Synthesis, Optimization, and Structure-Activity Relationships of Nicotinamide Phosphoribosyltransferase (NAMPT) Positive Allosteric Modulators (N-PAMs).

Depletion of nicotinamide adenine dinucleotide (NAD+) is associated with aging and disease, spurring the study of dietary supplements to replenish NAD+. The catabolism of NAD+ to nicotinamide (NAM) requires the salvage of NAM to replenish cellular NAD+, which relies on the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). Pharmacological activation of NAMPT provides an alternative to dietary supplements. Screening for activators of NAMPT identified small molecule NAMPT positive allosteric modulators (N-PAMs). N-PAMs bind to the rear channel of NAMPT increasing enzyme activity and alleviating feedback inhibition by NAM and NAD+. Synthesis of over 70 N-PAMs provided an excellent correlation between rear channel binding affinity and potency for enzyme activation, confirming the mechanism of allosteric activation via binding to the rear channel. The mechanism accounts for higher binding affinity leading to loss of efficacy. Enzyme activation translated directly to elevation of NAD+ measured in cells. Optimization led to an orally bioavailable N-PAM.

Mechanism of Allosteric Modulation of Nicotinamide Phosphoribosyltransferase to Elevate Cellular NAD.

In aging and disease, cellular nicotinamide adenine dinucleotide (NAD+) is depleted by catabolism to nicotinamide (NAM). NAD+ supplementation is being pursued to enhance human healthspan and lifespan. Activation of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ biosynthesis, has the potential to increase the salvage of NAM. Novel NAMPT-positive allosteric modulators (N-PAMs) were discovered in addition to the demonstration of NAMPT activation by biogenic phenols. The mechanism of activation was revealed through the synthesis of novel chemical probes, new NAMPT co-crystal structures, and enzyme kinetics. Binding to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD+ measurements in human cells. The mechanism of action of N-PAMs identifies, for the first time, the role of the rear channel in the regulation of NAMPT turnover coupled to productive and nonproductive NAM binding. The tight regulation of cellular NAMPT via feedback inhibition by NAM, NAD+, and adenosine 5'-triphosphate (ATP) is differentially regulated by N-PAMs and other activators, indicating that different classes of pharmacological activators may be engineered to restore or enhance NAD+ levels in affected tissues.

Human Escherichia coli O157:H7 genetic marker in isolates of bovine origin.

The antiterminator Q gene of bacteriophage 933W (Q933) was identified upstream of the stx2 gene in 90% of human disease-origin Escherichia coli O157:H7 isolates and in 44.5% of bovine isolates. Shiga toxin production was higher in Q933-positive isolates than Q933-negative isolates. This genetic marker may provide a useful molecular tool for epidemiologic studies.

Microbiological quality of ground beef from conventionally-reared cattle and "raised without antibiotics" label claims.

The contamination of the food supply with pathogens and antimicrobial-resistant bacteria has emerged as an important health concern. We compared the microbiological quality of 77 samples of ground beef from conventionally raised cattle with 73 samples of ground beef from cattle raised without antimicrobial agents. Contamination with coliforms (1.7 log CFU/g) and Escherichia coli (0.51 log CFU/g) and Shiga toxin 2-producing E. coli (6% prevalence) was similar in both sample groups. Neither Salmonella. E. coli O157, nor vancomycin-resistant enterococci were isolated from any sample. Prevalence of E. coli resistant to ampicillin (39%), amoxicillin/clavulanic acid (23%), ceftriaxone (5%), tetracycline (19%), streptomycin (19%), kanamycin (11%), sulfamethoxazole/trimethoprim (2%), and gentamicin (1%) was similar in both groups. E. coli resistant to ciprofloxacin was not identified. Resistance to ceftiofur and chloramphenicol was more prevalent in beef from conventionally raised cattle at 18 and 30%, respectively, compared to 5 and 12% prevalence in beef from cattle raised without antimicrobial agents. These results do not correlate with the frequency of subtherapeutic use of these two antibiotics in beef production. Other factors in addition to, or in lieu of, the subtherapeutic use of specific antimicrobial agents in the preharvest stages of beef production may contribute significantly to the occurrence of antimicrobial-resistant bacteria in ground beef.