Compound 2

Amino Acid Sequence

3D Structure (/structures/AF-P00338-F1-model_v6.cif)

Function

Interconverts simultaneously and stereospecifically pyruvate and lactate with concomitant interconversion of NADH and NAD+.

Amino Acid Sequence

3D Structure (/structures/AF-Q9UJM8-F1-model_v6.cif)

Function

Broad substrate specificity (S)-2-hydroxy-acid oxidase that preferentially oxidizes glycolate. The glyoxylate produced by the oxidation of glycolate can then be utilized by alanine-glyoxylate aminotransferase for the peroxisomal synthesis of glycine; this pathway appears to be an important step for the detoxification of glyoxylate which, if allowed to accumulate, may be metabolized to oxalate with formation of kidney stones . Can also catalyze the oxidation of glyoxylate, and long chain hydroxyacids such as 2-hydroxyhexadecanoate and 2-hydroxyoctanoate, albeit with much lower catalytic efficiency. Active in vitro with the artificial electron acceptor 2,6-dichlorophenolindophenol (DCIP), but O2 is believed to be the physiological electron acceptor, leading to the production of H2O2. Is not active on L-lactate and 2-hydroxybutanoate .

Mechanism

The autophagy-lysosomal pathway (ALP) is capable of degrading a broader range of substrates, including longer-lived and larger misfolded proteins and damaged organelles. ALP is classified into three subtypes: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, substrates are directly sequestered and degraded by lysosomes and late endosomes via membrane protrusion and invagination. In CMA, HSPA8/HSC70 recognizes specific substrates containing KFERQ-like sequences; these substrates subsequently bind to LAMP2A on the lysosomal surface and are delivered into lysosomes for degradation. In macroautophagy, substrates are encapsulated through the formation of double-membrane autophagosomes, which subsequently fuse with lysosomes to degrade the substrates.

Description

First, we cotreated cells with compound 2 and MG-132, a well-characterized proteasome inhibitor.37 No significant increase in LDHA protein levels was observed in cells treated with 2 and MG-132, compared to cells treated with compound 2 alone, therefore ruling out proteasome involvement in this process (Figure 4F). To determine whether compound 2 induces LDHA degradation via the autophagy-lysosomal pathway, we silenced autophagy-related gene 5 (Atg5) using siRNA (siAtg5). The encoded protein, ATG5, is essential for autophagosome formation and has previously been shown to be required for the action of compounds that engage this pathway.38 Transfection with siAtg5 reduced ATG5 expression by approximately 70% (Figure S75). Under these conditions, the reduction in LDHA protein levels induced by compound 2 was abolished, and LDHA protein levels were significantly higher than in cells treated with the scrambled siRNA (siScr) (Figure 4G). These results indicate that compound 2 promotes LDHA degradation via an autophagy-dependent lysosomal pathway.

Description

For hGO, most IC50 values ranged between 0.8 and 5 μM, whereas for hLDHA, many compounds exhibited nanomolar inhibitory activity. Compound 2 exhibited excellent IC50 values of 1.05 μM and 53 nM, respectively

Description

For hGO, most IC50 values ranged between 0.8 and 5 μM, whereas for hLDHA, many compounds exhibited nanomolar inhibitory activity. Compound 2 exhibited excellent IC50 values of 1.05 μM and 53 nM, respectively.

Description

We report the development of compound 2, a dual GO/LDHA inhibitor (Ki = 390 and 40 nM, respectively) that also promotes hydrophobic tag-mediated autophagic degradation of LDHA.

Description

We report the development of compound 2, a dual GO/LDHA inhibitor (Ki = 390 and 40 nM, respectively) that also promotes hydrophobic tag-mediated autophagic degradation of LDHA