Cryo-EM analyses of KIT and oncogenic mutants reveal structural oncogenic plasticity and a target for therapeutic intervention.

The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as acute myeloid leukemia, gastrointestinal stromal tumor, and mast cell leukemia, are driven by somatic gain-of-function KIT mutations. Here, we report cryo electron microscopy (cryo-EM) structural analyses of full-length wild-type and two oncogenic KIT mutants, which show that the overall symmetric arrangement of the extracellular domain of ligand-occupied KIT dimers contains asymmetric D5 homotypic contacts juxtaposing the plasma membrane. Mutational analysis of KIT reveals in D5 region an "Achilles heel" for therapeutic intervention. A ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stable D5 asymmetric conformation. A constitutively active ligand-independent oncogenic KIT mutant adopts a V-shaped conformation solely held by D5-mediated contacts. Binding of SCF to this mutant fully restores the conformation of wild-type KIT dimers, including the formation of salt bridges responsible for D4 homotypic contacts and other hallmarks of SCF-induced KIT dimerization. These experiments reveal an unexpected structural plasticity of oncogenic KIT mutants and a therapeutic target in D5.

Structure-Guided Identification of DNMT3B Inhibitors.

Methyltransferase 3 beta (DNMT3B) inhibitors that interfere with cancer growth are emerging possibilities for treatment of melanoma. Herein we identify small molecule inhibitors of DNMT3B starting from a homology model based on a DNMT3A crystal structure. Virtual screening by docking led to purchase of 15 compounds, among which 5 were found to inhibit the activity of DNMT3B with IC50 values of 13-72 µM in a fluorogenic assay. Eight analogues of 7, 10, and 12 were purchased to provide 2 more active compounds. Compound 11 is particularly notable as it shows good selectivity with no inhibition of DNMT1 and 22 µM potency toward DNMT3B. Following additional de novo design, exploratory synthesis of 17 analogues of 11 delivered 5 additional inhibitors of DNMT3B with the most potent being 33h with an IC50 of 8.0 µM. This result was well confirmed in an ultrahigh-performance liquid chromatography (UHPLC)-based analytical assay, which yielded an IC50 of 4.8 µM. Structure-activity data are rationalized based on computed structures for DNMT3B complexes.

Structural and pharmacological evaluation of a novel non-nucleoside reverse transcriptase inhibitor as a promising long acting nanoformulation for treating HIV.

Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48 h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.

DRONE: Direct Tracking of DNA Cytidine Deamination and Other DNA Modifying Activities.

Enzymes that catalyze DNA modifying activities including cytidine deamination and cytosine methylation play important biological roles and have been implicated pathologically in diseases such as cancer. Here, we report Direct Resolution of ONE dalton difference (DRONE), an ultra high performance liquid chromatography (UHPLC)-based analytical method to track a single dalton change in the cytosine-to-uracil conversion catalyzed by the human apolipoprotein B m-RNA editing catalytic polypeptide-like 3 (APOBEC3) cytidine deaminases, implicated in cancer and antiviral defense. Additionally, we demonstrate broad applicability by tracking other important DNA modifications and assessing epigenetic enzyme inhibition. We have extended our methodology to obtain data on two distinct deamination events in the same oligonucleotide substrate designed from a putative APOBEC substrate, diversifying the utility of the described method. DRONE provides an important foundation for in-depth analysis of DNA-modifying enzymes and versatile detection of novel DNA modifications of interest.

Structure-based design and profiling of novel 17ß-HSD14 inhibitors.

The human enzyme 17ß-hydroxysteroid dehydrogenase 14 (17ß-HSD14) oxidizes the hydroxyl group at position 17 of estradiol and 5-androstenediol using NAD+ as cofactor. However, the physiological role of the enzyme remains unclear. We recently described the first class of nonsteroidal inhibitors for this enzyme with compound 1 showing a high 17ß-HSD14 inhibitory activity. Its crystal structure was used as starting point for a structure-based optimization in this study. The goal was to develop a promising chemical probe to further investigate the enzyme. The newly designed compounds revealed mostly very high inhibition of the enzyme and for seven of them the crystal structures of the corresponding inhibitor-enzyme complexes were resolved. The crystal structures disclosed that a small change in the substitution pattern of the compounds resulted in an alternative binding mode for one inhibitor. The profiling of a set of the most potent inhibitors identified 13 (Ki = 9 nM) with a good selectivity profile toward three 17ß-HSDs and the estrogen receptor alpha. This inhibitor displayed no cytotoxicity, good solubility, and auspicious predicted bioavailability. Overall, 13 is a highly interesting 17ß-HSD14 inhibitor, which might be used as chemical probe for further investigation of the target enzyme.