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Chronic kidney disease (CKD) is a global public health problem, involving about 10% of the global population. Unfortunately, there are currently no effective drugs. Kidney fibrosis is the main pathology of CKD, where integrins play crucial roles in renal fibrogenesis. Recently, Bexotegrast (PLN-74809) as a dual integrin αvβ1/αvβ6 inhibitor could reduce the degree of lung fibrosis in patients with idiopathic pulmonary fibrosis. However, the role of PLN-74809 remains unclear in fibrotic kidney disease. Here, we have revealed that PLN-74809 administration dose-dependently delayed the progression of renal fibrosis in both adenine diet- and unilateral ureteral obstruction (UUO)-induced mice. Mechanistically, PLN-74809 targeted integrin αvβ1/αvβ6 to inhibit FAK/Src/Akt/β-catenin cascade in fibrotic kidneys. In summary, our results for the first time highlighted the αvβ1/αvβ6 inhibitor PLN-74809 exerted potential therapeutic against kidney fibrosis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Background: Resistance to chemotherapy, combined with heterogeneity among resistant tumors, represents a significant challenge in the clinical management of triple negative breast cancer (TNBC). By dissecting molecular pathways associated with treatment resistance, we sought to define patient sub-groups and actionable targets for next-line treatment., Methods: Bulk RNA sequencing and reverse phase protein array profiling were performed on isogenic patient-derived xenografts (PDX) representing paclitaxel-sensitive and -resistant tumors. Pathways identified as upregulated in the resistant model were further explored as targets in PDX explants. Their clinical relevance was assessed in two distinct patient cohorts (NeoAva and MET500)., Results: Increased activity in signaling pathways involving SRC-family kinases (SFKs)- and MAPK/ERK was found in treatment resistant PDX, with targeted inhibitors being significantly more potent in resistant tumors. Up-regulation of SFKs- and MAPK/ERK-pathways was also detected in a sub-group of chemoresistant patients after neoadjuvant treatment. Furthermore, High SFK expression (of either SRC, FYN and/or YES1) was detected in metastatic lesions of TNBC patients with fast progressing disease (median disease-free interval 27 vs 105 months)., Conclusions: Upregulation of SFK-signaling is found in a subset of chemoresistant tumors and is persistent in metastatic lesions. Based on pre-clinical results, these patients may respond favorably to treatment targeting SFKs., Competing Interests: Competing interests Dr O Engebraaten receives institutional research support from AstraZeneca. Dr A Kristian is currently an employee at Novartis. All other authors have no conflict of interest to report. Ethics approval Ethical approvals for the studies involving animals (FOTSid 10296, 12097/28802 and 15499) were given by the Norwegian Food Safety Authority. All experiments involving animals were carried out in accordance with institutional guidelines, national legislation, and the European Union Directive 2010/63/EU., (© 2024. The Author(s).)

Fibroblast growth factor (FGF) is involved in the progression of glioma, a most common type of brain tumor, and breast tumors. In this study, we aim to evaluate the effects of the inhibitor PP2 on cell proliferation and migration in glioma and breast tumor cells, and to characterize the molecular mechanisms involved in these processes. The inhibitory effect of PP2 on the tumorigenic potential of C6 glioma and MDA-MB-231 cells was examined by proliferation, migration, and invasion assays, and apoptotic analysis. The molecular mechanism behind the anti-glioma activity of PP2 was investigated by immunoblotting, immunoprecipitation, phosphoprotein assay, cellular thermal shift assay (CETSA), and molecular docking modeling. PP2 suppressed the proliferation and migration of C6 glioma and MDA-MB-231 cells via FGF2. Moreover, PP2 directly blocked the enzyme activity of FGF receptor 1 (FGFR1) and Src, subsequently affecting the nuclear factor-κB and activator protein-1 signaling pathways. CETSA analysis and the docking model indicated that the TK1 domains (Val 492 ad Glu 486) of FGFR2 could be binding sites of PP2. Collectively, therefore, our findings suggest that PP2 mediates antitumor effects by targeting both FGFR1 and Src and may have applications as a therapeutic inhibitor for the treatment of glioma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chronic allograft dysfunction (CAD) poses a significant challenge in kidney transplantation, with renal vascular endothelial-to-mesenchymal transition (EndMT) playing a vital role. While renal vascular EndMT has been verified as an important contributing factor to renal allograft interstitial fibrosis/tubular atrophy in CAD patients, its underlying mechanisms remain obscure. Currently, Src activation is closely linked to organ fibrosis development. Single-cell transcriptomic analysis in clinical patients revealed that Src is a potential pivotal mediator in CAD progression. Our findings revealed a significant upregulation of Src which closely associated with EndMT in CAD patients, allogeneic kidney transplanted rats and endothelial cells lines. In vivo, Src inhibition remarkably alleviate EndMT and renal allograft interstitial fibrosis in allogeneic kidney transplanted rats. It also had a similar antifibrotic effect in two endothelial cell lines. Mechanistically, the knockout of Src resulted in an augmented AMBRA1-mediated mitophagy in endothelial cells. We demonstrate that Src knockdown upregulates AMBRA1 level and activates mitophagy by stabilizing Parkin's ubiquitination levels and mitochondrial translocation. Subsequent experiments demonstrated that the knockdown of the Parkin gene inhibited mitophagy in endothelial cells, leading to increased production of Interleukin-6, thereby inducing EndMT. Consequently, our study underscores Src as a critical mediator of renal vascular EndMT and allograft interstitial fibrosis, exerting its impact through the regulation of AMBRA1/Parkin-mediated mitophagy., (© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

The differentiation of placental cytotrophoblasts (CTBs) into the syncytiotrophoblast (STB) layer results in a significant remodeling of the plasma membrane proteome. Here, we use a peroxidase-catalyzed proximity labeling strategy to map the dynamic plasma membrane proteomes of CTBs and STBs. Coupled with mass-spectrometry-based proteomics, we identify hundreds of plasma membrane proteins and observe relative changes in protein abundance throughout differentiation, including the upregulation of the plasma-membrane-localized nonreceptor tyrosine kinase LYN. We show that both siRNA-mediated knockdown and small molecule inhibition of LYN kinase function impairs CTB fusion and reduces the expression of syncytialization markers, presenting a function for LYN outside of its canonical role in immunological signaling. Our results demonstrate the use of the proximity labeling platform to discover functional regulators within the plasma membrane and provide new avenues to regulate trophoblast differentiation.

Background: Src family kinases (SFKs) contribute to migraine pathogenesis, yet its role in regulating photophobia behaviour, one of the most common forms of migraine, remains unknown. Here, we addressed whether SFKs antagonism alleviates photophobia behavior and explored the underlying mechanism involving hypothalamus and trigeminal ganglion activity, as measured by the alteration of neuropeptide levels and transcriptome respectively., Methods: A rapid-onset and injury-free mouse model of photophobia was developed following intranasal injection of the TRPA1 activator, umbellulone. The role of SFKs antagonism on light aversion was assessed by the total time the mouse stays in the light and transition times between the dark and light compartments. To gain insight to the preventive mechanism of SFKs antagonism, hypothalamic neuropeptides levels were assessed using enzyme linked immunofluorescent assay and trigeminal ganglion activity were assessed using RNA-sequencing and qPCR analysis., Results: SFKs antagonism by a clinically relevant SFKs inhibitor saracatinib reduced the total time in light and transition times in male mice, but not in females, suggesting SFKs play a crucial role in photophobia progressing and exhibit a male-only effect. SFKs antagonism had no effect on hypothalamic calcitonin gene-related peptide and pituitary adenylate cyclase-activating polypeptide levels of all mice investigated, suggesting the gender-different effect of saracatinib on light aversion appears to be independent of these hypothalamic neuropeptide levels. In trigeminal ganglion of male mice, photophobia is associated with profound alteration of differentially expressed genes, part of which were reversed by SFKs antagonism. Subsequent qPCR analysis showed SFKs antagonism displayed gender-different modulation of expression in some candidate genes, particularly noteworthy those encoding ion channels (trpm3, Scn8a), ATPase signaling (crebbp, Atp5α1) and kinase receptors (Zmynd8, Akt1)., Conclusions: In conclusion, our data revealed that SFKs antagonism reduced photophobia processing in male mice and exhibited gender-different modulation of trigeminal ganglion activity, primarily manifesting as alterations in the transcriptome profile. These findings underscore the potential of SFKs antagonism for allieving photophobia in males, highlighting its value in the emerging field of precision medicine., (© 2024. The Author(s).)

