scholarly journals Reaction-based fluorogenic probes for selective detection of cysteinyl oxidation in living cells

Author(s):  
Renan Ferreira ◽  
Ling Fu ◽  
Jing Yang ◽  
Kate Carroll

Abstract Measuring reactive oxygen, nitrogen and sulfur species in cells is established technology, but turn-on fluorescence tools for detecting the products of their reaction with protein cysteines remain essentially unknown. Toward this goal, here we describe fluorogenic probes for sulfenic acid, a redox modification of protein cysteines inextricably linked to signaling and oxidative stress. The probes, called CysOx1 and CysOx2, are reaction-based, exhibit excellent cell permeability, rapid reactivity, and high selectivity with minimal cytotoxicity. We applied CysOx2 in a cell-based 96-well plate assay to determine whether kinase inhibitors modulate protein S-sulfenylation as well as O-phosphorylation. Analysis of these data revealed an unexpected positive association of S-sulfenylation and inhibition of select kinases within the TK, AGC, and CMGC families including GSK3, a multitasking Ser/Thr kinase and emerging therapeutic target for neurodegenerative and mood disorders. Chemoproteomic mapping of sulfenic acid-modified cysteines in GSK3 inhibitor-treated cells shows that sites of S-oxidation localize to regulatory cysteines within key components of antioxidant defense systems. Our studies with CysOx probes offer up new insights into kinase-inhibitor dependent modulation of sulfenylome dynamics and should accelerate future efforts in the modern era of translational redox medicine.

2019 ◽  
Author(s):  
Hamad Yadikar ◽  
Isabel Torres ◽  
Gabrielle Aiello ◽  
Milin Kurup ◽  
Zhihui Yang ◽  
...  

ABSTRACTTauopathies are a class of neurodegenerative disorders characterized by abnormal deposition of post-translationally modified tau protein in the human brain. Tauopathies are associated with Alzheimer’s disease (AD), chronic traumatic encephalopathy (CTE), and other diseases. Hyperphosphorylation increases tau tendency to aggregate and forms neurofibrillary tangles (NFT), a pathological hallmark of AD. In this study, okadaic acid (OA, 100 nM), a protein phosphatase 1/2A inhibitor, was treated for 24h in mouse neuroblastoma (N2a) and differentiated rat primary neuronal cortical cell cultures (CTX) to induce tau-hyperphosphorylation and oligomerization as a cell-based tauopathy model. Following the treatments, the effectiveness of different kinase inhibitors was assessed using the tauopathy-relevant tau antibodies through tau-immunoblotting, including the sites: pSer202/pThr205 (AT8), pThr181 (AT270), pSer202 (CP13), pSer396/pSer404 (PHF-1), and pThr231 (RZ3). OA-treated samples induced tau phosphorylation and oligomerization at all tested epitopes, forming a monomeric band (46-67 kDa) and oligomeric bands (170 kDa and 240 kDa). We found that TBB (a casein kinase II inhibitor), AR and LiCl (GSK-3 inhibitors), cyclosporin A (calcineurin inhibitor), and Saracatinib (Fyn kinase inhibitor) caused robust inhibition of OA-induced monomeric and oligomeric p-tau in both N2a and CTX culture. Additionally, a cyclin-dependent kinase 5 inhibitor (Roscovitine) and a calcium chelator (EGTA) showed conflicting results between the two neuronal cultures.This study provides a comprehensive view of potential drug candidates (TBB, CsA, AR, and Saracatinib), and their efficacy against tau hyperphosphorylation and oligomerization processes. These findings warrant further experimentation, possibly including animal models of tauopathies, which may provide a putative Neurotherapy for AD, CTE, and other forms of tauopathy-induced neurodegenerative diseases.


Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2369 ◽  
Author(s):  
Cinzia Francini ◽  
Francesca Musumeci ◽  
Anna Fallacara ◽  
Lorenzo Botta ◽  
Alessio Molinari ◽  
...  

Protein kinases have emerged as crucial targets for cancer therapy over the last decades. Since 2001, 40 and 39 kinase inhibitors have been approved by FDA and EMA, respectively, and the majority are antineoplastic drugs. Morevoer, many candidates are currently in clinical trials. We previously reported a small library of 4-aminoimidazole and 2-aminothiazole derivatives active as Src family kinase (SFK) inhibitors. Starting from these results, we decided to perform an optimization study applying a mix and match strategy to identify a more potent generation of 4-aminoimidazoles. Firstly, a computational study has been performed, then compounds showing the best predicted docking scores were synthesized and screened in a cell-free assay for their SFK inhibitory activity. All the new chemical entities showed IC50s in the nanomolar range, with 2–130 fold increased activities compared to the previously reported inhibitors. Finally, the most active compounds have been tested on three cancer cell lines characterized by Src hyperactivation. Compounds 4k and 4l showed an interesting antiproliferative activity on SH-SY5Y neuroblastoma (NB) cell line. In this assay, the compounds resulted more potent than dasatinib, a tyrosine kinase inhibitor approved for the treatment of leukemias and in clinical trials for NB.


