ABSTRACT

We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (i) robust targeting of peptide N-termini and lysyl side chains, (ii) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (iii) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (iv) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally we provide exemplar data that extends the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.

Factor quinolinone inhibitors (FQIs), a first-in-class set of small molecule inhibitors targeted to the transcription factor LSF (TFCP2), exhibit promising cancer chemotherapeutic properties. FQI1, the initial lead compound identified, unexpectedly induced a concentration-dependent delay in mitotic progression. Here, we show that FQI1 can rapidly and reversibly lead to mitotic arrest, even when added directly to mitotic cells, implying that FQI1-mediated mitotic defects are not transcriptionally based. Furthermore, treatment with FQIs resulted in a striking, concentration-dependent diminishment of spindle microtubules, accompanied by a concentration-dependent increase in multi-aster formation. Aberrant γ-tubulin localization was also observed. These phenotypes suggest that perturbation of spindle microtubules is the primary event leading to the mitotic delays upon FQI1 treatment. Previously, FQIs were shown to specifically inhibit not only LSF DNA-binding activity, which requires LSF oligomerization to tetramers, but also other specific LSF-protein interactions. Other transcription factors participate in mitosis through non-transcriptional means, and we recently reported that LSF directly binds α-tubulin and is present in purified cellular tubulin preparations. Consistent with a microtubule role for LSF, here we show that LSF enhanced the rate of tubulin polymerization in vitro, and FQI1 inhibited such polymerization. To probe whether the FQI1-mediated spindle abnormalities could result from inhibition of mitotic LSF-protein interactions, mass spectrometry was performed using as bait an inducible, tagged form of LSF that is biotinylated by endogenous enzymes. The global proteomics analysis yielded expected associations for a transcription factor, notably with RNA processing machinery, but also to nontranscriptional components. In particular, and consistent with spindle disruption due to FQI treatment, mitotic, FQI1-sensitive interactions were identified between the biotinylated LSF and microtubule-associated proteins that regulate spindle assembly, positioning, and dynamics, as well as centrosome-associated proteins. Probing the mitotic LSF interactome using small molecule inhibitors therefore supported a non-transcriptional role for LSF in mediating progression through mitosis.

Introduction: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second leading cause of cancer mortality. The transcription factor Late SV40 Factor (LSF) functions as an oncogene in HCC, making it a potential protein target for HCC therapy. LSF overexpression correlates with pathogenesis of liver, colorectal and pancreatic cancers, for which there are limited molecularly targeted therapy options. A library of dihydroquinolinones, termed Factor Quinolinone Inhibitors (FQIs), inhibits LSF-DNA binding and specific LSF-protein interactions in in vitro and in cellular assays. The initial lead compound FQI1 causes dramatic mitotic defects in HCC cell lines but has no toxic consequences on immortalized human hepatocytes or primary mouse hepatocytes. Additionally, FQI1 has proven efficacious in endogenous HCC mouse models, with no evidence of associated toxicity. Methods: A series of dihydroquinolinone compounds were synthesized and tested for potency in two HCC cell lines, Huh7 and SNU423, by a cell proliferation assay. The FQI analogs, FQI34, N-oxide FQI34 and FQI37, were separated by chiral chromatography to the corresponding R and S enantiomers. Direct target engagement of the three lead compounds, FQI1, FQI34 and FQI37, is shown with cellular thermal stability assays on Huh7 cells. Results: More than 20 compounds were synthesized and characterized. Among them, FQI37 showed the most potent activity (GI50 = 70 nM) against Huh7 HCC cells. Structure-activity-relationship studies suggest that the amide portion of quinolinone core is important for optimal activity. Growth inhibition assays revealed enantiomeric specificity; the (S)-enantiomers are more potent than the (R)-compounds and the racemate. The cellular thermal shift assay in Huh7 cells demonstrated the direct target binding of FQIs to LSF in cells at micromolar concentrations. Growth inhibition assays also identified colorectal cancer and pancreatic cancer lines to be sensitive to the dihydroquinolinones treatment. Conclusions: Aryl-dihydroquinolinones are promising small molecule chemotherapies for LSF-driven cancers such as HCC, colorectal cancer, and pancreatic cancer. Citation Format: Niranjana Pokharel, John Kavouris, Jessica Biagi, Ulla Hansen, Scott E. Schaus. Assessing the sensitivity of LSF inhibitors against liver cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4021.