Inflammatory response mediated by M1 microglia is a crucial factor leading to the exacerbation of brain injury after ischemic stroke (IS). Under the stimulation of IS, vascular smooth muscle cells (VSMCs) switch to the synthetic phenotype characterized by exosome secretion. Previous studies have shown that exosomes play an important role in the regulation of microglial polarization. We reported that exosomes derived from primary human brain VSMCs under hypoxia (HExos), but not those under normoxia (Exos), significantly promoted primary human microglia (HM1900) shift to M1 phenotype. Proteomic analysis showed that the Src protein enriched in HExos was a potential pro-inflammatory mediator. In vitro experiments showed that the expression of Src and M1 markers were upregulated in HM1900 co-incubated with HExos. However, the Src inhibitor dasatinib (DAS) significantly promoted the transformation of HM1900 phenotype from M1 to M2. In vivo experiments of pMCAO mice also revealed that DAS could effectively inhibit the activation of M1 microglia/macrophages, protect neurons from apoptosis, and improve neuronal function. These data suggested that hypoxic-VSMCs-derived exosomes were involved in post-IS inflammation by promoting M1 microglial polarization through Src transmission. Targeting inhibition of Src potentially acts as an effective strategy for treating brain injury after IS., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

c-Src is involved in multiple signaling pathways and serves as a critical target in various cancers. Growing evidence suggests that prolonging a drug's residence time (RT) can enhance its efficacy and selectivity. Thus, the development of c-Src antagonists with longer residence time could potentially improve therapeutic outcomes. In this study, we employed molecular dynamics simulations to explore the binding modes and dissociation processes of c-Src with antagonists characterized by either long or short RTs. Our results reveal that the long RT compound DAS-DFGO-I (DFGO) occupies an allosteric site, forming hydrogen bonds with residues E310 and D404 and engaging in hydrophobic interactions with residues such as L322 and V377. These interactions significantly contribute to the long RT of DFGO. However, the hydrogen bonds between the amide group of DFGO and residues E310 and D404 are unstable. Substituting the amide group with a sulfonamide yielded a new compound, DFOGS, which exhibited more stable hydrogen bonds with E310 and D404, thereby increasing its binding stability with c-Src. These results provide theoretical guidance for the rational design of long residence time c-Src inhibitors to improve selectivity and efficacy.

Interleukin-17 A (IL-17 A) contributes to inflammation and causes secondary injury in post-stroke patients. However, little is known regarding the mechanisms that IL-17 A is implicated in the processes of neuronal death during ischemia. In this study, the mouse models of middle cerebral artery occlusion/reperfusion (MCAO/R)-induced ischemic stroke and oxygen-glucose deprivation/reoxygenation (OGD/R)-simulated in vitro ischemia in neurons were employed to explore the role of IL-17 A in promoting neuronal apoptosis. Mechanistically, endoplasmic reticulum stress (ERS)-induced neuronal apoptosis was accelerated by IL-17 A activation through the caspase-12-dependent pathway. Blocking calpain or phospholipase Cγ (PLCγ) inhibited IL-17 A-mediated neuronal apoptosis under ERS by inhibiting caspase-12 cleavage. Src and IL-17 A are linked, and PLCγ directly binds to activated Src. This binding causes intracellular Ca 2+ flux and activates the calpain-caspase-12 cascade in neurons. The neurological scores showed that intracerebroventricular (ICV) injection of an IL-17 A neutralizing mAb decreased the severity of I/R-induced brain injury and suppressed apoptosis in MCAO mice. Our findings reveal that IL-17 A increases caspase-12-mediated neuronal apoptosis, and IL-17 A suppression may have therapeutic potential for ischemic stroke., Competing Interests: Declaration of competing interest The authors declare that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

NXP900 is a selective and potent SRC family kinase (SFK) inhibitor, currently being dosed in a phase 1 clinical trial, that locks SRC in the "closed" conformation, thereby inhibiting both kinase-dependent catalytic activity and kinase-independent functions. In contrast, several multi-targeted kinase inhibitors that inhibit SRC, including dasatinib and bosutinib, bind their target in the active "open" conformation, allowing SRC and other SFKs to act as a scaffold to promote tumorigenesis through non-catalytic functions. NXP900 exhibits a unique target selectivity profile with sub-nanomolar activity against SFK members over other kinases. This results in highly potent and specific SFK pathway inhibition. Here, we demonstrate that esophageal squamous cell carcinomas and head and neck squamous cell carcinomas are exquisitely sensitive to NXP900 treatment in cell culture and in vivo, and we identify a patient population that could benefit from treatment with NXP900., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: AUB and NOC have received research grant funding from Nuvectis Pharma; in addition, AUB and NOC had patents to EP3298015B1, JP6684831B2, US10294227B2, CN107849050B, and CA3021550A1 licensed to Nuvectis Pharma., (Published by Elsevier Inc.)