2020 ◽  
Author(s):  
Tigist Y. Tamir ◽  
David H. Drewry ◽  
Carrow Wells ◽  
M. Ben Major ◽  
Alison D. Axtman

AbstractThe Published Kinase Inhibitor Set (PKIS) is a publicly-available chemogenomic library distributed to more than 300 laboratories by GlaxoSmithKline (GSK) between 2011–2015 and by SGC-UNC from 2015–2017. Screening this library of well-annotated, published kinase inhibitors has yielded a plethora of data in diverse therapeutic and scientific areas, funded applications, publications, and provided impactful pre-clinical results. Based on kinome-wide screening results, we report a thorough investigation of one PKIS compound, GW296115, as an inhibitor of several members of the Illuminating the Druggable Genome (IDG) list of understudied dark kinases. Specifically, GW296115 validates as a potent lead chemical tool that inhibits six IDG kinases with IC50 values less than 100nM. Focused studies establish that GW296115 is cell active, and directly engages BRSK2. Further evaluation showed that GW296115 downregulates BRSK2-driven phosphorylation and downstream signaling.Summary StatementGW296115 inhibits understudied kinases, including BRSK2, with IC50 values less than 100nM.


2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tigist Y. Tamir ◽  
David H. Drewry ◽  
Carrow Wells ◽  
M. Ben Major ◽  
Alison D. Axtman

Abstract The Published Kinase Inhibitor Set (PKIS) is a publicly-available chemogenomic library distributed to more than 300 laboratories by GlaxoSmithKline (GSK) between 2011 and 2015 and by SGC-UNC from 2015 to 2017. Screening this library of well-annotated, published kinase inhibitors has yielded a plethora of data in diverse therapeutic and scientific areas, funded applications, publications, and provided impactful pre-clinical results. GW296115 is a compound that was included in PKIS based on its promising selectivity following profiling against 260 human kinases. Herein we present more comprehensive profiling data for 403 wild type human kinases and follow-up enzymatic screening results for GW296115. This more thorough investigation of GW296115 has confirmed it as a potent inhibitor of kinases including BRSK1 and BRSK2 that were identified in the original panel of 260 kinases as well as surfaced other kinases that it potently inhibits. Based on these new kinome-wide screening results, we report that GW296115 is an inhibitor of several members of the Illuminating the Druggable Genome (IDG) list of understudied dark kinases. Specifically, our results establish GW296115 as a potent lead chemical tool that inhibits six IDG kinases with IC50 values less than 100 nM. Focused studies establish that GW296115 is cell active, and directly engages BRSK2. Further evaluation showed that GW296115 downregulates BRSK2-driven phosphorylation and downstream signaling. Therefore, we present GW296115 as a cell-active chemical tool that can be used to interrogate the poorly characterized function(s) of BRSK2.


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1373-1373 ◽  
Author(s):  
Thomas O’Hare ◽  
Christopher A. Eide ◽  
Jeffrey W. Tyner ◽  
Matthew J. Wong ◽  
Caitlyn A. Smith ◽  
...  

