Search

Protein fragmentation is a critical component of top-down proteomics, enabling gene-specific protein identification and full proteoform characterization. The factors that influence protein fragmentation include precursor charge, structure, and primary sequence, which have been explored extensively for collision-induced dissociation (CID). Recently, noticeable differences in CID-based fragmentation were reported for native versus denatured proteins, motivating the need for scoring metrics that are tailored specifically to native top-down mass spectrometry (nTDMS). To this end, position and intensity were tracked for 10,252 fragment ions produced by higher-energy collisional dissociation (HCD) of 159 native monomers and 70 complexes. We used published structural data to explore the relationship between fragmentation and protein topology and revealed that fragmentation events occur at a large range of relative residue solvent accessibility. Additionally, our analysis found that fragment ions at sites with an N-terminal aspartic acid or a C-terminal proline make up on average 40 and 27%, respectively, of the total matched fragment ion intensity in nTDMS. Percent intensity contributed by each amino acid was determined and converted into weights to (1) update the previously published C-score and (2) construct a native Fragmentation Propensity Score. Both scoring systems showed an improvement in protein identification or characterization in comparison to traditional methods and overall increased confidence in results with fewer matched fragment ions but with high probability nTDMS fragmentation patterns. Given the rise of nTDMS as a tool for structural mass spectrometry, we forward these scoring metrics as new methods to enhance analysis of nTDMS data.

Efforts at altering the dismal prognosis of pediatric midline gliomas focus on direct-delivery strategies like convection-enhanced delivery (CED), where a cannula is implanted into tumor. Successful CED treatments require confirmation of tumor coverage, dosimetry, and longitudinal in vivo pharmacokinetics monitoring. These properties would be best determined clinically with image guided dosimetry using theranostic compounds, agents with both therapeutic and imaging properties. In this study, we combine CED with novel, molecular-grade positron emission tomography (PET) imaging. We synthesized PETobinostat, a novel PET-imageable HDAC inhibitor, and showed its effectiveness against DIPG models in vitro and in vivo. Cell studies against a library of DIPG cells show nanomolar IC50, allowing for rapid in vivo translation. When injected in mice, PET shows the need of CED to achieve high brain concentrations, as systemic delivery yields inferior brain permeation. PET also shows that CED has significant mouse-to-mouse variability: imaging is used to modulate CED infusions to maximize tumor saturation over time. By determining condition-specific clearance half-life (ranging between 60 and 120 minutes), we maximized tumor permeation above therapeutic concentrations for at least 12 hours. This PET-guided approach resulted in decrease tumor cellularity (p= 0.001), increased apoptosis (p= 0.034), decreased dividing cells (p= 0.003), and recovery of histone-3 acetylation (p < 0.0001) when compared against vehicle and systemic-treated controls in tumor-bearing mice. Further, the PET-guided CED of PETobinostat resulted in survival prolongation (67.5 vs. 35 days, p = 0.0001) when compared to systemic administration of another potent HDAC inhibitor (Panobinostat). CED without PET guidance failed at improving survival (37.5 vs. 35 days, p = 0.74). No significant toxicity was observed following CED of PETobinostat. This work demonstrates how personalized image-guided drug delivery of a novel HDAC inhibitor may be useful in potentiating CED-based treatment platforms, and supports a foundation for the clinical translation of PETobinostat.

In vivo, real-time image-guided CED of a PET-imageable HDAC inhibitor is needed to improve survival in murine models of DIPG., Efforts at altering the dismal prognosis of pediatric midline gliomas focus on direct delivery strategies like convection-enhanced delivery (CED), where a cannula is implanted into tumor. Successful CED treatments require confirmation of tumor coverage, dosimetry, and longitudinal in vivo pharmacokinetic monitoring. These properties would be best determined clinically with image-guided dosimetry using theranostic agents. In this study, we combine CED with novel, molecular-grade positron emission tomography (PET) imaging and show how PETobinostat, a novel PET-imageable HDAC inhibitor, is effective against DIPG models. PET data reveal that CED has significant mouse-to-mouse variability; imaging is used to modulate CED infusions to maximize tumor saturation. The use of PET-guided CED results in survival prolongation in mouse models; imaging shows the need of CED to achieve high brain concentrations. This work demonstrates how personalized image-guided drug delivery may be useful in potentiating CED-based treatment algorithms and supports a foundation for clinical translation of PETobinostat.

Cancer cells reprogram their metabolism to support growth and to mitigate cellular stressors. The serine synthesis pathway has been identified as a metabolic pathway frequently altered in cancers and there has been considerable interest in developing pharmacological agents to target this pathway. Here, we report a series of indole amides that inhibit human 3-phosphoglycerate dehydrogenase (PHGDH), the enzyme that catalyzes the first committed step of the serine synthesis pathway. Using X-ray crystallography, we show that the indole amides bind the NAD(+) pocket of PHGDH. Through structure-based optimization we were able to develop compounds with low nanomolar affinities for PHGDH in an enzymatic IC(50) assay. In cellular assays, the most potent compounds inhibited de novo serine synthesis with low micromolar to sub-micromolar activities and these compounds successfully abrogated the proliferation of cancer cells in serine free media. The indole amide series reported here represent an important improvement over previously published PHGDH inhibitors as they are markedly more potent and their mechanism of action is better defined.

Cancer cells can adapt to nutrient poor conditions by rewiring their metabolism and using alternate fuel sources. Identifying these adaptive metabolic pathways may provide novel targets for cancer therapy. Here, we identify a subset of non-small cell lung cancer (NSCLC) cell lines that survive in the absence of glucose by internalizing and metabolizing extracellular protein via macropinocytosis. Macropinocytosis is increased in these glucose independent cells, and is regulated by phosphoinositide 3-kinase (PI3K) activation of Rac-Pak signaling. Furthermore, inhibition of Rac-dependent macropinocytosis blocks glucose-independent proliferation. We find that degradation of internalized protein produces amino acids, including alanine, which generates TCA cycle and glycolytic intermediates in the absence of glucose. In this process, the conversion of alanine to pyruvate by alanine transaminase 2 (ALT2) is critical for survival during glucose starvation. Collectively, Rac driven macropinocytosis of extracellular protein is an adaptive metabolic pathway used by a subset of lung cancers to survive states of glucose deprivation, and may serve as a potential drug target for cancer therapy.