Real-Time Monitoring of Solid-Liquid Slurries: Optimized Synthesis of Tetrabenazine.

Solid-liquid slurries are vital and increasingly prevalent in the pharmaceutical and chemical industries. Despite the importance of these heterogeneous systems, process control and optimization are fundamentally hindered by a lack of compatible real-time analytical techniques. We present herein an online HPLC monitoring platform enabling access to real-time compositional information on slurries. We demonstrate the system by investigating the heterogeneous synthesis reaction of tetrabenazine. Furthermore, we integrated our online HPLC platform with the orthogonal monitoring techniques of a pH probe and a microscopic imaging probe to provide additional mechanistic insight. These combined insights enable the optimization of tetrabenazine synthesis in terms of reaction time, byproduct formation, and diastereomeric purity of the final product.

An evolved oxazolidinone with selective potency against Mycobacterium tuberculosis and gram positive bacteria.

Innovation of new antibacterials that are effective against strains that have developed resistance to existing drugs would strengthen our ability to treat and subsequently control spread of pathogenic bacteria. Increasing incidence of infections with drug resistant bacteria has become a common occurrence in recent times. We have developed an evolved oxazolidinone, T145, which inhibits growth of Enterococcus faecalis, Staphylococcus aureus and Mycobacterium tuberculosis (Mtb) with sub µg/ml potencies that are potentially therapeutically valuable. The oxazolidinone is bactericidal against Mtb but bacteriostatic against E. faecalis and S. aureus. In addition to therapeutically valuable potency and bactericidal activity against Mtb, T145 minimizes selection of spontaneous resistant mutants, a trait that prolongs longevity of a drug in clinical use.

Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis.

Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked ß-N-acetyl-glucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2ß and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.

Structural basis of histone demethylation by LSD1 revealed by suicide inactivation.

Histone methylation regulates diverse chromatin-templated processes, including transcription. The recent discovery of the first histone lysine-specific demethylase (LSD1) has changed the long-held view that histone methylation is a permanent epigenetic mark. LSD1 is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that demethylates histone H3 Lys4 (H3-K4). However, the mechanism by which LSD1 achieves its substrate specificity is unclear. We report the crystal structure of human LSD1 with a propargylamine-derivatized H3 peptide covalently tethered to FAD. H3 adopts three consecutive gamma-turns, enabling an ideal side chain spacing that places its N terminus into an anionic pocket and positions methyl-Lys4 near FAD for catalysis. The LSD1 active site cannot productively accommodate more than three residues on the N-terminal side of the methyllysine, explaining its H3-K4 specificity. The unusual backbone conformation of LSD1-bound H3 suggests a strategy for designing potent LSD1 inhibitors with therapeutic potential.

Enantioselective synthesis of (-)-tetrabenazine via continuous crystallization-induced diastereomer transformation.

A multi-well continuous CIDT approach with inline racemization of the solution phase is presented. Using two in-house built PATs and a flow reactor, we were able to successfully crystallize an enantiopure salt of TBZ, the active metabolite of the tardive dyskinesia drug valbenazine. Despite discovering an undesired racemic solid phase, inline racemization combined with careful control of crystallization conditions allowed for multigram quantities of enantiopure material to be harvested using our setup. Critically, this control was made possible by the use of PATs to observe and quantify the composition of both the solid and solution phases.

Comparative analysis of small molecules and histone substrate analogues as LSD1 lysine demethylase inhibitors.

LSD1 is a flavin-dependent histone demethylase that oxidatively removes methyl groups from Lys-4 of histone H3. LSD1 belongs to the amine oxidase enzyme superfamily which utilize molecular oxygen to transform amines to imines that are hydrolytically cleaved to formaldehyde. In prior studies, it has been shown that monoamine oxidase inhibitory scaffolds such as propargylamines and cyclopropylamines can serve as mechanism-based inactivators of LSD1. Propargylamine-histone H3 peptide analogues are potent LSD1 inhibitors, whereas small molecule antidepressant MAO acetylenic inhibitors like pargyline do not inhibit LSD1. In contrast, the small molecule MAO cyclopropylamine inhibitor tranylcypromine is a time-dependent LSD1 inhibitor but exo-cyclopropylamine-peptide substrate analogue is not. To provide further insight into small molecule versus peptide relationships in LSD1 inhibition, herein we further our analysis of warheads in peptide scaffolds to include the chlorovinyl, endo-cyclopropylamine, and hydrazine-functionalities as LSD1 inactivators. We find that chlorovinyl-H3 is a mechanism-based LSD1 inactivator whereas endo-cyclopropylamine-H3 does not show time-dependent inactivation. The hydrazine-H3 was shown to be the most potent LSD1 suicide inhibitor yet reported, more than 20-fold more efficient in inhibiting demethylation than propargylamine-H3 derivatives. We re-explored MAO antidepressant agent phenelzine (phenethylhydrazine), previously reported to be a weak LSD1 inhibitor, and found that it is far more potent than previously appreciated. We show that phenelzine can block histone H3K4Me demethylation in cells, validating it as a pharmacologic tool and potential lead structure for anticancer therapy.

