N-Propargylglycine: a unique suicide inhibitor of proline dehydrogenase with anticancer activity and brain-enhancing mitohormesis properties

Abstract

Proline dehydrogenase (PRODH) is a mitochondrial inner membrane flavoprotein critical for cancer cell survival under stress conditions and newly recognized as a potential target for cancer drug development. Reversible (competitive) and irreversible (suicide) inhibitors of PRODH have been shown in vivo to inhibit cancer cell growth with excellent host tolerance. Surprisingly, the PRODH suicide inhibitor N-propargylglycine (N-PPG) also induces rapid decay of PRODH with concordant upregulation of mitochondrial chaperones (HSP-60, GRP-75) and the inner membrane protease YME1L1, signifying activation of the mitochondrial unfolded protein response (UPRmt) independent of anticancer activity. The present study was undertaken to address two aims: (i) use PRODH overexpressing human cancer cells (ZR-75-1) to confirm the UPRmt inducing properties of N-PPG relative to another equipotent irreversible PRODH inhibitor, thiazolidine-2-carboxylate (T2C); and (ii) employ biochemical and transcriptomic approaches to determine if orally administered N-PPG can penetrate the blood–brain barrier, essential for its future use as a brain cancer therapeutic, and also potentially protect normal brain tissue by inducing mitohormesis. Oral daily treatments of N-PPG produced a dose-dependent decline in brain mitochondrial PRODH protein without detectable impairment in mouse health; furthermore, mice repeatedly dosed with 50 mg/kg N-PPG showed increased brain expression of the mitohormesis associated protease, YME1L1. Whole brain transcriptome (RNAseq) analyses of these mice revealed significant gene set enrichment in N-PPG stimulated neural processes (FDR p < 0.05). Given this in vivo evidence of brain bioavailability and neural mitohormesis induction, N-PPG appears to be unique among anticancer agents and should be evaluated for repurposing as a pharmaceutical capable of mitigating the proteotoxic mechanisms driving neurodegenerative disorders.

Brain transcriptomic, metabolic and mitohormesis properties associated with N-propargylglycine treatment: A prevention strategy against neurodegeneration

“…1A ). In agreement with our previous study in WT mice ( Scott et al, 2021 ), N -PPG also induced some evidence of mitohormesis in HD brain samples. Treated HD brains exhibited nominal (but insignificant) declines in Prodh protein levels associated with increases in Yme1l1 protease ( Fig.…”

“…However, until very recently no systemically well-tolerated, mitochondria-targeted, brain-penetrating, and mitohormesis-inducing drug candidate has been proposed and tested in vivo . In pursuing N -propargylglycine ( N -PPG) as a unique suicide inhibitor of mitochondrial proline dehydrogenase (Prodh) and potential anticancer therapeutic, we observed that, in both human cancer cells and normal mouse tissues, its irreversible inhibition and structural distortion of Prodh protein induces UPR mt and thereby activates mitohormesis, a property independent of N -PPG’s anticancer activity but consistent with its lack of any obvious host toxicity when administered orally for 9 days at 50 mg/kg ( Scott et al, 2019 ; Scott et al, 2021 ).…”

“…Prior to immunblotting, protein content was determined by Bradford Coomassie Assay (BCA) kit (Pierce, Rockford, IL) and lysates diluted into 2X Laemmli sample buffer. Immunoblotting was performed as described ( Scott et al, 2019 ; Scott et al, 2021 ) using polyvinylidene fluoride (PVDF) membranes blocked with 5 % non-fat milk in TBST (Tris-buffered saline with 0.1 % Tween-20) incubated with primary and then secondary antibodies conjugated to horse radish peroxidase (HRP), and the resulting immunoblot signals were scanned for densitometry.…”

“…The resulting Trizol solution was processed according to the manufacture’s protocol with RNA yields of at least 100 μg of RNA, from which at least 12 μg/sample was shipped as instructed on dry ice to Gene-Wiz (Azenta Life Sciences, South Plainfield, NJ) for full transcriptome sequencing (RNAseq). As described ( Scott et al, 2021 ), raw and normalized log2-scaled gene expression values (transcripts per million) were reported back. The Genewiz bioinformatics workflow used Trimmomatic (v.0.36) to remove poor quality sequences ( Bolger et al, 2014 ), and trimmed reads were then aligned to the Mus musculus GRCm38 (mm10) reference genome using STAR (v.2.5.2b) ( Dobin et al, 2013 ).…”

Perspectives, past, present and future: the proline cycle/proline-collagen regulatory axis

“…A number of agents have already been proposed and tested. Inhibitors and the enzymes they target include: tetrahydrofuroic acid, N -propargylglycine (Scott et al 2021 ) inhibitors of PRODH; NFLP, inhibitor of PYCR; halofuginone, inhibitor of prolyl t-RNA synthase; budesonide, inhibitor of collagen synthesis. These are published reports of inhibitors of the proline regulated axis and its downstream effectors (D'Aniello et al 2020 ).…”