Here, we investigate an unusual antiviral mechanism developed in the bacterium Streptomyces griseus. SgrAI is a type II restriction endonuclease that forms run-on oligomer filaments when activated and possesses both accelerated DNA cleavage activity and expanded DNA sequence specificity. Mutations disrupting the run-on oligomer filament eliminate the robust antiphage activity of wild-type SgrAI, and the observation that even relatively modest disruptions completely abolish this anti-viral activity shows that the greater speed imparted by the run-on oligomer filament mechanism is critical to its biological function. Simulations of DNA cleavage by SgrAI uncover the origins of the kinetic advantage of this newly described mechanism of enzyme regulation over more conventional mechanisms, as well as the origin of the sequestering effect responsible for the protection of the host genome against damaging DNA cleavage activity of activated SgrAI. IMPORTANCE This work is motivated by an interest in understanding the characteristics and advantages of a relatively newly discovered enzyme mechanism involving filament formation. SgrAI is an enzyme responsible for protecting against viral infections in its host bacterium and was one of the first such enzymes shown to utilize such a mechanism. In this work, filament formation by SgrAI is disrupted, and the effects on the speed of the purified enzyme as well as its function in cells are measured. It was found that even small disruptions, which weaken but do not destroy filament formation, eliminate the ability of SgrAI to protect cells from viral infection, its normal biological function. Simulations of enzyme activity were also performed and show how filament formation can greatly speed up an enzyme’s activation compared to that of other known mechanisms, as well as to better localize its action to molecules of interest, such as invading phage DNA.

Filament or run-on oligomer formation by enzymes is now recognized as a widespread phenomenon with potentially unique enzyme regulatory properties and biological roles. SgrAI is an allosteric type II restriction endonuclease that forms run-on oligomeric filaments with activated DNA cleavage activity and altered DNA sequence specificity. In this two-part work, we measure individual steps in the run-on oligomer filament mechanism to address specific questions of cooperativity, trapping, filament growth mechanisms, and sequestration of activity using fluorophore-labeled DNA, kinetic FRET measurements, and reaction modeling with global data fitting. The final models and rate constants show that the assembly step involving association of SgrAI-DNA complexes into the run-on oligomer filament is relatively slow (3-4 orders of magnitude slower than diffusion limited) and rate-limiting at low to moderate concentrations of SgrAI-DNA. The disassembly step involving dissociation of complexes of SgrAI-DNA from each other in the run-on oligomer filament is the next slowest step but is fast enough to limit the residence time of any one copy of SgrAI or DNA within the dynamic filament. Further, the rate constant for DNA cleavage is found to be 4 orders of magnitude faster in the run-on oligomer filament than in isolated SgrAI-DNA complexes and faster than dissociation of SgrAI-DNA complexes from the run-on oligomer filament, making the reaction efficient in that each association into the filament likely leads to DNA cleavage before filament dissociation.

Filament or run-on oligomer formation by metabolic enzymes is now recognized as a widespread phenomenon having potentially unique enzyme regulatory properties and biological roles, and its dysfunction is implicated in human diseases such as cancer, diabetes, and developmental disorders. SgrAI is a bacterial allosteric type II restriction endonuclease that binds to invading phage DNA, may protect the host DNA from off-target cleavage activity, and forms run-on oligomeric filaments with enhanced DNA-cleavage activity and altered DNA sequence specificity. However, the mechanisms of SgrAI filament growth, cooperativity in filament formation, sequestration of enzyme activity, and advantages over other filament mechanisms remain unknown. In this first of a two-part series, we developed methods and models to derive association and dissociation rate constants of DNA-bound SgrAI in run-on oligomers and addressed the specific questions of cooperativity and filament growth mechanisms. We show that the derived rate constants are consistent with the run-on oligomer sizes determined by EM analysis and are most consistent with a noncooperative growth mode of the run-on oligomer. These models and methods are extended in the accompanying article to include the full DNA-cleavage pathway and address specific questions related to the run-on oligomer mechanism including the sequestration of DNA-cleavage activity and trapping of products.

No abstract

Glioblastoma multiforme (GBM) is an aggressive stage IV brain cancer that is difficult to detect, typically inoperable, and associated with a poor prognosis. Challenges in targeting GBM arise from the involvement of various pro-survival pathways, being comprised of four different cellular states, and displaying heterogeneity between each tumor. Marketed FDA-approved drugs for GBM have demonstrated limited efficacy, toxicity issues, resistance, and poor BBB penetrance. Further probing into pathway breakdowns led to the discovery of the DYRKs (dual-specificity tyrosine-regulated kinase), CLKs (CDC-like kinases), PDGFRA/B (platelet-derived growth factor receptor), and PI3Kα (phosphoinositide 3-kinase) involvement and overexpression. The upregulation of these kinases are found to be prominently involved in GBM cell proliferation, survival, replication, aggression, metastasis, and resistance. Herein, we believe that the development of pleiotropic competitive inhibitors for the treatment of GBM could be a promising therapeutic option based on literature precedence and current data from research. DYR726, a water-soluble, pleiotropic inhibitor [IC50 PDGFRA/B/PI3Kα/CLk2/3DYRK1a/2/3 ( 53.0/48.0/6.2/15.13/137/4/38.6/11.2 nM)] has demonstrated in vitro cellular efficacy across various GBM cell lines. When treated in non-cancerous cell lines, DYR726 displayed an optimal therapeutic window, which is not observed in any glioma drugs currently in clinical trials. At 1µM concentrations, complete dissociation of the formation of neural stem cells and neurosphere growth in glioma cells are observed. Reduction in metastasis and proliferation are found when treated with DYR726 over three weeks. In vitro efficacy against current approved FDA treatment options and clinical candidates has been extensively tested across numerous GBM cell lines. When benched against one of the best PI3K kinase inhibitors in the clinic, Buparlisib, DYR726 was comparable, indicating a disruption in the RTK signaling survival pathway that is commonly associated with GBM. DYR726 performed favorably against Avapritinib (clinical trials for gliomas are beginning at Stanford) and when treated against FDA-approved combination treatment Dabrafenib+Trametinib (for 2% of glioma patients), it performed identically, suggesting that DYR726 can inhibit the cells at the same rate as the new FDA drug. Within patient-derived cell lines, it behaves identically to Buparlisib, and DYR726 has been shown to outperform AstraZenecas’ Osimertinib (which was significantly less potent) and Tafinlars’ Dabrafenib (displayed no effect). Based on the significance of efficacy, this series is on the CRUK Glasgow Cancer Center grant renewal as a potential Phase 0 within the next five years. Citation Format: Alessandra Fistrovich, Vasudah Tandon, Carly Cabel, Laura Basantes, Curtis Thorne, William Montfort, Sourav Banerjee, Christopher Hulme. The development of pleotropic small molecule kinase inhibitors (SKMIs) for the treatment of glioblastoma multiforme (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3100.