LSD1 and the chemistry of histone demethylation.

The recent discovery that histone demethylation can be catalyzed by the flavin-dependent amine oxidase LSD1 has ushered in a new chapter in the chromatin-remodeling community. Herein, we discuss the rapid progress of the histone demethylase field including the recent identification of the non-heme iron-dependent histone demethylases (JmjC family), the basis for LSD1 substrate site specificity and the newly emerging potential for inhibition of these enzymes in structural and functional analysis.

Synthesis of novel oxazolidinone antimicrobial agents.

The oxazolidinone class of antimicrobials represents a promising advance in the fight against resistant Gram-positive bacterial infections. Four novel oxazolidinone antimicrobial compounds, each containing a benzodioxin ring system, have been prepared. The general synthesis of each compound begins with the construction of a benzodioxin ring system containing a nitro substituent that ultimately becomes the nitrogen of the oxazolidinone ring. Three of the compounds utilize high yielding 'click chemistry' in their final step. The antimicrobial activities of the new oxazolidinones have been measured and the MIC against Staphylococcus aureus for one of the antimicrobials was determined to be 2-3 microg/mL, which is comparable to the well-known oxazolidinone, linezolid.

De novo discovery of serotonin N-acetyltransferase inhibitors.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is a member of the GCN5 N-acetyltransferase (GNAT) superfamily and catalyzes the penultimate step in the biosynthesis of melatonin; a large daily rhythm in AANAT activity drives the daily rhythm in circulating melatonin. We have used a structure-based computational approach to identify the first druglike and selective inhibitors of AANAT. Approximately 1.2 million compounds were virtually screened by 3D high-throughput docking into the active site of X-ray structures for AANAT, and in total 241 compounds were tested as inhibitors. One compound class, containing a rhodanine scaffold, exhibited low micromolar competitive inhibition against acetyl-CoA (AcCoA) and proved to be effective in blocking melatonin production in pineal cells. Compounds from this class are predicted to bind as bisubstrate inhibitors through interactions with the AcCoA and serotonin binding sites. Overall, this study demonstrates the feasibility of using virtual screening to identify small molecules that are selective inhibitors of AANAT.

Structure of Hepatitis C Virus Envelope Glycoprotein E1 Antigenic Site 314-324 in Complex with Antibody IGH526.

Hepatitis C virus (HCV) is a positive-strand RNA virus within the Flaviviridae family. The viral "spike" of HCV is formed by two envelope glycoproteins, E1 and E2, which together mediate viral entry by engaging host receptors and undergoing conformational changes to facilitate membrane fusion. While E2 can be readily produced in the absence of E1, E1 cannot be expressed without E2 and few reagents, including monoclonal antibodies (mAbs), are available for study of this essential HCV glycoprotein. A human mAb to E1, IGH526, was previously reported to cross-neutralize different HCV isolates, and therefore, we sought to further characterize the IGH526 neutralizing epitope to obtain information for vaccine design. We found that mAb IGH526 bound to a discontinuous epitope, but with a major component corresponding to E1 residues 314-324. The crystal structure of IGH526 Fab with this E1 glycopeptide at 1.75Å resolution revealed that the antibody binds to one face of an α-helical peptide. Single mutations on the helix substantially lowered IGH526 binding but did not affect neutralization, indicating either that multiple mutations are required or that additional regions are recognized by the antibody in the context of the membrane-associated envelope oligomer. Molecular dynamics simulations indicate that the free peptide is flexible in solution, suggesting that it requires stabilization for use as a candidate vaccine immunogen.