibited better growth inhibition of FLT3-mutated MOLM-13 cells than FLT3 inhibitors sorafenib (2) and ABT-869 (3). In particular, compound 8d exhibited the ability to regress tumors in mouse xenograft model using MOLM-13 cells.

Discovery and evaluation of 3-phenyl-1H-5-pyrazolylamine-based derivatives as potent, selective and efficacious inhibitors of FMS-like tyrosine kinase-3 (FLT3).

Preclinical investigations and early clinical trial studies suggest that FLT3 inhibitors offer a viable therapy for acute myeloid leukemia. However, early clinical data for direct FLT3 inhibitors provided only modest results because of the failure to fully inhibit FLT3. We have designed and synthesized a novel class of 3-phenyl-1H-5-pyrazolylamine-derived compounds as FLT3 inhibitors which exhibit potent FLT3 inhibition and high selectivity toward different receptor tyrosine kinases. The structure-activity relationships led to the discovery of two series of FLT3 inhibitors, and some potent compounds within these two series exhibited comparable potency to FLT3 inhibitors sorafenib (3) and ABT-869 (4) in both wt-FLT3 enzyme inhibition and FLT3-ITD inhibition on cell growth (MOLM-13 and MV4;11 cells). In particular, the selected compound 12a exhibited the ability to regress tumors in mouse xenograft models using MOLM-13 and MV4;11 cells.

Novel trans-2-aryl-cyclopropylamine analogues as potent and selective dipeptidyl peptidase IV inhibitors.

A series of trans-2-aryl-cyclopropylamine derived compounds were synthesized and evaluated their biological activities against DPP-IV. The structure-activity relationships (SAR) led to the discovery of novel series of DPP-IV inhibitors, having IC(50) values of <100 nM with excellent selectivity over the closely related enzymes, DPP8, DPP-II and FAP. The studies identified a potent and selective DPP-IV inhibitor 24b, which exhibited the ability to both significantly inhibit plasma DPP-IV activity in rats and improve glucose tolerance in lean mice and diet induced obese mice.