Development of a Bioluminescent High-Throughput Screening Assay for Nicotinamide Mononucleotide Adenylyltransferase (NMNAT).

Nicotinamide mononucleotide adenylyltransferase (NMNAT; EC 2.7.7.1) catalyzes the reversible production of NAD+ from NMN+ and ATP and is a potential drug target for cancer and neurodegenerative diseases. A sensitive bioluminescent assay format suitable to high-throughput screening (HTS) and mechanistic follow-up has not been reported and is of value to identify new modulators of NMNATs. To this end, we report the development of a bioluminescent assay using Photinus pyralis ATP-dependent luciferase and luciferin for NMNAT1 in a 384-well plate format. We also report a mechanistic follow-up paradigm using this format to determine time dependence and competition with substrates. The assay and follow-up paradigm were used to screen 912 compounds from the National Cancer Institute (NCI) Mechanistic Diversity Set II and the Approved Oncology Set VI against NMNAT1. Twenty inhibitors with greater than 35% inhibition at 20 µM were identified. The follow-up studies showed that seven actives were time-dependent inhibitors of NMNAT1. 2,3-Dibromo-1,4-naphthoquinone was the most potent, time-dependent inhibitor with IC50 values of 0.76 and 0.26 µM for inhibition of the forward and reverse reactions of the enzyme, respectively, and was shown to be NMN and ATP competitive. The bioluminescent NMNAT assay and mechanistic-follow-up will be of use to identify new modulators of NAD biosynthesis.

A Four-Point Screening Method for Assessing Molecular Mechanism of Action (MMOA) Identifies Tideglusib as a Time-Dependent Inhibitor of Trypanosoma brucei GSK3ß.

BACKGROUND: New therapeutics are needed for neglected tropical diseases including Human African trypanosomiasis (HAT), a progressive and fatal disease caused by the protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense. There is a need for simple, efficient, cost effective methods to identify new molecules with unique molecular mechanisms of action (MMOAs). The mechanistic features of a binding mode, such as competition with endogenous substrates and time-dependence can affect the observed inhibitory IC50, and differentiate molecules and their therapeutic usefulness. Simple screening methods to determine time-dependence and competition can be used to differentiate compounds with different MMOAs in order to identify new therapeutic opportunities. METHODOLOGY/PRINCIPAL FINDINGS: In this work we report a four point screening methodology to evaluate the time-dependence and competition for inhibition of GSK3ß protein kinase isolated from T. brucei. Using this method, we identified tideglusib as a time-dependent inhibitor whose mechanism of action is time-dependent, ATP competitive upon initial binding, which transitions to ATP non-competitive with time. The enzyme activity was not recovered following 100-fold dilution of the buffer consistent with an irreversible mechanism of action. This is in contrast to the T. brucei GSK3ß inhibitor GW8510, whose inhibition was competitive with ATP, not time-dependent at all measured time points and reversible in dilution experiments. The activity of tideglusib against T. brucei parasites was confirmed by inhibition of parasite proliferation (GI50 of 2.3 µM). CONCLUSIONS/SIGNIFICANCE: Altogether this work demonstrates a straightforward method for determining molecular mechanisms of action and its application for mechanistic differentiation of two potent TbGSK3ß inhibitors. The four point MMOA method identified tideglusib as a mechanistically differentiated TbGSK3ß inhibitor. Tideglusib was shown to inhibit parasite growth in this work, and has been reported to be well tolerated in one year of dosing in human clinical studies. Consequently, further supportive studies on the potential therapeutic usefulness of tideglusib for HAT are justified.

Discovery of S-[5-amino-1-(4-fluorophenyl)-1H-pyrazol-4-yl]-[3-(2,3-dihydroxypropoxy)phenyl]methanone (RO3201195), an orally bioavailable and highly selective inhibitor of p38 MAP kinase.

A novel class of highly selective inhibitors of p38 MAP kinase was discovered from high throughput screening. The synthesis and optimization of a series of 5-amino-N-phenyl-1H-pyrazol-4-yl-3-phenylmethanones is described. An X-ray crystal structure of this series bound in the ATP binding pocket of unphosphorylated p38alpha established the presence of a unique hydrogen bond between the exocyclic amine of the inhibitor and threonine 106 which likely contributes to the selectivity for p38. The crystallographic information was used to optimize the potency and physicochemical properties of the series. The incorporation of the 2,3-dihydroxypropoxy moiety on the pyrazole scaffold resulted in a compound with excellent drug-like properties including high oral bioavailability. These efforts identified 63 (RO3201195) as an orally bioavailable and highly selective inhibitor of p38 which was selected for advancement into Phase I clinical trials.

Rational design of 6-methylsulfonylindoles as selective cyclooxygenase-2 inhibitors.

The introduction of 3-arylmethyl, 3-aryloxy and 3-arylthio moieties into a 6-methylsulfonylindole framework using rational drug design led to potent, selective COX-2 inhibitors having efficacy in a rat carrageenan air pouch model. Incorporation of a conformationally more rigid 3-aroyloxy substituent onto the 6-methylsulfonylindole scaffold led to selective, but considerably less potent COX-2 inhibitors. Variation of the hydrophilicity and size of the indole 2-substituent of 3-arylthio-6-methylsulfonylindole inhibitors led to modulation of the COX-2 human whole blood (HWB) potency and selectivity.