Chemical and Biochemical Reactivity of the Reduced Forms of Nicotinamide Riboside.

All life forms require nicotinamide adenine dinucleotide, NAD+, and its reduced form NADH. They are redox partners in hundreds of cellular enzymatic reactions. Changes in the intracellular levels of total NAD (NAD+ + NADH) and the (NAD+/NADH) ratio can cause cellular dysfunction. When not present in protein complexes, NADH and its phosphorylated form NADPH degrade through intricate mechanisms. Replenishment of a declining total NAD pool can be achieved with biosynthetic precursors that include one of the reduced forms of nicotinamide riboside (NR+), NRH. NRH, like NADH and NADPH, is prone to degradation via oxidation, hydration, and isomerization and, as such, is an excellent model compound to rationalize the nonenzymatic metabolism of NAD(P)H in a biological context. Here, we report on the stability of NRH and its propensity to isomerize and irreversibly degrade. We also report the preparation of two of its naturally occurring isomers, their chemical stability, their reactivity toward NRH-processing enzymes, and their cell-specific cytotoxicity. Furthermore, we identify a mechanism by which NRH degradation causes covalent peptide modifications, a process that could expose a novel type of NADH-protein modifications and correlate NADH accumulation with "protein aging." This work highlights the current limitations in detecting NADH's endogenous catabolites and in establishing the capacity for inducing cellular dysfunction.

An Ultra-High-Throughput Screen for Catalytic Inhibitors of Serine/Threonine Protein Phosphatases Types 1 and 5 (PP1C and PP5C).

Although there has been substantial success in the development of specific inhibitors for protein kinases, little progress has been made in the identification of specific inhibitors for their protein phosphatase counterparts. Inhibitors of PP1 and PP5 are desired as probes for research and to test their potential for drug development. We developed and miniaturized (1536-well plate format) nearly identical homogeneous, fluorescence intensity (FLINT) enzymatic assays to detect inhibitors of PP1 or PP5. The assays were used in an ultra-high-throughput screening (uHTS) campaign, testing >315,000 small-molecule compounds. Both assays demonstrated robust performance, with a Z' of 0.92 ± 0.03 and 0.95 ± 0.01 for the PP1 and PP5 assays, respectively. Screening the same library with both assays aided the identification of class inhibitors and assay artifacts. Confirmation screening and hit prioritization assays used [32P/33P]-radiolabel protein substrates, revealing excellent agreement between the FLINT and radiolabel assays. This screening campaign led to the discovery of four novel unrelated small-molecule inhibitors of PP1 and ~30 related small-molecule inhibitors of PP5. The results suggest that this uHTS approach is suitable for identifying selective chemical probes that inhibit PP1 or PP5 activity, and it is likely that similar assays can be developed for other PPP-family phosphatases.

Crystal structures and mutagenesis of PPP-family ser/thr protein phosphatases elucidate the selectivity of cantharidin and novel norcantharidin-based inhibitors of PP5C.

Cantharidin is a natural toxin and an active constituent in a traditional Chinese medicine used to treat tumors. Cantharidin acts as a semi-selective inhibitor of PPP-family ser/thr protein phosphatases. Despite sharing a common catalytic mechanism and marked structural similarity with PP1C, PP2AC and PP5C, human PP4C was found to be insensitive to the inhibitory activity of cantharidin. To explore the molecular basis for this selectivity, we synthesized and tested novel C5/C6-derivatives designed from quantum-based modeling of the interactions revealed in the co-crystal structures of PP5C in complex with cantharidin. Structure-activity relationship studies and analysis of high-resolution (1.25Å) PP5C-inhibitor co-crystal structures reveal close contacts between the inhibitor bridgehead oxygen and both a catalytic metal ion and a non-catalytic phenylalanine residue, the latter of which is substituted by tryptophan in PP4C. Quantum chemistry calculations predicted that steric clashes with the bulkier tryptophan side chain in PP4C would force all cantharidin-based inhibitors into an unfavorable binding mode, disrupting the strong coordination of active site metal ions observed in the PP5C co-crystal structures, thereby rendering PP4C insensitive to the inhibitors. This prediction was confirmed by inhibition studies employing native human PP4C. Mutation of PP5C (F446W) and PP1C (F257W), to mimic the PP4C active site, resulted in markedly suppressed sensitivity to cantharidin. These observations provide insight into the structural basis for the natural selectivity of cantharidin and provide an avenue for PP4C deselection. The novel crystal structures also provide insight into interactions that provide increased selectivity of the C5/C6 modifications for PP5C versus other PPP-family phosphatases.