Frank Preugschat

A Steady-state and Pre-steady-state Kinetic Analysis of the NTPase Activity Associated with the Hepatitis C Virus NS3 Helicase Domain

The helicase domain of hepatitis C virus NS3 (genotype 1b) was expressed in Escherichia coli and purified to homogeneity. The purified protein catalyzed the hydrolysis of nucleoside triphosphates (NTP) and the unwinding of duplex RNA in the presence of divalent metal ion. The enzyme was not selective for the NTP substrate. For example, UTP and acyclovir triphosphate were hydrolyzed efficiently by the enzyme. The rate of NTP hydrolysis was stimulated up to 27-fold by oligomeric nucleic acids (NA). Furthermore, NA bound to the enzyme with concomitant quenching of the intrinsic protein fluorescence. The dissociation constants of the enzyme for selected NA in the absence of NTP were between 10 and 35 μM at pH 7.0 and 25°C. The enzyme had maximal affinity for NA with 12 or more nucleotides. A detailed steady-state and pre-steady-state kinetic analysis of ATP hydrolysis was made with (dU)18 as the effector. The kcat values for ATP hydrolysis in the presence and absence of (dU)18 were 80 s−1 and 2.7 s−1, respectively. The association (dissociation) rate constants for the enzyme and (dU)18 in the presence and absence of ATP were 5.7 μM−1 s−1 (3.9 s−1) and 290 μM−1 s−1 (2.27 s−1), respectively. The association (dissociation) rate constants for the enzyme and ATP in the presence and absence of (dU)18 were 0.4 μM−1 s−1 (<0.5 s−1) and 0.9 μM−1 s−1 (<10−1 s−1), respectively. These data were consistent with a random kinetic mechanism.

Product Release Is the Major Contributor tok cat for the Hepatitis C Virus Helicase-catalyzed Strand Separation of Short Duplex DNA

Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3′ single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5′-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3′-fluorescein-tagged 21-mer (F21).k cat for this reaction was 0.12 s−1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs (∼2) were disrupted concertedly. The maximal value of the rate constant (k eff) describing the late phase of the reaction (strand separation) was 0.5 s−1, which was 4-fold greater than k cat. Release of HF31 from E·HF31 in the presence of ATP (0.21 s−1) was the major contributor tok cat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.

Discovery, Synthesis, and Biological Evaluation of Thiazoloquin(az)olin(on)es as Potent CD38 Inhibitors

A series of thiazoloquin(az)olinones were synthesized and found to have potent inhibitory activity against CD38. Several of these compounds were also shown to have good pharmacokinetic properties and demonstrated the ability to elevate NAD levels in plasma, liver, and muscle tissue. In particular, compound 78c was given to diet induced obese (DIO) C57Bl6 mice, elevating NAD > 5-fold in liver and >1.2-fold in muscle versus control animals at a 2 h time point. The compounds described herein possess the most potent CD38 inhibitory activity of any small molecules described in the literature to date. The inhibitors should allow for a more detailed assessment of how NAD elevation via CD38 inhibition affects physiology in NAD deficient states.

Discovery of 4-Amino-8-quinoline Carboxamides as Novel, Submicromolar Inhibitors of NAD-Hydrolyzing Enzyme CD38.

Starting from the micromolar 8-quinoline carboxamide high-throughput screening hit 1a, a systematic exploration of the structure-activity relationships (SAR) of the 4-, 6-, and 8-substituents of the quinoline ring resulted in the identification of approximately 10-100-fold more potent human CD38 inhibitors. Several of these molecules also exhibited pharmacokinetic parameters suitable for in vivo animal studies, including low clearances and decent oral bioavailability. Two of these CD38 inhibitors, 1ah and 1ai, were shown to elevate NAD tissue levels in liver and muscle in a diet-induced obese (DIO) C57BL/6 mouse model. These inhibitor tool compounds will enable further biological studies of the CD38 enzyme as well as the investigation of the therapeutic implications of NAD enhancement in disease models of abnormally low NAD.

Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility

Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet.

Expression and Characterization of the HCV NS3 Helicase Domain

The hepatitis C virus (HCV) NS3 protein has two distinct biochemical domains. The N-terminal 20 kDa has serine protease activity (see Chapter 31 ) and the C-terminal 50 kDa has both nucleoside triphosphatase (NTPase) and helicase activities (1-4).