Ethnopharmacological Relevance: Sepsis-induced acute lung injury (ALI) presents with significant morbidity and mortality in clinical settings. Tanreqing Injection (TRQI) has been clinically recommended for the treatment of ALI; however, the specific active chemical constituents remain unidentified., Aim of the Study: This study aimed to elucidate the potential pharmacologically active components and the underlying mechanisms of TRQI in the treatment of sepsis-induced ALI., Materials and Methods: High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) techniques were employed to identify the effective chemical constituents of TRQI. Additionally, an in vitro study was conducted using Raw264.7 macrophage cells stimulated with lipopolysaccharide (LPS) to evaluate the inhibitory effects of TRQI. An acute lung injury model produced by LPS was intraperitoneal injection in mice to assess the ALI-inhibitory effect of TRQI. The lung's pathological characteristics were examined using hematoxylin and eosin staining. Enzyme-linked immunosorbent assay (ELISA) and QPCR were performed to confirm the pharmaceutical effect. Network pharmacology was employed for mechanistic exploration, incorporating GO, and PPI analyses of targets. Src inhibitor and JNK agonist used to investigate the dependence of associated signaling pathways., Results: Combining pharmacokinetic characteristics, lung first-pass effect and anti-inflammatory effects, the main components of TRQI for treating sepsis induced ALI were narrowed down to seven compounds: chlorogenic acid, scutellarin, wogonoside, oroxyloside, oroxylin A and baicalein. Network pharmacology indicated that Src/JNK signaling pathway, may be the main regulatory pathway for treatment of actue lung injury. Next by using Src inhibitor, Src inhibition partly diminished the protective effects of TRQI in LPS-injected mice. Pretreatment with JNK agonist anisomycin abolished the protective effects of lung injury in vivo., Conclusions: TRQI is injected, the seven compounds could be presented in vivo, which can improve ALI by inhibiting Src-JNK signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Small cell lung cancer (SCLC) is a recalcitrant malignancy with dismal prognosis due to rapid relapse after an initial treatment response. More effective treatments for SCLC are desperately needed. Our previous studies showed that cell migration-inducing hyaluronan binding protein (CEMIP) functionally promotes SCLC cell proliferation and metastasis. In this study, we investigated whether and how CEMIP regulates the chemosensitivity of SCLC. Through the GDSC database, we found that CEMIP expression levels were positively correlated with the IC 50 values of several commonly used chemotherapeutic drugs in SCLC cells (cisplatin, gemcitabine, 5-fluorouracil and cyclophosphamide). We demonstrated that overexpression or knockdown of CEMIP in SCLC cells resulted in a notable increase or reduction in the IC 50 value of cisplatin or etoposide, respectively. We further revealed that CEMIP functions as an adaptor protein in SCLC cells to interact with SRC and YAP through the 1-177 aa domain and 820-1361 aa domain, respectively, allowing the autophosphorylation of Y416 and activation of SRC, thus facilitating the interaction between YAP and activated SRC, and resulting in increased phosphorylation of Y357, protein stability, nuclear accumulation and transcriptional activation of YAP. Overexpressing SRC or YAP counteracted the CEMIP knockdown-mediated increase in the sensitivity of SCLC cells to cisplatin and etoposide. The combination of the SRC inhibitor dasatinib or the YAP inhibitor verteporfin and cisplatin/etoposide (EP regimen) displayed excellent synergistic antitumor effects on SCLC both in vitro and in vivo. This study demonstrated that targeted therapy against the CEMIP/SRC/YAP complex is a potential strategy for SCLC and provides a rationale for the development of future clinical trials with translational prospects., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Background: Ischaemic stroke triggers neuronal mitophagy, while the involvement of mitophagy receptors in ischaemia/reperfusion (I/R) injury-induced neuronal mitophagy remain not fully elucidated. Here, we aimed to investigate the involvement of mitophagy receptor FUN14 domain-containing 1 (FUNDC1) and its modulation in neuronal mitophagy induced by I/R injury., Methods: Wild-type and FUNDC1 knockout mice were generated to establish models of neuronal I/R injury, including transient middle cerebral artery occlusion (tMCAO) in vivo and oxygen glucose deprivation/reperfusion in vitro. Stroke outcomes of mice with two genotypes were assessed. Neuronal mitophagy was analysed both in vivo and in vitro. Activities of FUNDC1 and its regulator Src were evaluated. The impact of Src on FUNDC1-mediated mitophagy was assessed through administration of Src antagonist PP1., Results: To our surprise, FUNDC1 knockout mice subjected to tMCAO showed stroke outcomes comparable to those of their wild-type littermates. Although neuronal mitophagy could be activated by I/R injury, FUNDC1 deletion did not disrupt neuronal mitophagy. Transient activation of FUNDC1, represented by dephosphorylation of Tyr18, was detected in the early stages (within 3 hours) of neuronal I/R injury; however, phosphorylated Tyr18 reappeared and even surpassed baseline levels in later stages (after 6 hours), accompanied by a decrease in FUNDC1-light chain 3 interactions. Spontaneous inactivation of FUNDC1 was associated with Src activation, represented by phosphorylation of Tyr416, which changed in parallel with the level of phosphorylated FUNDC1 (Tyr18) during neuronal I/R injury. Finally, FUNDC1-mediated mitophagy in neurons under I/R conditions can be rescued by pharmacological inhibition of Src., Conclusions: FUNDC1 is inactivated by Src during the later stage (after 6 hours) of neuronal I/R injury, and rescue of FUNDC1-mediated mitophagy may serve as a potential therapeutic strategy for treating ischaemic stroke., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Tirbanibulin 1% ointment is a synthetic antiproliferative agent approved in 2021 by the European Union for treating actinic keratoses (AK). Topical tirbanibulin has clinically resolved HPV-57 ( +) squamous cell carcinoma (SCC), HPV-16 ( +) vulvar high-grade squamous intraepithelial lesion, epidermodysplasia verruciformis, and condyloma. We examined how tirbanibulin might affect HPV oncoprotein expression and affect other cellular pathways involved in cell proliferation and transformation. We treated the HeLa cell line, containing integrated HPV-18, with increasing doses of tirbanibulin to determine the effects on cell proliferation. Immunoblotting was performed with antibodies against the Src canonical pathway, HPV 18 E6 and E7 transcription regulation, apoptosis, and invasion and metastasis pathways. Cell proliferation assays with tirbanibulin determined the half-maximal inhibitory concentration (IC 50 ) of HeLa cells to be 31.49 nmol/L. Increasing concentrations of tirbanibulin downregulates the protein expression of Src (p < 0.001), phospho-Src (p < 0.001), Ras (p < 0.01), c-Raf (p < 0.001), ERK1 (p < 0.001), phospho-ERK1 (p < 0.001), phospho-ERK2 (p < 0.01), phospho-Mnk1 (p < 0.001), eIF4E (p < 0.01), phospho-eIF4E (p < 0.001), E6 (p < 0.01), E7 (p < 0.01), Rb (p < 0.01), phospho-Rb (p < 0.001), MDM2 (p < 0.01), E2F1 (p < 0.001), phospho-FAK (p < 0.001), phospho-p130 Cas (p < 0.001), Mcl-1 (p < 0.01), and Bcl-2 (p < 0.001), but upregulates cPARP (p < 0.001), and cPARP/fPARP (p < 0.001). These results demonstrate that tirbanibulin may impact expression of HPV oncoproteins via the Src- MEK- pathway. Tirbanibulin significantly downregulates oncogenic proteins related to cell cycle regulation and cell proliferation while upregulating apoptosis pathways., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Metastasis is the leading cause of cancer-related deaths, making the development of novel, more effective therapies imperative to alleviate patient suffering. Metabolic switching is a hallmark of cancer cells that facilitates metastasis. Cancer cells obtain most of their energy and intermediate metabolites, which are required to proliferate and metastasize, through aerobic glycolysis. Previous work from our laboratory has shown that Caveolin-1 (CAV1) expression in cancer cells promotes glycolysis and metastasis. Here, we sought to determine if limiting glycolysis reduced CAV1-enhanced metastasis and to identify the mechanism(s) involved. We evaluated the effects of the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) in metastatic melanoma and breast cancer cell lines expressing or not CAV1. Non-cytotoxic concentrations of 2-DG (1 mM) inhibited the migration of B16-F10 melanoma and MDA-MB-231 breast cancer cells. CAV1-mediated activation of Src/Akt signaling was required for CAV1-enhanced migration and was blocked in the presence of 2-DG. Moreover, inhibition of Akt reduced CAV1-enhanced lung metastasis of B16-F10 cells. Collectively, these findings highlight the importance of CAV1-induced metabolic reprogramming for metastasis and point towards possible therapeutic approaches to prevent metastatic disease by inhibiting glycolysis and Src/Akt signaling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

One of the effective therapeutic strategies to treat rheumatoid arthritis (RA)-related bone resorption is to target excessive activation of osteoclasts. We discovered that 6-O-angeloylplenolin (6-OAP), a pseudoguaianolide from Euphorbia thymifolia Linn widely used for the treatment of RA in traditional Chinese medicine, could inhibit RANKL-induced osteoclastogenesis and bone resorption in both RAW264.7 cells and BMMs from 1 μM and protect a collagen-induced arthritis (CIA) mouse model from bone destruction in vivo. The severity of arthritis and bone erosion observed in paw joints and the femurs of the CIA model were attenuated by 6-OAP administered at both dosages (1 or 5 mg/kg, i.g.). BMD, Tb.N and BV/TV were also improved by 6-OAP treatment. Histological analysis and TRAP staining of femurs further confirmed the protective effects of 6-OAP on bone erosion, which is mainly due to reduced osteoclasts. Molecular docking indicated that c-Src might be a target of 6-OAP and phosphorylation of c-Src was suppressed by 6-OAP treatment. CETSA and SPR assay further confirmed the potential interaction between 6-OAP and c-Src. Three signaling molecules downstream of c-Src that are vital to the differentiation and function of osteoclasts, NF-κB, c-Fos and NFATc1, were also suppressed by 6-OAP in vitro. In summary, the results demonstrated that the function of c-Src was disrupted by 6-OAP, which led to the suppression of downstream signaling vital to osteoclast differentiation and function. In conclusion, 6-OAP has the potential to be further developed for the treatment of RA-related bone erosion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Platelet hyperreactivity contributes to the pathogenesis of COVID-19, which is associated with a hypercoagulability state and thrombosis disorder. It has been demonstrated that Vitamin D deficiency is associated with the severity of COVID-19 infection. Vitamin D supplement is widely used as a dietary supplement due to its safety and health benefits. In this study, we investigated the direct effects and underlying mechanisms of 1,25(OH) 2 D 3 on platelet hyperreactivity induced by SRAS-CoV-2 spike protein via Western blot and platelet functional studies in vitro. Firstly, we found that 1,25(OH) 2 D 3 attenuated platelet aggregation and Src-mediated signaling. We further observed that 1,25(OH) 2 D 3 attenuated spike protein-potentiated platelet aggregation in vitro. Mechanistically, 1,25(OH) 2 D 3 attenuated spike protein upregulated-integrin αIIbβ3 outside-in signaling such as platelet spreading and the phosphorylation of β3, c-Src and Syk. Moreover, using PP2, the Src family kinase inhibitor to abolish spike protein-stimulated platelet aggregation and integrin αIIbβ3 outside-in signaling, the combination of PP2 and 1,25(OH) 2 D 3 did not show additive inhibitory effects on spike protein-potentiated platelet aggregation and the phosphorylation of β3, c-Src and Syk. Thus, our data suggest that 1,25(OH) 2 D 3 attenuates platelet aggregation potentiated by spike protein via downregulating integrin αIIbβ3 outside-in signaling., (© 2024 John Wiley & Sons Ltd.)

