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In this study, we designed and synthesized novel 6‐(thiophen‐2‐yl) indolizino[3,2‐c]quinolines (IQs) guided by density functional theory (DFT) calculations and explored the optical properties of the IQ derivatives together with fractional atomic orbital contribution (FAOC) analysis. In accordance with the DFT predictions, the designed compound, IQ‐C9, with a methoxycarbonyl (MC) group attached to the C9 position of the IQ scaffold, displayed improved fluorescence quantum yields (QY) in solution and in the solid state along with bathochromic shifts compared to previous analogues. TEM images demonstrated that IQ‐C9 forms well‐defined nanoparticle structures (dimensions: 20×50 nm, aspect ratio: 2.3) in aqueous solution. Confocal images of IQ‐C9‐treated MCF7 cells revealed colocalization of IQ‐C9 with lipid droplets, highly dynamic subunits within cells involved in various metabolic disorders and cancers. Our results demonstrated that the analyses based on the FAOC coefficients and molecular orbital (MO) energy levels can be used as a computational method to predict spectral shifts for fluorophores and to design novel fluorescent probes for tracking dynamic cellular processes. [ABSTRACT FROM AUTHOR]

Rational design and synthetic feasibility are critical factors for the photoaffinity labelling (PAL) approach, which can identify protein targets of bioactive small molecules under native cellular conditions. In this study, we developed photoaffinity labelling probes derived from 1,3,4‐oxadiazin‐5(6H)‐ones (OPALs) for LL‐2003, a previously reported potential anticancer agent against IGF‐1R and Src. Our photoaffinity labelling strategy enabled successful photo‐crosslinking of the probes (OPAL‐6 and OPAL‐8) with the target proteins in both mammalian cell lysates and live MCF7, A549, HepG2 and HeLa cells in situ. In vitro and in situ labelling demonstrated different patterns and expression levels of the proteome, and the strongest band for Src appeared in the A549 cell line. An in‐gel fluorescence scan combined with MS/MS analysis of the IGF‐1R overexpressed insect proteome labelled by OPAL‐6 and OPAL‐8 identified the binding location of the synthesized probes. [ABSTRACT FROM AUTHOR]

Keywords: target identification; photoaffinity labelling; fluorescence probes; proteomics; proteins The labelling strategy enabled successful photo-crosslinking with target proteins in both insect cell lysates and mammalian live cells, demonstrating the efficient and selective labelling of Src. Target identification, photoaffinity labelling, fluorescence probes, proteomics, proteins. [Extracted from the article]

Rationale: The type I insulin-like growth factor receptor (IGF-1R) signaling pathway plays key roles in the development and progression of numerous types of human cancers, and Src and AXL have been found to confer resistance to anti-IGF-1R therapies. Hence, co-targeting Src and AXL may be an effective strategy to overcome resistance to anti-IGF-1R therapies. However, pharmacologic targeting of these three kinases may result in enhanced toxicity. Therefore, the development of novel multitarget anticancer drugs that block IGF-1R, Src, and AXL is urgently needed. Methods: We synthesized a series of phenylpyrazolo[3,4- d ]pyrimidine (PP)-based compounds, wherein the PP module was conjugated with 2,4-bis-arylamino-1,3-pyrimidines (I2) via a copper(I)-catalyzed alkyne-azide cycloaddition reaction. To develop IGF-1R/Src/AXL-targeting small molecule kinase inhibitors, we selected LL6 as an active compound and evaluated its antitumor and antimetastatic effects in vitro and in vivo using the MTT assay, colony formation assays, migration assay, flow cytometric analysis, a tumor xenograft model, the Kras G12D/+ -driven spontaneous lung tumorigenesis model, and a spontaneous metastasis model using Lewis lung carcinoma (LLC) allografts. We also determined the toxicity of LL6 in vitro and in vivo . Results: LL6 induced apoptosis and suppressed viability and colony-forming capacities of various non-small cell lung cancer (NSCLC) cell lines and their sublines with drug resistance. LL6 also suppressed the migration of NSCLC cells at nontoxic doses. Administration of LL6 in mice significantly suppressed the growth of NSCLC xenograft tumors and metastasis of LLC allograft tumors with outstanding toxicity profiles. Furthermore, the multiplicity, volume, and load of lung tumors in Kras G12D/+ transgenic mice were substantially reduced by the LL6 treatment. Conclusions: Our results show the potential of LL6 as a novel IGF-1R/Src/AXL-targeting small molecule kinase inhibitor, providing a new avenue for anticancer therapies., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Ellman's reagent has caused substantial confusion and concern as a probe for thiol-mediated uptake because it is the only established inhibitor available but works neither efficiently nor reliably. Here we use fluorescent cyclic oligochalcogenides that enter cells by thiol-mediated uptake to systematically screen for more potent inhibitors, including epidithiodiketopiperazines, benzopolysulfanes, disulfide-bridged γ-turned peptides, heteroaromatic sulfones and cyclic thiosulfonates, thiosulfinates and disulfides. With nanomolar activity, the best inhibitors identified are more than 5000 times better than Ellman's reagent. Different activities found with different reporters reveal thiol-mediated uptake as a complex multitarget process. Preliminary results on the inhibition of the cellular uptake of pseudo-lentivectors expressing SARS-CoV-2 spike protein do not exclude potential of efficient inhibitors of thiol-mediated uptake for the development of new antivirals., Competing Interests: A U.S. Patent application has been filed (No. 63/073863)., (This journal is © The Royal Society of Chemistry.)