Enrichment of DNA replication intermediates by EdU pull down.

Analysis of replication fork structures in electron microscopy (EM) can provide important mechanistic insights in DNA replication studies. A major challenge in this type of analysis is the paucity of replication intermediates. At any given time only a small fraction of the restriction fragments of genomic DNA will contain a replication fork. To address this issue, we have developed an EdU-pull-down procedure to enrich for replicating DNA. Cells are exposed to a brief pulse of EdU, a cleavable biotin moiety is attached to EdU by copper-catalyzed azide-alkyne cycloaddition (CuAAC), in conditions that minimize the damage to DNA. Biotinylated DNA is purified with streptavidin beads, in conditions that facilitate association of long DNA filaments. Finally, the DNA is eluted by cleaving the biotin moiety. This approach can enrich over 150-times for replicating DNA and about 50-times in replication fork structures, as verified by EM. This procedure could benefit analysis of replication intermediates in EM as well as other techniques for the study of replicating DNA.

Discovery of Reversible Inhibitors of KDM1A Efficacious in Acute Myeloid Leukemia Models.

Lysine-specific demethylase 1 (LSD1 or KDM1A) is a FAD-dependent enzyme that acts as a transcription corepressor or coactivator by regulating the methylation status of histone H3 lysines K4 and K9, respectively. KDM1A represents an attractive target for cancer therapy. While, in the past, the main medicinal chemistry strategy toward KDM1A inhibition was based on the optimization of ligands that irreversibly bind the FAD cofactor within the enzyme catalytic site, we and others have also identified reversible inhibitors. Herein we reported the discovery of 5-imidazolylthieno[3,2-b]pyrroles, a new series of KDM1A inhibitors endowed with picomolar inhibitory potency, active in cells and efficacious after oral administration in murine leukemia models.

Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 1: High-Throughput Screening and Preliminary Exploration.

Lysine specific demethylase 1 KDM1A (LSD1) regulates histone methylation and it is increasingly recognized as a potential therapeutic target in oncology. We report on a high-throughput screening campaign performed on KDM1A/CoREST, using a time-resolved fluorescence resonance energy transfer (TR-FRET) technology, to identify reversible inhibitors. The screening led to 115 hits for which we determined biochemical IC50, thus identifying four chemical series. After data analysis, we have prioritized the chemical series of N-phenyl-4H-thieno[3, 2-b]pyrrole-5-carboxamide for which we obtained X-ray structures of the most potent hit (compound 19, IC50 = 2.9 µM) in complex with the enzyme. Initial expansion of this chemical class, both modifying core structure and decorating benzamide moiety, was directed toward the definition of the moieties responsible for the interaction with the enzyme. Preliminary optimization led to compound 90, which inhibited the enzyme with a submicromolar IC50 (0.162 µM), capable of inhibiting the target in cells.

Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 2: Structure-Based Drug Design and Structure-Activity Relationship.

The balance of methylation levels at histone H3 lysine 4 (H3K4) is regulated by KDM1A (LSD1). KDM1A is overexpressed in several tumor types, thus representing an emerging target for the development of novel cancer therapeutics. We have previously described ( Part 1, DOI 10.1021.acs.jmedchem.6b01018 ) the identification of thieno[3,2-b]pyrrole-5-carboxamides as novel reversible inhibitors of KDM1A, whose preliminary exploration resulted in compound 2 with biochemical IC50 = 160 nM. We now report the structure-guided optimization of this chemical series based on multiple ligand/KDM1A-CoRest cocrystal structures, which led to several extremely potent inhibitors. In particular, compounds 46, 49, and 50 showed single-digit nanomolar IC50 values for in vitro inhibition of KDM1A, with high selectivity in secondary assays. In THP-1 cells, these compounds transcriptionally affected the expression of genes regulated by KDM1A such as CD14, CD11b, and CD86. Moreover, 49 and 50 showed a remarkable anticlonogenic cell growth effect on MLL-AF9 human leukemia cells.

New insights into selective PDE4D inhibitors: 3-(Cyclopentyloxy)-4-methoxybenzaldehyde O-(2-(2,6-dimethylmorpholino)-2-oxoethyl) oxime (GEBR-7b) structural development and promising activities to restore memory impairment.

Phosphodiesterase type 4D (PDE4D) has been indicated as a promising target for treating neurodegenerative pathologies such as Alzheimer's Disease (AD). By preventing cAMP hydrolysis, PDE4 inhibitors (PDE4Is) increase the cAMP response element-binding protein (CREB) phosphorylation, synaptic plasticity and long-term memory formation. Pharmacological and behavioral studies on our hit GEBR-7b demonstrated that selective PDE4DIs could improve memory without causing emesis and sedation. The hit development led to new molecule series, herein reported, characterized by a catechol structure bonded to five member heterocycles. Molecular modeling studies highlighted the pivotal role of a polar alkyl chain in conferring selective enzyme interaction. Compound 8a showed PDE4D3 selective inhibition and was able to increase intracellular cAMP levels in neuronal cells, as well as in the hippocampus of freely moving rats. Furthermore, 8a was able to readily cross the blood-brain barrier and enhanced memory performance in mice without causing any emetic-like behavior. These data support the view that PDE4D is an adequate molecular target to restore memory deficits in different neuropathologies, including AD, and also indicate compound 8a as a promising candidate for further preclinical development.

New selective phosphodiesterase 4D inhibitors differently acting on long, short, and supershort isoforms.

The lack of selective inhibitors toward the long, short, or supershort phosphodiesterases (PDE4s) prevented researchers from carefully defining the connection between different enzyme isoforms, their brain localization, and their role in neurodegenerative diseases such as Alzheimer's disease (AD). In the search for new therapeutic agents for treating memory and learning disorders, we synthesized new rolipram related PDE4 inhibitors, which had some selectivity toward the long form PDE4D3. The first series was synthesized as racemate and then resolved by semipreparative HPLC on chiral supports. Herein we report the synthetic pathways to obtain compounds 1a-c, 2a-c, 3a-c, 4a-f, 5a,b, 6a,b, 7a,b, the chiral analytical study to resolve compounds 1a-c, 2a-c, 3a-c, the molecular docking study for compound 1c, and the biological results and some SAR considerations that provide some insights and hints for the structural requirements for PDE4D subtype selectivity and enzyme inhibition.

Relative stereochemistry of a diterpene from Salvia cinnabarina.

The relative stereochemistry of 3,4-secoisopimara-4(18),7,15-triene-3-oic acid, a diterpenoid with antispasmodic, hypotensive and antibacterial activities isolated from Salvia cinnabarina, was determined by an X-ray diffraction analysis of a single crystal of a suitable crystalline derivative.