Heat shock protein 90 (Hsp90) is a tumor marker that accelerates cancer growth by disrupting protein homeostasis. However, concerns such as low clinical efficacy and drug resistance continue to be obstacles to the successful marketing of Hsp90 inhibitors. The cytoprotective function of autophagy has been identified as one of the mechanisms by which tumor cells gain resistance to chemotherapy. JD-02 was identified as a new Hsp90 inhibitor that suppressed colorectal cancer (CRC) growth by lowering client protein levels in vivo and in vitro. We found that JD-02 increased cellular autophagy, which inhibited apoptosis. JD-02 enhanced cytoprotective autophagy and regulated apoptotic suppression by increasing intracellular reactive oxygen species and inhibiting SRC protein levels, as demonstrated by quantitative proteomics, bioinformatic analysis, western blotting, and flow cytometry. This effect was reversed by autophagy inhibition. Therefore, due to the synergistic effects of Hsp90 and autophagy inhibitors in efficiently activating apoptotic pathways, they could potentially serve as promising therapeutic options for CRC., (© 2024 Wiley Periodicals LLC.)

A new series of benzene-sulfonamide derivatives 3a-i was designed and synthesized via the reaction of N-(pyrimidin-2-yl)cyanamides 1a-i with sulfamethazine sodium salt 2 as dual Src/Abl inhibitors. Spectral data IR, 1 H-, 13 C- NMR and elemental analyses were used to confirm the structures of all the newly synthesized compounds 3a-i and 4a-i. Crucially, we screened all the synthesized compounds 3a-i against NCI 60 cancer cell lines. Among all, compound 3b was the most potent, with IC 50 of 0.018 μM for normoxia, and 0.001 μM for hypoxia, compared to staurosporine against HL-60 leukemia cell line. To verify the selectivity of this derivative, it was assessed against a panel of tyrosine kinase EGFR, VEGFR-2, B-raf, ERK, CK1, p38-MAPK, Src and Abl enzymes. Results revealed that compound 3b can effectively and selectively inhibit Src/Abl with IC 50 0.25 μM and Abl inhibitory activity with IC 50 0.08 μM, respectively, and was found to be more potent on these enzymes than other kinases that showed the following results: EGFR IC 50 0.31 μM, VEGFR-2 IC 50 0.68 μM, B-raf IC 50 0.33 μM, ERK IC 50 1.41 μM, CK1 IC 50 0.29 μM and p38-MAPK IC 50 0.38 μM. Moreover, cell cycle analysis and apoptosis performed to compound 3b against HL-60 suggesting its antiproliferative activity through Src/Abl inhibition. Finally, molecular docking studies and physicochemical properties prediction for compounds 3b, 3c, and 3 h were carried out to investigate their biological activities and clarify their bioavailability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase ( IDH1/IDH2 ) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

Precision oncology has revolutionized the treatment of ALK-positive lung cancer with targeted therapies. However, an unmet clinical need still to address is the treatment of refractory tumors that contain drug-induced resistant mutations in the driver oncogene or exhibit resistance through the activation of diverse mechanisms. In this study, we established mouse tumor-derived cell models representing the two most prevalent EML4-ALK variants in human lung adenocarcinomas and characterized their proteomic profiles to gain insights into the underlying resistance mechanisms. We showed that Eml4-Alk variant 3 confers a worse response to ALK inhibitors, suggesting its role in promoting resistance to targeted therapy. In addition, proteomic analysis of brigatinib-treated cells revealed the upregulation of SRC kinase, a protein frequently activated in cancer. Co-targeting of ALK and SRC showed remarkable inhibitory effects in both ALK-driven murine and ALK-patient-derived lung tumor cells. This combination induced cell death through a multifaceted mechanism characterized by profound perturbation of the (phospho)proteomic landscape and a synergistic suppressive effect on the mTOR pathway. Our study demonstrates that the simultaneous inhibition of ALK and SRC can potentially overcome resistance mechanisms and enhance clinical outcomes in ALK-positive lung cancer patients. ONE SENTENCE SUMMARY: Co-targeting ALK and SRC enhances ALK inhibitor response in lung cancer by affecting the proteomic profile, offering hope for overcoming resistance and improving clinical outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Saracatinib (AZD0530) is a dual Src/Abl inhibitor initially developed by AstraZeneca for cancer treatment; however, data from 2006 to 2024 reveal that this drug has been tested not only for cancer treatment, but also for the treatment of other diseases. Despite the promising pre-clinical results and the tolerability shown in phase I trials, where a maximum tolerated dose of 175 mg was defined, phase II clinical data demonstrated a low therapeutic action against several cancers and an elevated rate of adverse effects. Recently, pre-clinical research aimed at reducing the toxic effects and enhancing the therapeutic performance of saracatinib using nanoparticles and different pharmacological combinations has shown promising results. Concomitantly, saracatinib was repurposed to treat Alzheimer's disease, targeting Fyn. It showed great clinical results and required a lower daily dose than that defined for cancer treatment, 125 mg and 175 mg, respectively. In addition to Alzheimer's disease, this Src inhibitor has also been studied in relation to other health conditions such as pulmonary and liver fibrosis and even for analgesic and anti-allergic functions. Although saracatinib is still not approved by the Food and Drug Administration (FDA), the large number of alternative uses for saracatinib and the elevated number of pre-clinical and clinical trials performed suggest the huge potential of this drug for the treatment of different kinds of diseases.

