ABSTRACT
PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases through clinical genetics and gene disruption in mice. New selective PAD4 inhibitors binding a calcium-deficient form of the PAD4 enzyme have validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation for, to our knowledge, the first time. The therapeutic potential of PAD4 inhibitors can now be explored.
Subject(s)
Benzimidazoles/pharmacology , Enzyme Inhibitors/pharmacology , Hydrolases/antagonists & inhibitors , Neutrophils/drug effects , Animals , Benzimidazoles/chemical synthesis , Binding, Competitive , Calcium/metabolism , Citrulline/metabolism , Enzyme Inhibitors/chemical synthesis , HEK293 Cells , Histones/metabolism , Humans , In Vitro Techniques , Mice , Models, Molecular , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , Small Molecule Libraries , Substrate SpecificityABSTRACT
Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.
Subject(s)
DNA/chemistry , Drug Design , Protein Kinase Inhibitors/chemical synthesis , Small Molecule Libraries/chemical synthesis , Animals , Aurora Kinases , Combinatorial Chemistry Techniques , DNA/genetics , Models, Molecular , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
PTEN tumor suppressor inactivation is the earliest step in endometrial carcinogenesis, occurring in morphologically unremarkable endometrial glands in half of normal women. We test the hypothesis that sex hormones positively or negatively select for these "latent precancers" by examining their emergence, persistence, and regression rates under differing hormonal conditions. Perimenopausal and postmenopausal women had an intake endometrial biopsy and underwent hormonal therapy with progestin-impregnated intrauterine device (IUD; n = 21), cyclic oral progestins (n = 28), or surveillance only (n = 22) with follow-up biopsies. For comparison, premenopausal naturally cycling endometrial biopsies were studied as single time points in 87 patients and multiple surveillance time points in 34 patients. Biopsies in which any PTEN protein-null glands were found by immunohistochemistry were scored as containing a latent endometrial precancer. All groups had a similar proportion of latent precancers at intake but differed after therapy. Emergence rates were highest (21%) for the naturally cycling premenopausal group compared with just 9% for untreated perimenopausal women. The IUD group had the highest rate of regression, with a 62% pretherapy and 5% post-therapy rate of latent precancers. This contrasted to nonsignificant changes for the oral progestin and untreated control groups. Delivery of high doses of progestins locally to the endometrium by IUD leads to ablation of preexisting PTEN-inactivated endometrial latent precancers and is a possible mechanism for reduction of long-term endometrial cancer risk known to occur in response to this hormone.
