Disruption of R2TP-mediated PIKK maturation with de novo designed binders

Gonzalez-Rodriguez, N.; Lopez-Perrote, A.; Reglero, C.; Alegrio-Louro, J.; Failde-Soler, M.; Chacon-Sanchez, C.; Blanco-Romero, E.; Casajus-Pelegay, E.; Lopez-Pernas, G.; Hernandez, I.; Murga, M.; Fernandez-Capetillo, O.; Llorca, O.; Fernandez-Leiro, R.

doi:10.64898/2026.10.26.761954

bioRxiv · 10.64898/2026.10.26.761954

Disruption of R2TP-mediated PIKK maturation with de novo designed binders

Gonzalez-Rodriguez, N.·Lopez-Perrote, A.·Reglero, C.·Alegrio-Louro, J.·Failde-Soler, M.·Chacon-Sanchez, C.·Blanco-Romero, E.·Casajus-Pelegay, E.·Lopez-Pernas, G.·Hernandez, I.·Murga, M.·Fernandez-Capetillo, O.·Llorca, O.·Fernandez-Leiro, R.

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Abstract

Phosphatidylinositol 3-kinase-related kinases (PIKKs), including ATM, ATR, DNA-PKcs, SMG1, TRRAP and mTOR, are regulators of gene expression, DNA damage response and metabolism. Their maturation depends on R2TP, a co-chaperone complex comprising the RUVBL1/2 AAA+ ATPase, RPAP3 and PIH1D1. While small-molecule RUVBL1/2 inhibitors have shown promise in cancer models, the presence of RUVBL1/2 in other complexes raises concerns about specificity. Here, we identified the RUVBL1/2-RPAP3 interface as a vulnerability, exploiting it using de novo designed protein binders that occupy the RPAP3-binding site. These subnanomolar-affinity binders dismantle R2TP, depleting levels of all PIKKs while sparing other RUVBL1/2-containing complexes, including chromatin remodelers INO80, SRCAP and TIP60. CryoEM, proteomic, biochemical and cellular analyses revealed that these binders outcompete RPAP3, trapping RUVBL1/2 in a catalytically inactive conformation. Our findings establish selective disruption of R2TP by artificial proteins as a strategy to control PIKK abundance.

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Gonzalez-Rodriguez, N., Lopez-Perrote, A., Reglero, C., Alegrio-Louro, J., Failde-Soler, M., Chacon-Sanchez, C., Blanco-Romero, E., Casajus-Pelegay, E., Lopez-Pernas, G., Hernandez, I., Murga, M., Fernandez-Capetillo, O., Llorca, O., Fernandez-Leiro, R.. 2026-10-28. Disruption of R2TP-mediated PIKK maturation with de novo designed binders. https://doi.org/10.64898/2026.10.26.761954

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