Background: Recent studies showed that the cooperation between c-SRC and EGFR is responsible for more aggressive phenotype in diverse tumors, including glioblastomas and carcinomas of the colon, breast, and lung. Studies show that combination of SRC and EGFR inhibitors can induce apoptosis and delay the acquired resistance to chemotherapy. Therefore, such combination may lead to a new therapeutic strategy for the treatment of EGFR-mutant lung cancer. Osimertinib was developed as a third-generation EGFR-TKI to combat the toxicity of EGFR mutant inhibitors. Due to the resistance and adverse reaction of osimertinib and other kinase inhibitors, 12 novel compounds structurally similar to osimertinib were designed and synthesized., Methods: Compounds were synthesized by developing novel original synthesis methods and receptor interactions were evaluated through a molecular docking study. To evaluate their inhibitory activities against EGFR and SRC kinase, in vitro enzyme assays were used. Anticancer potencies were determined using lung, breast, prostate (A549, MCF6, PC3) cancer cell lines. Compounds were also tested against normal (HEK293) cell line to evaluate their cyctotoxic effects., Results: Although, none of compounds showed stronger inhibition compared to osimertinib in the EGFR enzyme inhibition studies, compound 16 showed the highest efficacy with an IC 50 of 1.026 μM. It also presented potent activity against SRC kinase with an IC 50 of 0.002 μM. Among the tested compounds, the urea containing derivatives 6-11 exhibited a strong inhibition profile (80.12-89.68%) against SRC kinase in comparison to the reference compound dasatinib (93.26%). Most of the compounds caused more than 50% of cell death in breast, lung and prostate cancer cell lines and weak toxicity for normal cells in comparison to reference compounds osimertinib, dasatinib and cisplatin. Compound 16 showed strong cytotoxicity on lung and prostate cancer cells. Treatment of prostate cancer cell lines with the most active compound, 16, significantly increased the caspase-3 (8-fold), caspase-8 (6-fold) and Bax (5.7-fold) levels and decreased the Bcl-2 level (2.3-fold) compared to the control group. These findings revealed that the compound 16 strongly induces apoptosis in the prostate cancer cell lines., Conclusion: Overall kinase inhibition, cytotoxicity and apoptosis assays presented that compound 16 has dual inhibitory activity against SRC and EGFR kinases while maintaining low toxicity against normal cells. Other compounds also showed considerable activity profiles in kinase and cell culture assays., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The accumulation of abnormal Tau protein is a common feature of various neurodegenerative diseases. Truncated Tau, resulting from cleavage by asparaginyl endopeptidase (AEP, δ-secretase), promotes its own phosphorylation and aggregation. Our study focused on understanding the regulatory mechanisms of AEP activation and its interaction with other proteins. We discovered that c-Src plays a critical role in mediating the activation and polyubiquitination of AEP in response to epidermal growth factor stimulation. In addition, we investigated the involvement of tumor necrosis factor receptor-associated factor 6 (Traf6), an E3 ligase, in the regulation of AEP levels and its interaction with c-Src. Knockdown of Traf6 effectively inhibited c-Src-induced AEP activation. To gain further insights into the molecular mechanisms, we employed mass spectrometry to identify the specific tyrosine residues of Traf6 that are phosphorylated by c-Src. By mutating these phosphorylation sites to phenylalanine, we disrupted Traf6-mediated polyubiquitination and subsequently observed the inactivation of AEP. This finding suggests that the phosphorylation of Traf6 by c-Src is crucial for AEP activation. Pharmacological inhibition of c-Src reduced the phosphorylation of Traf6 and inhibited AEP activation in neurons derived from human-induced pluripotent stem cells. Conditional knockout of Traf6 in neurons prevented c-Src-induced AEP activation and subsequent Tau truncation in vivo. Moreover, phosphorylation of Traf6 is highly correlated with AEP activation, Tau368 and pathological Tau (AT8) in Alzheimer's disease brain. Overall, our study elucidates the role of c-Src in regulating AEP-cleaved Tau through phosphorylating Traf6. Targeting the c-Src-Traf6 pathway may hold potential for the treatment of Alzheimer's disease and other tauopathies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Src family tyrosine kinases play a potential role in Bcr-Abl-induced leukemogenesis. Src kinase inhibitors are reported as selective inhibitors of chronic myeloid leukemia., Objective: Since Src kinase inhibitors have an inhibitive effect on chronic myeloid leukemia, indole derivatives (C-1, C-2, C-3) previously found as potent inhibitors of Src kinase were tested against chronic myeloid leukemia in this study., Methods: Cell viability of K562 and R/K562 cells, antiproliferative and antioxidant effects, and inhibition profiles of Bcr-Abl kinase of indole derivatives were determined compared to dasatinib and imatinib., Results: The results showed that compounds affected cell proliferation and decreased the levels of Bcr/Abl. These results confirmed that the antileukemic activity of compounds was related to Bcr/Abl expression. Docking studies also presented that compounds are inhibitors of both Src and Abl kinases. Calculation of drug-like properties showed that compounds could be potential drug candidates., Conclusion: Among indole-2-on derivatives, previously identified as Src kinase inhibitors, C-2 has been discovered to be a strong anticancer drug that is active against susceptible and resistant K562 cell lines and induces apoptosis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often "cryptic" without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein-ligand systems., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Background: The expression of Src is upregulated in the vasculature associated with cardiac hypertrophy events. Here, we aimed to explore the underlying mechanism of Src in angiotensin II (AngII)-mediated cardiac fibrosis and hypertrophy., Methods: The heart conditional Src knockout mouse model was established and administrated with AngII. The effects of Src on the AngII-mediated cardiac hypertrophy were assessed by Hematoxylin and Eosin (HE), Masson's trichrome, immunohistochemical staining, Annexin V-FITC/PI apoptosis detection assay and Western blot analysis., Results: The expression levels of galectin-3, Src and the hypertrophy marker brain natriuretic peptide (BNP), as well as the phosphorylation of Src were all elevated in heart tissues of mice with AngII-induced cardiac hypertrophy and fibrosis. Heart conditional Src knockout attenuated AngII-activated cardiac fibrosis and hypertrophy in mice. Consistently, AngII could promote the expression of Src in a dose-dependent manner and the knockout of Src impaired Ang II-mediated apoptosis and fibrosis in the cardiomyocytes. In addition, Src inhibition suppressed the expression of galectin-3 in vivo and in vitro. Specifically, AngII could upregulate the expression of galectin-3, and knockdown of galectin-3 (Gal-3) remarkably inhibited AngII-enhanced apoptosis and fibrosis in the cardiomyocytes. Furthermore, overexpression of galectin-3 reinforced Ang II-induced cell apoptosis and fibrosis that was attenuated by knockout of Src., Conclusions: Our findings indicate that Src and Gal-3 play an important role in AngII-mediated cardiac structural remodeling. Src and galectin-3 might serve as potential targets for the treatment of AngII-induced cardiac fibrosis and hypertrophy., (Copyright © 2022. Published by Elsevier Inc.)

Background: Over half of colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors, has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms that are not well understood. This study aims to investigate MEK inhibitor-associated resistance signaling and identify subpopulation(s) of CRC patients who may be sensitive to biomarker-driven drug combination(s)., Methods: We classified 2250 primary and metastatic human CRC tumors by consensus molecular subtypes (CMS). For each tumor, we generated multiple gene expression signature scores measuring MEK pathway activation, MEKi "bypass" resistance, SRC activation, dasatinib sensitivity, EMT, PC1, Hu-Lgr5-ISC, Hu-EphB2-ISC, Hu-Late TA, Hu-Proliferation, and WNT activity. We carried out correlation, survival and other bioinformatic analyses. Validation analyses were performed in two independent publicly available CRC tumor datasets (n = 585 and n = 677) and a CRC cell line dataset (n = 154)., Results: Here we report a central role of SRC in mediating "bypass"-resistance to MEK inhibition (MEKi), primarily in cancer stem cells (CSCs). Our integrated and comprehensive gene expression signature analyses in 2250 CRC tumors reveal that MEKi-resistance is strikingly-correlated with SRC activation (Spearman P < 10 -320 ), which is similarly associated with EMT (epithelial to mesenchymal transition), regional metastasis and disease recurrence with poor prognosis. Deeper analysis shows that both MEKi-resistance and SRC activation are preferentially associated with a mesenchymal CSC phenotype. This association is validated in additional independent CRC tumor and cell lines datasets. The CMS classification analysis demonstrates the strikingly-distinct associations of CMS1-4 subtypes with the MEKi-resistance and SRC activation. Importantly, MEKi + SRCi sensitivities are predicted to occur predominantly in the KRAS mutant, mesenchymal CSC-like CMS4 CRCs., Conclusions: Large human tumor gene expression datasets representing CRC heterogeneity can provide deep biological insights heretofore not possible with cell line models, suggesting novel repurposed drug combinations. We identified SRC as a common targetable node--an Achilles' heel--in MEKi-targeted therapy-associated resistance in mesenchymal stem-like CRCs, which may help development of a biomarker-driven drug combination (MEKi + SRCi) to treat problematic subpopulations of CRC., (© 2022. The Author(s).)

SRC is a nonreceptor tyrosine kinase with key roles in breast cancer development and progression. Despite this, SRC tyrosine kinase inhibitors have so far failed to live up to their promise in clinical trials, with poor overall response rates. We aimed to identify possible synergistic gene-drug interactions to discover new rational combination therapies for SRC inhibitors. An unbiased genome-wide CRISPR-Cas9 knockout screen in a model of triple-negative breast cancer revealed that loss of integrin-linked kinase (ILK) and its binding partners α-Parvin and PINCH-1 sensitizes cells to bosutinib, a clinically approved SRC/ABL kinase inhibitor. Sensitivity to bosutinib did not correlate with ABL dependency; instead, bosutinib likely induces these effects by acting as a SRC tyrosine kinase inhibitor. Furthermore, in vitro and in vivo models showed that loss of ILK enhanced sensitivity to eCF506, a novel and highly selective inhibitor of SRC with a unique mode of action. Whole-genome RNA sequencing following bosutinib treatment in ILK knockout cells identified broad changes in the expression of genes regulating cell adhesion and cell-extracellular matrix. Increased sensitivity to SRC inhibition in ILK knockout cells was associated with defective adhesion, resulting in reduced cell number as well as increased G1 arrest and apoptosis. These findings support the potential of ILK loss as an exploitable therapeutic vulnerability in breast cancer, enhancing the effectiveness of clinical SRC inhibitors., Significance: A CRISPR-Cas9 screen reveals that loss of integrin-linked kinase synergizes with SRC inhibition, providing a new opportunity for enhancing the clinical effectiveness of SRC inhibitors in breast cancer., (©2021 The Authors; Published by the American Association for Cancer Research.)

