Fig 1.
Schematic of NAD+ biosynthetic pathway and NAMPT enzymatic assay.
Fig 2.
Validation of Autodock Vina screening of NCI diversity set III.
(A) Ribbon image of NAMPT dimer (PDB ID: 2GVG) Black box indicates search space centered around K389 residue. (B) Overlays of Autodock redocking compared to interactions reported in 2GVG molecule. Top panel shows known inhibitor FK866. NAMPT product NMN shown in bottom panel. (C) Table comparing Binding Energy and Root Mean Square Deviation (RMSD) reported in literature to Autodock measured parameters.
Fig 3.
NAMPT activity assay biochemical screen of Autodock Vina ranked carbon skeletons.
Unique, high binding molecules within the NCI Diversity Set III library screen were tested for modulation of NAMPT activity. Candidates were diluted to 20uM final concentration and evaluated a minimum of 2 independent trials. Molecules that consistently produced greater NMN product are circled (left of panel). Known inhibitor FK866 was included as a negative control, circle to bottom right.
Fig 4.
Novel NAMPT activator compound 20.
(A) Organic structure of polyphenolic compound 20, 9-hydroxyphenylfluoron (NSC ID 9037, PubChem CID: 72722). (B) NAMPT activity as a function of increasing concentrations of compound 20. Graph represents the average of three independent trials each containing three replicates per data point. Data analyzed using the GraphPad Prism 8.0 software with a non-linear regression, asymmetric sigmoidal, 5PL equation and fitted with the least-squares method.
Fig 5.
NAMPT activity as a function of substrate NAM concentration.
Black circles represent NAMPT activity without compound 20 present while gray squares are with 20uM of compound 20 present. Each graph represents the average of three independent trials each containing three replicates per data point. Data was analyzed using the GraphPad prism software with a non-linear regression, asymmetric sigmoidal, 5PL equation and fitted with the least-squares method.
Fig 6.
Novel NAMPT activator compound 2.
(A) Organic structure of polyphenolic compound 2, Myricitrin (NSC ID 19803, PubChem CID 5281673). (B) NAMPT activity as a function of increasing concentrations of compound 2. Graph represents the average of three independent trials each containing three replicates per data point. Data was analyzed using the GraphPad prism software with a non-linear regression asymmetric sigmoidal, 5PL equation and fitted with the least-squares method.
Fig 7.
Derivatives of compound 2 highlight important functional groups.
Compound 2 is shown in the center panel with critical hydroxyl groups circled. Derivatives that were tested for their ability to modulate NAMPT activity are shown surrounding compound 2.
Fig 8.
Luteolin derivative of compound 2 activates NAMPT.
NAMPT activity upon exposure to 20uM of indicated compound. Compound 2 is shown as positive control, no modulating compound addition (F100/F0) acts as negative control. Graph represents the average of three independent trials each containing three replicates per data point.
Fig 9.
NAMPT activation by natural products.
(A) NAMPT Activity background control, using only natural products as the source of NAMPT enzyme. (B) NAMPT activity using recombinant human NAMPT enzyme supplemented with natural products. Each bar represents the average of three independent trials, normalized for intrinsic fluorescence of natural product.
Table 1.
Autodock calculations for binding to NAMPT search spaces.
Fig 10.
Theoretical interaction between NAMPT K389 cavity and novel activators.
Molecular images were visualized using the educational version of PyMOL version 1.2 from Schrodinger, LLC. in the context of an undergraduate research experience. The active NAMPT dimer is represented in the top panel, with the K389 cavity highlighted in the gray box. Autodock Vina results of compound 20 docked into the search space suggests hydrogen bonding between compound 20 and individual NAMPT monomer units. (Left Panel) Autodock Vina results of compound 2 docked into the search space suggests hydrogen bonding between compound 2 and individual NAMPT monomer units. (Right Panel) There is no suggestion that the molecules will bind simultaneously.