A DUAL MTOR/NAD+ ACTING GEROTHERAPY

Abstract

The geroscience hypothesis states that a therapy that prevents the underlying aging process should prevent multiple aging related diseases. The mTOR (mechanistic target of rapamycin)/insulin and NAD+ (nicotinamide adenine dinucleotide) pathways are two of the most validated aging pathways. Yet, it's largely unclear how they might talk to each other in aging. In genome-wide CRISPRa screening with a novel class of N-O-Methyl-propanamide-containing compounds we named BIOIO-1001, we identified lipid metabolism centering on SIRT3 as a point of intersection of the mTOR/insulin and NAD+ pathways. In vivo testing indicated that BIOIO-1001 reduced high fat, high sugar diet-induced metabolic derangements, inflammation, and fibrosis, each being characteristic of non-alcoholic steatohepatitis (NASH). An unbiased screen of patient datasets suggested a potential link between the anti-inflammatory and anti-fibrotic effects of BIOIO-1001 in NASH models to those in amyotrophic lateral sclerosis (ALS). Directed experiments subsequently determined that BIOIO-1001 was protective in both sporadic and familial ALS models. Both NASH and ALS have no treatments and suffer from a lack of convenient biomarkers to monitor therapeutic efficacy. A potential strength in considering BIOIO-1001 as a therapy is that the blood biomarker that it modulates, namely plasma triglycerides, can be conveniently used to screen patients for responders. More conceptually, to our knowledge BIOIO-1001 is a first therapy that fits the geroscience hypothesis by acting on multiple core aging pathways and that can alleviate multiple conditions after they have set in.

Figure 1.. Genome-wide screening with BIOIO-1001 identifies SIRT3 as a point of intersection between the MTOR and NAD+ pathways.

A. Chemical structure of BIOIO-1001. B. Schematic of a CRISPRa screen with BIOIO-1001. Screening was performed in duplicate with 50μM of BIOIO-1001 or vehicle (DMSO) in K562 cells. C. Genetic map of top hits from the BIOIO-1001 CRISPRa screen. Sensitizing and resistance hits (p < 0.05, based on the average phenotype of top three sgRNAs out of 10) are shown in red and blue, respectively. The putative mechanism of action of BIOIO-1001 is where the hits flip from resistance to sensitizing to resistance or vice versa (highlighted in gray) in both the mTORC1 and NAD+ pathways. The common target of both pathways is fatty acid oxidation, and specifically the mTORC1 and NAD+ targets, LPIN1 and SIRT3, respectively. Asterisk on LPIN1 indicates it was sensitizing but its p value was greater than 0.05. D. Relative SIRT3 mRNA expression in SIRT3 CRISPRa K562 cells. SIRT3 mRNA levels were assessed by RT-qPCR. One-way ANOVA for all SIRT3 sgRNAs vs. control sgRNA. E. Relative ATP levels in SIRT3 overexpressing and control cells in the presence of the indicated concentrations of BIOIO-1001 or vehicle (DMSO). ATP levels were measured after 72 hours treatment in cells from D. One-way ANOVA for all SIRT3 sgRNAs vs. control sgRNA. F. Transcriptionally profiling of livers harvested from mice treated with BIOIO-1001. Mice fed a high fat diet for 12 weeks were gavaged once daily with 30mg/kg/day of BIOIO-1001 or vehicle (n=6) for one week prior to euthanasia. Genes that were increased in expression 10-fold compared to vehicle treated condition were included in the gene ontology analysis. Only genes with counts per million (CPM) in the vehicle treated condition of greater than 0.1 were included. G. Wildtype and SIRT3 deficient primary mouse hepatocytes treated with BIOIO-1001, BIOIO-1002, or vehicle at the indicated concentrations for 24 hours. Gene expression of the indicated genes were measured. One-way ANOVA for all SIRT3 sgRNAs vs. control sgRNA. Asterisk (*) indicates p < 0.05 (n=4). H. Predicted small molecule docking pose of BIOIO-1001 with SIRT3. Shown is the highest confidence docking pose of BIOIO-1001 (red) with human SIRT3 protein (PDB: #5514, solvent and PEG removed). NAD-Ribose (blue) is visible bound in the active site of SIRT3. Zinc ion (gray) shown.

Figure 2.. BIOIO-1001 reverses liver injury and stellate cell activation in a mouse model of NASH.

A. Schematic depicting the time course of diet and drug treatment. Mice were fed a low fat or HTF-C diet for 16 weeks and then treated with vehicle (veh), PIO, or BIOIO-1001 for 3 weeks by gavage. B-F. Liver triglycerides (B, circulating ALT activity (C), circulating AST (D), hepatic NAS (E) and fibrosis scoring (F) from mice treated in A. One-way ANOVA was performed to compare the four groups (n=6 except, NAS and fibrosis where n=10-13). G-I. Expression of genes encoding markers of inflammation and stellate cell activation in mice treated as in A. J. Wildtype and SIRT3 deficient primary mouse hepatocytes treated with BIOIO-1001 or vehicle at the indicated concentrations for 24 hours. Gene expression of the indicated genes were measured. P-values based on multiple unpaired t-tests.

Figure 3.. BIOIO-1001 promotes cell and organismal survival in genetic and sporadic models of ALS.

A. Transcriptional profiling (RNAseq) in liver, heart, and subcutaneous fat using BIOIO-1001. Top gene signatures from dbGaP via Enrichr were derived from those genes that were 1.25X increased due to 30mg/kg BIOIO-1001 as in Figure 2F (127 genes out of 40,757). B. The indicated concentrations of BIOIO-1001 or DMSO were given to iPSCs derived from a patient with sporadic ALS. 31-day motor neuron survival as judged by ISL1 (motor neuron marker, in red)/DAPI (nuclear marker, in blue) ratios were normalized to day 28 motor neuron survival. SMI-32, another neuronal marker is in green. C. Cell viability in human iPSCs containing a G93A SOD1 mutation. Cells were treated with the indicated concentrations of BIOIO-1001 or vehicle for 24hrs. D. Schematic depicting the time course of BIOIO-1001 treatment in the hereditary (genetic) ALS mouse model, G93A Sod1. E. Plasma triglycerides collected from animals treated as in D after four weeks of treatment. F, G. Paralysis and survival analysis of G93A Sod1 mutant mice treated with BIOIO-1001 or vehicle as schematized in D.