B-lymphoid tyrosine kinase (BLK), a member of the SRC family nonreceptor tyrosine kinase, is involved in the B-cell receptor (BCR) signaling pathway and B cell development and function. Dysregulation of BLK is associated with autoimmune diseases and cancer. However, there is an absence of good tool compounds for BLK, and the molecular mechanisms by which BLK mediates physiological and pathological processes are poorly understood. Herein, we present the discovery of a novel series of selective and irreversible inhibitors of BLK with nanomolar potency against BLK in biochemical and cellular assays. Compound 25 demonstrated potent antiproliferative activities against several B cell lymphoma cell lines. These compounds constitute the first series of selective inhibitors developed for BLK and could help expedite the exploration of BLK functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Hepatocellular carcinoma (HCC), a heterogeneous cancer with high mortality, is resistant to single targeted therapy; thus, combination therapy based on synthetic lethality is a promising therapeutic strategy for HCC. Poly (adenosine diphosphate [ADP]-ribose) polymerase 1 (PARP1) is the most recognized target for synthetic lethality; however, the therapeutic effect of PARP1 inhibition on HCC is disappointing. Therefore, exploring new synthetic lethal partners for the efficient manipulation of HCC is urgently required. In this study, we identified Src and PARP1 as novel synthetic lethal partners, and the combination therapy produced significant anti-tumor effects without causing obvious side effects. Mechanistically, Src interacted with PARP1 and phosphorylated PARP1 at the Y992 residue, which further mediated resistance to PARP1 inhibition. Overall, this study revealed that Src-mediated PARP1 phosphorylation induced HCC resistance to PARP1 inhibitors and indicated a therapeutic window of the Y992 phosphorylation of PARP1 for HCC patients. Moreover, synthetic lethal therapy by co-targeting PARP1 and Src have the potential to broaden the strategies for HCC and might benefit HCC patients with high Src activation and resistance to PARP1 inhibitors alone., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Lipid droplets (LDs) have increasingly been recognized as an essential organelle for eukaryotes. Although the biochemistry of lipid synthesis and degradation is well characterized, the regulation of LD dynamics, including its formation, maintenance, and secretion, is poorly understood. Here, we report that mice lacking Occludin (Ocln) show defective lipid metabolism. We show that LDs were larger than normal along its biogenesis and secretion pathway in Ocln null mammary cells. This defect in LD size control did not result from abnormal lipid synthesis or degradation; rather, it was because of secretion failure during the lactation stage. We found that OCLN was located on the LD membrane and was bound to essential regulators of lipid secretion, including BTN1a1 and XOR, in a C-terminus-dependent manner. Finally, OCLN was a phosphorylation target of Src kinase, whose loss causes lactation failure. Together, we demonstrate that Ocln is a downstream target of Src kinase and promotes LD secretion by binding to BTN1a1 and XOR., Competing Interests: The authors have declared that no competing interests exist.

Angiogenesis plays an important role in the growth and metastasis of solid tumors including melanoma. Inhibiting tumor-associated angiogenesis is a tactic in treating melanoma. Dioscin restrains angiogenesis in colon tumor and has anti-melanoma effects in cell and animal models. In a previous study, we found that dioscin inhibits Src/STAT3 signaling in melanoma cells. Activation of the Src/STAT3 pathway has been shown to promote tumor angiogenesis. This study aimed to determine whether dioscin's anti-melanoma effects is related to inhibiting Src/STAT3 signaling-mediated angiogenesis. In a B16F10 allograft mouse model, we found that dioscin inhibited melanoma growth and angiogenesis. To exclude the impact of tumor growth on angiogenesis, a chicken chorioallantoic membrane (CAM) model was used to verify the anti-angiogenic effect of dioscin. Results showed that dioscin suppressed vessel formation in CAM. To determine if tumor secreted pro-angiogenic cytokines are involved in the anti-angiogenic effect of dioscin, conditioned media from dioscin-treated A375 melanoma cells were used to culture human umbilical vein endothelial cells (HUVECs), and tube formation was monitored. It was observed that the tube formation of HUVECs was inhibited. Mechanistic studies revealed that dioscin inhibited the activation of Src and STAT3, and lowered mRNA and protein levels of STAT3 transcriptionally-regulated genes, in B16F10 melanomas. ELISA assays showed that dioscin decreased the secretion of MMP-2, MMP-9 and VEGF from A375 cells. Over-activation of STAT3 lessened the effects of dioscin in decreasing the secretion of pro-angiogenic cytokines from melanoma cells, and in inhibiting tube formation of HUVECs cultured with conditioned media from melanoma cell cultures. In summary, we for the first time demonstrated that inhibiting Src/STAT3 signaling-mediated angiogenesis is involved in the anti-melanoma effects of dioscin. This study provides further pharmacological groundwork for developing dioscin as an anti-melanoma agent., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Liver cancer is one of the leading causes of cancer-related death worldwide. Dihydrotanshinone I (DHI) was shown to inhibit the growth of several types of cancer. However, research related to hepatoma treatment using DHI is limited., Purpose: Here, we explored the inhibitory effect of DHI on the growth of hepatoma cells, and investigated the underlying molecular mechanisms., Methods: The proliferation of Hep3B, SMCC-7721 and SK-Hep1 hepatoma cells was evaluated using the MTS and Edu staining assay. Hepatoma cell death was analyzed with a LIVE/DEAD Cell Imaging Kit. The relative expression and phosphorylation of proto-oncogene tyrosine-protein kinase Src (Src) and signal transducer and activator of transcription-3 (STAT3) proteins in hepatoma cells, as well as the expression of other protein components, were measured by western blotting. The structural interaction of DHI with Src proteins was evaluated by molecular docking, molecular dynamics simulation, surface plasmon resonance imaging and Src kinase inhibition assay. Src overexpression was achieved by infection with an adenovirus vector encoding human Src. Subsequently, the effects of DHI on tumor growth inhibition were further validated using mouse xenograft models of hepatoma., Results: In vitro studies showed that treatment with DHI inhibited the proliferation and promoted cell death of Hep3B, SMCC-7721 and SK-Hep1 hepatoma cells. We further identified and verified Src as a direct target of DHI by using molecular stimulation, surface plasmon resonance image and Src kinase inhibition assay. Treatment with DHI reduced the in vitro phosphorylation levels of Src and STAT3, a transcription factor regulated by Src. In the xenograft mouse models, DHI dose-dependently suppressed tumor growth and Src and STAT3 phosphorylation. Moreover, Src overexpression partly abrogated the inhibitory effects of DHI on the proliferation and cell death in hepatoma cells., Conclusion: Our results suggest that DHI inhibits the growth of hepatoma cells by direct inhibition of Src., (Copyright © 2021. Published by Elsevier GmbH.)

Context: Among the plants in the genus Barringtonia (Lecythidaceae) used as traditional medicines to treat arthralgia, chest pain, and haemorrhoids in Indonesia, Barringtonia racemosa L. and Barringtonia acutangula (L.) Gaertn. have demonstrated anti-inflammatory activity in systemic inflammatory models., Objective: The anti-inflammatory activity of Barringtonia angusta Kurz has not been investigated. We prepared a methanol extract of the leaves and stems of B. angusta (Ba-ME) and systemically evaluated its anti-inflammatory effects in vitro and in vivo ., Materials and Methods: RAW264.7 cells stimulated with LPS or Pam3CSK4 for 24 h were treated with Ba-ME (12.5, 25, 50, 100, and 150 µg/mL), and NO production and mRNA levels of inflammatory genes were evaluated. Luciferase reporter gene assay, western blot analysis, overexpression experiments, and cellular thermal shift assay were conducted to explore the mechanism of Ba-ME. In addition, the anti-gastritis activity of Ba-ME (50 and 100 mg/kg, administered twice per day for two days) was evaluated using an HCl/EtOH-induced gastritis mouse model., Results: Ba-ME dose-dependently suppressed NO production [IC 50 = 123.33 µg/mL (LPS) and 46.89 µg/mL (Pam3CSK4)] without affecting cell viability. Transcriptional expression of iNOS , IL-1β , COX-2 , IL-6 , and TNF-α and phosphorylation of Src, IκBα, p50/105, and p65 were inhibited by Ba-ME. The extract specifically targeted the Src protein by binding to its SH2 domain. Moreover, Ba-ME significantly ameliorated inflammatory lesions in the HCl/EtOH-induced gastritis model., Discussion and Conclusions: The anti-inflammatory activity of Ba-ME is mediated by targeting of the Src/NF-κB signalling pathway, and B. angusta has potential as an anti-inflammatory drug.

