Identification of Direct Target Engagement Biomarkers for Kinase-Targeted Therapeutics

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3′-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1Ser410 and p-PDK1Thr513 are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1Ser241), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers

Prognostic implications of serial high-sensitivity cardiac troponin testing among patients with COVID-19:A Danish nationwide registry-based cohort study

BACKGROUND: Although troponin elevation is associated with worse outcomes among patients with coronavirus disease 2019 (COVID-19), prognostic implications of serial troponin testing are lacking. We investigated the association between serial troponin measurements and adverse COVID-19 outcomes. METHODS: Using Danish registries, we identified COVID-19 patients with a high-sensitivity troponin measurement followed by a second measurement within 1–24 h. All measurements during follow-up were also utilized in subsequent time-varying analyses. We assessed all-cause mortality associated with the absence/presence of myocardial injury (≥1 troponin measurement >99th percentile upper reference limit) and absence/presence of dynamic troponin changes (>20% relative change if first measurement elevated, >50% relative change if first measurement normal). RESULTS: Of 346 included COVID-19 patients, 56% had myocardial injury. Overall, 20% had dynamic troponin changes. In multivariable Cox regression models, myocardial injury was associated with all-cause mortality (HR = 2.56, 95%CI = 1.46–4.51), as were dynamic troponin changes (HR = 1.66, 95%CI = 1.04–2.64). We observed a low incidence of myocardial infarction (4%) and invasive coronary procedures (4%) among patients with myocardial injury. CONCLUSIONS: Myocardial injury and dynamic troponin changes determined using serial high-sensitivity troponin testing were associated with poor prognosis among patients with COVID-19. The risk of developing myocardial infarction requiring invasive management during COVID-19 hospitalization was low

Abstract B018: MTA-cooperative PRMT5 inhibitors selectively modulate RNA splicing in MTAP-deleted cancer cells across histologies

Abstract PRMT5 is a type II arginine methyltransferase that forms an active complex with methylosome protein 50 (MEP50) to catalyze the symmetric dimethylation (SDMA) of arginine residues in proteins that regulate biological roles including modulation of apoptosis, DNA damage response and RNA processing.  Some of the best characterized PRMT5 substrates are SNRPB, SNRPD1 and SNRPD3, which are necessary for the formation of the spliceosome and therefore RNA splicing fidelity.  The development of MTA-cooperative PRMT5 inhibitors, including the clinical stage inhibitors TNG908 and TNG462, have been shown in preclinical studies to selectively inhibit PRMT5 in MTAP-deleted cancer cells while sparing normal, MTAP-intact cells.  Consistent with this selective, on-target mechanism, treatment with TNG908 in preclinical studies results in increased aberrant RNA splicing in MTAP-deleted cells relative to MTAP-intact cells.  As MTAP loss occurs in 10-15% of all human cancer, the identification of a signature of alternative splicing events may report pharmacodynamic activity of PRMT5 inhibitors and potentially predict patient response.  Similar splicing alterations caused by PRMT5 inhibition were identified in preclinical MTAP-deleted cancer models representing glioblastoma, non-small cell lung cancer, among others, suggesting the events are histology-agnostic.  Additionally, titration of exogenous MTA, an endogenous inhibitor of PRMT5, recapitulated the observed splicing events in preclinical MTAP-proficient cells.  Similar findings were observed utilizing a pharmacological inhibitor of MTAP suggesting that the identified alternative splicing events are dependent on accumulation of MTA.  Collectively, these data suggest that a PRMT5-dependent RNA splicing signature can be developed to identify solid tumors that may best respond to MTA-cooperative PRMT5 inhibitors as well as monitor their pharmacodynamic activity. Citation Format: Matthew R Tonini, Samuel R Meier, Shangtao Liu, Kevin M Cottrell, John P Maxwell, Jannik N Andersen, Alan Huang, Luisa Cimmino, Kimberly J Briggs. MTA-cooperative PRMT5 inhibitors selectively modulate RNA splicing in MTAP-deleted cancer cells across histologies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B018

Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs

At present there are no drugs for the treatment of chronic liver fibrosis that have been approved by the Food and Drug Administration of the United States. Telmisartan, a small-molecule antihypertensive drug, displays antifibrotic activity, but its clinical use is limited because it causes systemic hypotension. Here, we report the scalable and convergent synthesis of macromolecular telmisartan prodrugs optimized for preferential release in diseased liver tissue. We have optimized the release of active telmisartan in fibrotic liver to be depot-like (that is, a constant therapeutic concentration) through the molecular design of telmisartan brush-arm star polymers, and show that these lead to improved efficacy and to the avoidance of dose-limiting hypotension in both metabolically and chemically induced mouse models of hepatic fibrosis, as determined by histopathology, enzyme levels in the liver, intact-tissue protein markers, hepatocyte necrosis protection and gene-expression analyses. In rats and dogs, the prodrugs are retained long term in liver tissue, and have a well-tolerated safety profile. Our findings support the further development of telmisartan prodrugs that enable infrequent dosing in the treatment of liver fibrosis.National Institutes of Health (U.S.) (Grant 1R01CA220468-01)National Institutes of Health (U.S.) (Fellowship 1F32EB023101