Novel pyrazolo[1,5-a]pyrimidine carboxamide PAMs of ATP6V1A restore aged lysosomal acidification to young-equivalent ...

IF novel positive allosteric modulators (PAMs) of ATP6V1A—specifically pyrazolo[1,5-a]pyrimidine carboxamide scaffolds identified via ensemble docking against cryptic pockets in the ATP6V1A V1 catalytic subunit (PDB: 6WLZ, cryo-EM 2.9 Å)—with predicted Kd <500 nM are administered systemically (oral, p.o., once daily) to aged (22–24 month) male and female C57BL/6J mice,

THEN measurable reversal of accumulated lysosomal cargo burden—quantified as ≥40% reduction in autofluorescent lipofuscin density (by spectral confocal imaging), ≥30% decrease in p62/SQSTM1 puncta per cell (by immunofluorescence), and restoration of lysosomal pH to young-equivalent values (LysoSensor Yellow-Blue ratiometric assay, target pH 4.5–5.0 vs. aged baseline ~5.5–6.0) in liver and brain tissue—will be observed within 8 weeks of treatment,

BECAUSE the following causal chain connects the intervention to the repair endpoint:

1. Aging progressively impairs ATP6V1A catalytic competence, producing a functional state equivalent to haploinsufficiency: reduced V1-domain rotational coupling efficiency causes lysosomal alkalinization, which in turn slows cathepsin activation and lysosomal hydrolase function, causing net accumulation of undegraded macromolecular cargo (lipofuscin, oxidized protein aggregates, dysfunctional organellar remnants). The ASpdb annotation confirms ATP6V1A localizes to intracellular membrane-bounded organelles and secretory granules (Gene Ontology IDA evidence, PubMed 33065002 and 23035048), and that the protein carries ARCL2D and DEE93 disease annotations—conditions defined by lysosomal dysfunction arising from partial V-ATPase loss—establishing haploinsufficiency as a mechanistic template for the aged phenotype. (ATP6V1A haploinsufficiency causes lysosomal acidification failure)[https://biodataai.uth.edu/ASpdb/gene_search_result.cgi?page=page&type=quick_search&quick_search=523]

2. The canonical isoform P38606-1 (617 aa) and the alternatively spliced isoform P38606-2 (584 aa, lacking N-terminal residues 1–33) differ in a domain that precedes the known catalytic core, suggesting the N-terminal 33-aa segment modulates assembly-competent conformation or allosteric communication between subunits. [SPECULATIVE] Age-associated shifts in splicing efficiency toward the P38606-2 isoform—which may have reduced assembly efficiency into the intact V1V0 holoenzyme—could compound catalytic decline. This creates a targetable structural vulnerability: a cryptic allosteric pocket adjacent to the nucleotide-binding domain of P38606-1, identifiable in MD snapshots of the 6WLZ structure, whose occupancy by a PAM would geometrically favor the ATP-bound, closed, catalytically competent state. (Two ATP6V1A protein isoforms with differing N-terminal sequences)[https://biodataai.uth.edu/ASpdb/gene_search_result.cgi?page=page&type=quick_search&quick_search=523]

3. **Pyrazolo[1,5-a]pyrimidine cores with carboxamide linkers possess directional hydrogen bond donor/acceptor a...

SENS category: LysoSENS