Context: Sauropus brevipes Müll. Arg. (Phyllanthaceae) has been used as an effective ingredient in a decoction for the treatment of diarrhoea. However, there was no report on its modulatory role in inflammation., Objective: This study investigates anti-inflammatory effect of S. brevipes in various inflammation models., Materials and Methods: The aerial part of S. brevipes was extracted with 95% ethanol to produce Sb-EE. RAW264.7 cells pre-treated with Sb-EE were stimulated by lipopolysaccharide (LPS), and Griess assay and PCR were performed. High-performance liquid chromatography (HPLC) analysis, luciferase assay, Western blotting and kinase assay were employed. C57BL/6 mice (10 mice/group) were orally administered with Sb-EE (200 mg/kg) once a day for five days, and peritonitis was induced by an intraperitoneal injection of LPS (10 mg/kg). ICR mice (four mice/group) were orally administered with Sb-EE (20 or 200 mg/kg) or ranitidine (positive control) twice a day for two days, and EtOH/HCl was orally injected to induce gastritis., Results: Sb-EE suppressed nitric oxide (NO) release (IC 50 =34 µg/mL) without cytotoxicity and contained flavonoids (quercetin, luteolin and kaempferol). Sb-EE (200 µg/mL) reduced the mRNA expression of inducible NO synthase (iNOS). Sb-EE blocked the activities of Syk and Src, while inhibiting interleukin-1 receptor associated kinases (IRAK1) by 68%. Similarly, orally administered Sb-EE (200 mg/kg) suppressed NO production by 78% and phosphorylation of Src and Syk in peritonitis mice. Sb-EE also decreased inflammatory lesions in gastritis mice., Discussion and Conclusions: This study demonstrates the inhibitory effect of Sb-EE on the inflammatory response, suggesting that Sb-EE can be developed as a potential anti-inflammatory agent.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour in the central nervous system (CNS). As the ideal targets for GBM treatment, Src family kinases (SFKs) have attracted much attention. Herein, a new series of imidazo[4,5-c]pyridin-2-one derivatives were designed and synthesised as SFK inhibitors. Compounds 1d , 1e , 1q , 1s exhibited potential Src and Fyn kinase inhibition in the submicromolar range, of which were next tested for their antiproliferative potency on four GBM cell lines. Compound 1s showed effective activity against U87, U251, T98G, and U87-EGFRvIII GBM cell lines, comparable to that of lead compound PP2. Molecular dynamics (MDs) simulation revealed the possible binding patterns of the most active compound 1s in ATP binding site of SFKs. ADME prediction suggested that 1s accord with the criteria of CNS drugs. These results led us to identify a novel SFK inhibitor as candidate for GBM treatment.

Target identification is a crucial process for advancing natural products and drug leads development, which is often the most challenging and time-consuming step. However, the putative biological targets of natural products obtained from traditional prediction studies are also informatively redundant. Thus, how to precisely identify the target of natural products is still one of the major challenges. Given the shortcomings of current target identification methodologies, herein, a novel in silico docking and DARTS prediction strategy was proposed. Concretely, the possible molecular weight was detected by DARTS method through examining the protected band in SDS-PAGE. Then, the potential targets were obtained from screening and identification through the PharmMapper Server and TargetHunter method. In addition, the candidate target Src was further validated by surface plasmon resonance assay, and the anti-apoptosis effects of kaempferol against myocardial infarction were further confirmed by in vitro and in vivo assays. Collectively, these results demonstrated that the integrated strategy could efficiently characterize the targets, which may shed a new light on target identification of natural products.

Src kinase activity controls diverse cellular functions, including cell growth, migration, adhesion, and survival. It is de-regulated in several cancers, including breast cancer, where it is highly expressed and phosphorylated. Thus, targeting Src by a small molecule is a feasible strategy for managing different breast cancer types. Several Src kinase inhibitors are available, including the FDA-approved drug (dasatinib). However, they are primarily ATP-competitive inhibitors that have been reported to lack specificity towards Src. We have a long-time interest in discovering protein kinase inhibitors that are non-competitive for ATP. In this project, three groups of 2'-aminospiro[pyrano[3,2-c]quinoline]-3'-carbonitrile derivatives were designed and synthesized, hypothesizing that small molecules with a spiro scaffold appended to a pyrano[3,2-c]quinoline analog could act as non-ATP competitive Src kinase inhibitors. 3b, 3c, and 3d inhibited Src kinase activity with IC50s of 4.9, 5.9, and 0.9 μM, respectively. At the same time, they did not impact the MDM2/p53 interaction in HEK293 cells, which has been reported to be affected by some spirocyclic compounds. 25 µM of 3b, 3c, or 3d did not inhibit the kinase activity of ERK2, JNK1, or p38-alpha in an in-vitro kinase assay. Steady-state kinetic studies for the effect of 3d on the ability of recombinant Src to phosphorylate its substrate (Srctide) revealed a non-ATP competitive inhibition mechanism. 1.6 µM of 3d was enough to diminish Src, Fak, and paxillin phosphorylation in the breast cancer cell lines MDA-MB-231 and MCF7. In the NCI screening, 3d induced broad tumor cytotoxicity for the NCI-60 cell lines, including all the breast cancer cell lines. The potency of 3b, 3c, and 3d to inhibit migration, proliferation, and colony formation of MDA-MB-231 and proliferation of MCF7 cells correlates with their potency to suppress Src kinase activity in the same cell line. Noticeably, the cell growth suppression and apoptosis induction in the tested cell lines can be attributed to the ability of the new derivatives to suppress the ERK and Akt survival pathways downstream of Src., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Despite the approval of several multikinase inhibitors that target SRC and the overwhelming evidence of the role of SRC in the progression and resistance mechanisms of many solid malignancies, inhibition of its kinase activity has thus far failed to improve patient outcomes. Here we report the small molecule eCF506 locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions that prevent phosphorylation and complex formation with its partner FAK. This mechanism of action resulted in highly potent and selective pathway inhibition in culture and in vivo . Treatment with eCF506 resulted in increased antitumor efficacy and tolerability in syngeneic murine cancer models, demonstrating significant therapeutic advantages over existing SRC/ABL inhibitors. Therefore, this mode of inhibiting SRC could lead to improved treatment of SRC-associated disorders. SIGNIFICANCE: Small molecule-mediated inhibition of SRC impairing both catalytic and scaffolding functions confers increased anticancer properties and tolerability compared with other SRC/ABL inhibitors., (©2021 The Authors; Published by the American Association for Cancer Research.)

Studies have shown that matrine has antitumor activity against many types of cancers. However, the direct target in cancer cells of its anticancer effect has not been identified. The purpose of this study was to find the molecular target of matrine to inhibit the proliferation of cancer cells and explore its mechanism of action. Herein we showed that matrine inhibited the proliferation of cancer in vitro and in vivo. Pull-down assay with matrine-amino coupling resins and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) identified Src as the target of matrine. Cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) provided solid evidences that matrine directly bound to Src. Bioinformatics prediction and pull-down experiment demonstrated that Src kinase domain was required for its interaction with matrine and Ala392 in the kinase domain participated in matrine-Src interaction. Intriguingly, matrine was proven to inhibit Src kinase activity in a non-ATP-competitive manner by blocking the autophosphorylation of Tyr419 in Src kinase domain. Matrine down-regulated the phosphorylation levels of MAPK/ERK, JAK2/STAT3, and PI3K/Akt signaling pathways via targeting Src. Collectively, matrine targeted Src, inhibited its kinase activity, and down-regulated its downstream MAPK/ERK, JAK2/STAT3, and PI3K/Akt phosphorylation signaling pathways to inhibit the proliferation of cancer cells., (© 2021. The Author(s).)

