Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities

Abstract

For inhibitor design, as in most research, the best system is question dependent. We suggest structurally defined allostery to design specific inhibitors that target regions beyond active sites. We choose systems allowing efficient quality structures with conformational changes as optimal for structure-based design to optimize inhibitors. We maintain that evolutionarily related targets logically provide molecular avatars, where this Sanskrit term for descent includes ideas of functional relationships and of being a physical embodiment of the target’s essential features without requiring high sequence identity. Appropriate biochemical and cell assays provide quantitative measurements, and for biomedical impacts, any inhibitor’s activity should be validated in human cells. Specificity is effectively shown empirically by testing if mutations blocking target activity remove cellular inhibitor impact. We propose this approach to be superior to experiments testing for lack of cross-reactivity among possible related enzymes, which is a challenging negative experiment. As an exemplary avatar system for protein and DNA allosteric conformational controls, we focus here on developing separation-of-function inhibitors for meiotic recombination 11 nuclease activities. This was achieved not by targeting the active site but rather by geometrically impacting loop motifs analogously to ribosome antibiotics. These loops are neighboring the dimer interface and active site act in sculpting dsDNA and ssDNA into catalytically competent complexes. One of our design constraints is to preserve DNA substrate binding to geometrically block competing enzymes and pathways from the damaged site. We validate our allosteric approach to controlling outcomes in human cells by reversing the radiation sensitivity and genomic instability in BRCA mutant cells.

MRE11-RAD50-NBS1 Complex Is Sufficient to Promote Transcription by RNA Polymerase II at Double-Strand Breaks by Melting DNA Ends

“…Next, we determined whether MRN favors the transcriptional activity of RNAPII beyond promoting its recruitment to DSBs. Previous cellular experiments showed that mirin, a molecule that restricts the access of MRE11 to dsDNA and inhibits its nuclease activity ( Moiani et al., 2018 ; Shibata et al., 2014 ), inhibited the transcription of dilncRNA ( Michelini et al., 2017 ), suggesting that the nuclease activity of MRE11 may be involved. We hypothesized that the ability of MRNs to resect DSBs to generate 3′ protruding DNA ends could provide an ideal DNA substrate for transcription by RNAPII ( Kadesch and Chamberlin, 1982 ).…”

“…To strengthen our conclusion that MRN stimulates RNAPII activity by melting DNA using an independent approach, we used small-molecule allosteric inhibitors of MRN activities ( Shibata et al., 2014 ). While PFM39 inhibits exonuclease activity, PFM01 attenuates the endonuclease activity of MRN ( Moiani et al., 2018 ; Shibata et al., 2014 ), and mirin was shown to reduce both activities ( Deshpande et al., 2016 ). We next performed our transcription assays with MRN and RNAPII in the presence of mirin, PFM39, or PFM01 ( Shibata et al., 2014 ).…”

“…Recent studies have reported specific allosteric inhibitors of MRN, which bind adjacent to active sites ( Moiani et al., 2018 ; Shibata et al., 2014 ). Mirin was shown to inhibit ATM activation ( Dupré et al., 2008 ), but MRN nuclease activity is dispensable for ATM activation ( Paull, 2015 ).…”

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

“…Furthermore, structural information on MR has emerged from prokaryotes that can provide more accessible structures than have been analyzed for their eukaryotic counterparts. In fact, prokaryotic Mre11 is used as a molecular avatar (an embodiment of human MRE11–essential features) for design of inhibitors against human MRE11 (47). Excitingly, high-resolution structures of eukaryotic MRN protein domains are also being solved (33, 38, 39).…”

Evolving SAXS versatility: solution X-ray scattering for macromolecular architecture, functional landscapes, and integrative structural biology

“…Moreover, ensemble readout from SAXS is well suited to detecting selective stabilization of transient conformations by ligand interactions. Development of allosteric modulators of protein ensembles has come increasingly into focus for drug targeting, as these ligands avoid competitive interplay with endogenous ligands [ 132 – 134 ]. The move to target small-molecules toward protein complexes and assemblies to more effectively modulate signaling pathways is well aligned to these advantages of SAXS-based approaches for screening and structure-function analysis.…”