7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase

Reviewer #1 (Public Review):

Summary:
Zink et al set out to identify selective inhibitors of the pyridoxal phosphatase (PDXP). Previous studies had demonstrated improvements in cognition upon removal of PDXP, and here the authors reveal that this correlates with an increase in pyridoxal phosphate (PLP; PDXP substrate and an active coenzyme form of vitamin B6) with age. Since several pathologies are associated with decreased vitamin B6, the authors propose that PDXP is an attractive therapeutic target in the prevention/treatment of cognitive decline. Following high throughput and secondary small molecule screens, they identify two selective inhibitors. They follow up on 7, 8 dihydroxyflavone (DHF). Following structure-activity relationship and selectivity studies, the authors then solve a co-crystal structure of 7,8 DHF bound to the active site of PDXP, supporting a competitive mode of PDXP inhibition. Finally, they find that treating hippocampal neurons with 7,8 DHF increases PLP levels in a WT but not PDXP KO context. The authors note that 7,8 DHF has been used in numerous rodent neuropathology models to improve outcomes. 7, 8 DHF activity was previously attributed to activation of the receptor tyrosine kinase TrkB, although this appears to be controversial. The present study raises the possibility that it instead/also acts through modulation of PLP levels via PDXP, and is an important area for future work.

Strengths:
The strengths of the work are in the comprehensive, thorough, and unbiased nature of the analyses revealing the potential for therapeutic intervention in a number of pathologies.

Weaknesses:
Potential weaknesses include the poor solubility of 7,8 DHF that might limit its bioavailability given its relatively low potency (IC50= 0.8 uM), which was not improved by SAR. However, the compound has an extended residence time and the co-crystal structure could aid the design of more potent molecules and would be of interest to those in the pharmaceutical industry. The images related to crystal structure could be improved.

Reviewer #2 (Public Review):

Summary: In this study, the authors performed a screening for PDXP inhibitors to identify compounds that could increase levels of pyridoxal 5'- phosphate (PLP), the co-enzymatically active form of vitamin B6. For the screening of inhibitors, they first evaluated a library of about 42,000 compounds for activators and inhibitors of PDXP and secondly, they validated the inhibitor compounds with a counter-screening against PGP, a close PDXP relative. The final narrowing down to 7,8-DHF was done using PLP as a substrate and confirmed the efficacy of this flavonoid as an inhibitor of PDXP function. Physiologically, the authors show that, by acutely treating isolated wild-type hippocampal neurons with 7,8-DHF they could detect an increase in the ratio of PLP/PL compared to control cultures. This effect was not seen in PDXP KO neurons.

Strengths: The screening and validation of the PDXP inhibitors have been done very well because the authors have performed crystallographic analysis, a counter screening, and mutation analysis. This is very important because such rigor has not been applied to the original report of 7,8 DHF as an agonist for TrkB. Which is why there is so much controversy on this finding.

Weaknesses: As mentioned in the summary report the study may benefit from some in vivo analysis of PLP levels following 7,8-DHF treatment, although I acknowledge that it may be challenging because of the working out of the dosage and timing of the procedure.

Reviewer #3 (Public Review):

This is interesting biology. Vitamin B6 deficiency has been linked to cognitive impairment. It is not clear whether supplements are effective in restoring functional B6 levels. Vitamin B6 is composed of pyridoxal compounds and their phosphorylated forms, with pyridoxal 5-phosphate (PLP) being of particular importance. The levels of PLP are determined by the balance between pyridoxal kinase and phosphatase activities. The authors are testing the hypothesis that inhibition of pyridoxal phosphatase (PDXP) would arrest the age-dependent decline in PLP, offering an alternative therapeutic strategy to supplements. Published data illustrating that ablation of the Pdxp gene in mice led to increases in PLP levels and improvement in learning and memory trials are consistent with this hypothesis.

In this report, the authors conduct a screen of a library of ~40k small molecules and identify 7,8-dihydroxyflavone (DHF) as a candidate PDXP inhibitor. They present an initial characterization of this micromolar inhibitor, including a co-crystal structure of PDXP and 7,8-DHF. In addition, they demonstrate that treatment of cells with 7,8 DHP increases PLP levels. Overall, this study provides further validation of PDXP as a therapeutic target for the treatment of disorders associated with vitamin B6 deficiency and provides proof-of-concept for inhibition of the target with small-molecule drug candidates.

Strengths include the biological context, the focus on an interesting and under-studied class of protein phosphatases that includes several potential therapeutic targets, and the identification of a small molecule inhibitor that provides proof-of-concept for a new therapeutic strategy. Overall, the study has the potential to be an important development for the phosphatase field in general.

Weaknesses include the fact that the compound is very much an early-stage screening hit. It is an inhibitor with micromolar potency for which mechanisms of action other than inhibition of PDXP have been reported. Extensive further development will be required to demonstrate convincingly the extent to which its effects in cells are due to on-target inhibition of PDXP.