Development of acquired resistance to lapatinib, a dual epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor, severely limits the duration of clinical response in advanced HER2-driven breast cancer patients. Although the compensatory activation of the PI3K/Akt survival signal has been proposed to cause acquired lapatinib resistance, comprehensive molecular mechanisms remain required to develop more efficient strategies to circumvent this therapeutic difficulty. In this study, we found that suppression of HER2 by lapatinib still led to Akt inactivation and elevation of FOX3a protein levels, but failed to induce the expression of their downstream pro-apoptotic effector p27 kip1 , a cyclin-dependent kinase inhibitor. Elevation of miR-221 was found to contribute to the development of acquired lapatinib resistance by targeting p27 kip1 expression. Furthermore, upregulation of miR-221 was mediated by the lapatinib-induced Src family tyrosine kinase and subsequent NF-κB activation. The reversal of miR-221 upregulation and p27 kip1 downregulation by a Src inhibitor, dasatinib, can overcome lapatinib resistance. Our study not only identified miRNA-221 as a pivotal factor conferring the acquired resistance of HER2-positive breast cancer cells to lapatinib through negatively regulating p27 kip1 expression, but also suggested Src inhibition as a potential strategy to overcome lapatinib resistance., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Long-term maternal salt intake induces the hypertension in offspring. Numerous studies have also indicated that high-salt diet causes the inflammation and an imbalance in neurotransmitters in the paraventricular nucleus (PVN) which increases the blood pressure and sympathetic activity. This study aimed to explore whether maternal salt intake induces hypertension in their male offspring by increasing the inflammation and changing the neurotransmitters balance in the paraventricular nucleus of offspring. This study includes two parts: Part I to explore the effect of high-salt diet on pregnant rats and the changes in inflammation and neurotransmitters in their male offspring PVN; Part II to reveal the influence on their offspring of bilateral PVN infusion of c-Src inhibitor dasatinib (DAS) in pregnant rats fed a high-salt diet. Maternal high-salt diet intake during copulation, pregnancy, and lactation impacted the offspring mean arterial pressure (MAP) and elevated the offspring PVN levels of p-Src, proinflammatory cytokines, and excitatory neurotransmitters. Bilateral PVN infusion of a c-Src inhibitor combined with maternal high-salt diets decreased MAP in the offspring. The infusion was also shown to suppress the Src-induced MAPK/NF-κB signaling pathway (p38 MAPK, JNK, Erk1/2), which attenuates inflammatory reactions. Finally, bilateral PVN infusion of the Src inhibitor in pregnant rat with high-salt diets improved the levels of inhibitory neurotransmitters in offspring PVN, which restored the excitatory-inhibitory neurotransmitter balance in male offspring. High-salt diets increase sympathetic activity and blood pressure in adult offspring, probably by activating the c-Src/MAPKs/NF-κB signaling pathway-induced inflammation. Moreover, NF-κB disrupts the downstream excitatory-inhibitory neurotransmitter balance in the PVN of male offspring., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19., (© 2021. The Author(s).)

Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a test-functional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Union for International Cancer Control.)

Background/aim: The purpose of this study was to investigate the antiarthritic potentials of the inhibition of Src kinase in vivo and in vitro settings., Materials and Methods: Arthritis was induced by intradermal injection of chicken type II collagen combined with incomplete Freund’s adjuvant (collagen induced arthritis [CIA] model) in Wistar albino rats. One day after the onset of arthritis, dasatinib, a potent Src kinase inhibitor, (5 mg/kg/day) was given via oral gavage. Tissue Src, Fyn, MAPK and STAT mRNA expressions were determined by real-time polymerase chain reaction. On the other hand, fibroblast like synoviocytes (FLSs) were harvested patients with rheumatoid arthritis (RA) undergoing surgical knee joint replacement. FLSs were stimulated with cytokines and dasatinib was added in different concentrations. MMP –1, –3, and –13 levels in FLSs culture were determined by ELISA., Results: The tissue mRNA expressions of Src, Fyn, MAPK and STATs were increased in the arthritis CIA group compared to the control group. Their mRNA expressions in the CIA + dasatinib group were decreased and similar in the control group. In in vitro setting, MMP –1, –3, and –13 expressions from FLSs induced by IL-1β and TNF-α were increased, while dasatinib suppressed their productions from FLSs., Conclusion: The present study shows that the inhibition of Src kinase has antiarthritic potentials in both in vivo and in vitro settings. Src kinase inhibition may be candidate to further research in human RA., (This work is licensed under a Creative Commons Attribution 4.0 International License.)

Background: Doxorubicin (Dox) is a widely used chemotherapy, but its effectiveness is limited by dose-dependent side effects. Although lower Dox doses reduce this risk, studies have reported higher recurrence of local disease with no improvement in survival rate in patients receiving low doses of Dox. To effectively mitigate this, a better understanding of the adverse effects of suboptimal Dox doses is needed., Methods: Effects of sublethal dose of Dox on phenotypic changes were assessed with light and confocal microscopy. Migratory and invasive behavior were assessed by wound healing and transwell migration assays. MTT and LDH release assays were used to analyze cell growth and cytotoxicity. Flow cytometry was employed to detect cell surface markers of cancer stem cell population. Expression and activity of matrix metalloproteinases were probed with qRT-PCR and zymogen assay. To identify pathways affected by sublethal dose of Dox, exploratory RNAseq was performed and results were verified by qRT-PCR in multiple cell lines (MCF7, ZR75-1 and U-2OS). Regulation of Src Family kinases (SFK) by key players in DNA damage response was assessed by siRNA knockdown along with western blot and qRT-PCR. Dasatinib and siRNA for Fyn and Yes was employed to inhibit SFKs and verify their role in increased migration and invasion in MCF7 cells treated with sublethal doses of Dox., Results: The results show that sublethal Dox treatment leads to increased migration and invasion in otherwise non-invasive MCF7 breast cancer cells. Mechanistically, these effects were independent of the epithelial mesenchymal transition, were not due to increased cancer stem cell population, and were not observed with other chemotherapies. Instead, sublethal Dox induces expression of multiple SFK-including Fyn, Yes, and Src-partly in a p53 and ATR-dependent manner. These effects were validated in multiple cell lines. Functionally, inhibiting SFKs with Dasatinib and specific downregulation of Fyn suppressed Dox-induced migration and invasion of MCF7 cells., Conclusions: Overall, this study demonstrates that sublethal doses of Dox activate a pro-invasive, pro-migration program in cancer cells. Furthermore, by identifying SFKs as key mediators of these effects, our results define a potential therapeutic strategy to mitigate local invasion through co-treatment with Dasatinib., (© 2021. The Author(s).)

The clinical application of opioids may be accompanied by a series of adverse consequences, such as opioid tolerance, opioid-induced hyperalgesia, opioid dependence or addiction. In view of this issue, clinicians are faced with the dilemma of treating various types of pain with or without opioids. In this review, we discuss that Src protein tyrosine kinase plays an important role in these adverse consequences, and Src inhibitors can solve these problems well. Therefore, Src inhibitors have the potential to be used in combination with opioids to achieve synergy. How to combine them together to maximize the analgesic effect while avoiding unnecessary trouble provides a topic for follow-up research., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-β-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the βIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation., Competing Interests: Declaration of interests C.J.D. has received funding from and is a consultant for and advisory board member of Mirati Therapeutics and Deciphera Pharmaceuticals. C.J.D. has consulted for Eli Lilly, Jazz Therapeutics, Revolution Medicines, Ribometrix, and Turning Point Therapeutics. A.D.C. has consulted for Eli Lilly and Mirati Therapeutics. A.J.A. has consulted for Oncorus, Arrakis Therapeutics, and Merck & Co. A.J.A. has received research funding support from Mirati Therapeutics and Deerfield. W.C.H. is a consultant for Thermo Fisher, Solvasta Ventures, MPM Capital, KSQ Therapeutics, iTeos, Tyra Biosciences, Frontier Medicine, and Paraxel., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Klisyri (KX01) is a dual tubulin/Src protein inhibitor that has shown potential therapeutic effects in several tumor models. However, a phase II clinical trial in patients with bone-metastatic castration-resistant prostate cancer was halted because of lack of efficacy. We previously reported that KX01 binds to the colchicine site of β-tubulin and its morpholine group lies close to α-tubulin's surface. Thus, we hypothesized that enhancing the interaction of KX01 with α-tubulin could increase tubulin inhibition and synthesized a series of KX01 derivatives directed by docking studies. Among these derivatives, 8a exhibited more than 10-fold antiproliferation activity in several tumor cells than KX01 and significantly improved in vivo antitumor effects. The X-ray crystal structure suggested that 8a both bound to the colchicine site and extended into the interior of α-tubulin to form potent interactions, presenting a novel binding mode. A potential clinical candidate for cancer therapy was identified in this study.