New drugs for pharmacological extension of replicative life span in normal and progeroid cells

New compounds with anti-aging properties were identified using modified biochemical HTS assay for quantification of SAβG and ATP in pre-senesced cells. a The schema describes SAβG/ATP assay that was applied to pre-senesced NHDF. b Two most potent compounds, VA and N2N1, were titrated using SAβG/ATP assay. All data were normalized on untreated samples and presented as a corresponding percentile of the controls. The numbers below plots show the ratio of normalized SAβG activity divided by normalized ATP level detected in the same wells. Absolute numbers used for calculation of the ratios are shown in Table S1. Results shown are mean？±？standard deviation calculated from three independent experiments (three wells per treatments in 384-well plate). c The chemical structures of lead compounds and possible mechanism of catalytic electrons transfer in redox reactions. d Schematic representation of the approach to measure the RLS in tissue culture. RLS is measured in population doublings (PD). e Identification of most effective concentration of VA and N2N1 in RLS extension assay applied to pre-senesced NHDF. VA was most effective at 30？μM, N2N1 at 1？μM. Results shown are mean？±？standard deviation calculated from three independent experiments (three flasks per treatment). f VA (30？μM) or N2N1 (1？μM) were tested if they can increase RLS in normal human fibroblasts, stromal cells derived from normal bone marrow and, progeroid cells derived from the patients with Bloom or Werner syndromes. NHDF, stromal and Bloom syndrome cells responded to the treatment; Werner syndrome cells did not. Results shown are mean？±？standard deviation calculated from three independent experiments (three flasks per treatment). g NHDF were tested after treatment with anti-aging compounds for the expression of classic markers of aging or senescence. Both VA and N2N1 decreased the expression of SAβG detected by classic histological method, down-regulated p16, p21 and γH2A.X, and up-regulated lamin B1 protein. No difference in Nrf2 protein expression was observed. All blots derived from the same experiment and were processed in parallel. h Increase of chronological life span (CLS) in mice or C. elegans by N2N1 or VA treatment. For the mice experiment N2N1 (100？μM) or VA (1？mM) were given in drinking water. Same concentrations of N2N1 and VA were used for the C. elegans CLS experiment. The Logrank test was used to verify the null hypothesis that there is no difference between control and treated groups (VA or N2N1). The calculated p values (0.007 for VA and 0.005 for N2N1) do not allow to