Abstract Imatinib effectively inhibits the tyrosine kinase activity of Bcr-Abl, the molecular driver of CML. Emergence of imatinib resistance due to mutations within the Bcr-Abl kinase domain (KD) has prompted the development of new Abl kinase inhibitors. A particularly important target is Bcr-Abl(T315I), which accounts for 15–20% of patients with resistance. To address this unresolved need, we profiled the novel Abl kinase inhibitor SGX70393 against native and mutant Bcr-Abl. Methods: We assessed the efficacy of SGX70393 in cellular and biochemical assays against a panel of KD mutants. Cell proliferation assays and Bcr-Abl tyrosine phosphorylation immunoblot analyses were performed for parental Ba/F3 cells, Ba/F3 cells expressing unmutated Bcr-Abl, or Ba/F3 cells expressing a single Bcr-Abl KD mutation (M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, F311L, T315I, F317L, M351T, F359V, V379I, L387M, H396P, or H396R). The resistance profile of SGX70393 was also evaluated using a recently developed accelerated, cell-based mutagenesis assay (Bradeen, et al. Blood, June 2006; doi:10.1182). Results: SGX70393 inhibited growth of cells expressing Bcr-Abl(T315I) (IC50: 7.3 nM) or unmutated Bcr-Abl (IC50: 12 nM). Sensitivity of Bcr-Abl mutants to SGX70393 partitioned into three categories: high (IC50<25 nM: M244V, T315I, F359V, V379I, L387M, H396P, and H396R), medium (IC50<300 nM: Q252H, Y253H, E255K, and F311L), and low (IC50>500 nM: G250E, Y253F, E255V, and F317L). A cell-based mutagenesis screen for Bcr-Abl kinase domain escape mutants emerging in the presence of SGX70393 revealed a concentration-dependent reduction in surviving clones, with five previously reported Bcr-Abl mutations (L248M; G250E; Y253F; E255V; F317V) accounting for almost all resistance. Conclusions: (a) SGX70393 is a potent inhibitor of native and T315I mutant Bcr-Abl. (b) SGX70393 coverage extends to most clinically relevant mutants except mutations of the p-loop and F317.


Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1131
Author(s):  
Mohamed H. Elsherbeny ◽  
Ahmed Elkamhawy ◽  
Hossam Nada ◽  
Magda H. Abdellattif ◽  
Kyeong Lee ◽  
...  

Although the sea ecosystem offers a broad range of bioactivities including anticancer, none of the FDA-approved antiproliferative protein kinase inhibitors are derived from a marine source. In a step to develop new marine-inspired potent kinase inhibitors with antiproliferative activities, a new series of hybrid small molecules (5a–5g) was designed and synthesized based on chemical moieties derived from two marine natural products (Meridianin E and Leucettamine B). Over a panel of 14 cancer-related kinases, a single dose of 10 µM of the parent hybrid 5a possessing the benzo[d][1,3]dioxole moiety of Leucettamine B was able to inhibit the activity of FMS, LCK, LYN, and DAPK1 kinases with 82.5 ± 0.6, 81.4 ± 0.6, 75.2 ± 0.0, and 55 ± 1.1%, respectively. Further optimization revealed the most potent multiple kinase inhibitor of this new series (5g) with IC50 values of 110, 87.7, and 169 nM against FMS, LCK, and LYN kinases, respectively. Compared to imatinib (FDA-approved multiple kinase inhibitor), compound 5g was found to be ~ 9- and 2-fold more potent than imatinib over both FMS and LCK kinases, respectively. In silico docking simulation models of the synthesized compounds within the active site of FMS, LCK, LYN, and DAPK1 kinases offered reasonable explanations of the elicited biological activities. In an in vitro anticancer assay using a library of 60 cancer cell lines that include blood, lung, colon, CNS, skin, ovarian, renal, prostate, and breast cancers, it was found that compound 5g was able to suppress 60 and 70% of tumor growth in leukemia SR and renal RXF 393 cell lines, respectively. Moreover, an ADME study indicated a suitable profile of compound 5g concerning cell permeability and blood-brain barrier (BBB) impermeability, avoiding possible CNS side effects. Accordingly, compound 5g is reported as a potential lead towards novel antiproliferative marine-derived kinase modulators.


2019 ◽  
Vol 4 (1-2) ◽  
pp. 41-45 ◽  
Author(s):  
Takeo Koshida ◽  
Sylvia Wu ◽  
Hitoshi Suzuki ◽  
Rimda Wanchoo ◽  
Vanesa Bijol ◽  
...  

Dasatinib is the second-generation tyrosine kinase inhibitor used in the treatment of chronic myeloid leukemia. Proteinuria has been reported with this agent. We describe two kidney biopsy–proven cases of dasatinib-induced thrombotic microangiopathy that responded to stoppage of dasatinib and using an alternate tyrosine kinase inhibitor. Certain specific tyrosine kinase inhibitors lead to endothelial injury and renal-limited thrombotic microangiopathy. Hematologists and nephrologists need to be familiar with this off-target effect of dasatinib.


2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Abdullahi Bello Umar ◽  
Adamu Uzairu ◽  
Gideon Adamu Shallangwa ◽  
Sani Uba

Abstract Background V600E-BRAF is a major protein target involved in various types of human cancers. However, the acquired resistance of the V600E-BRAF kinase to the vemurafenib and the side effects of other identified drugs initiate the search for efficient inhibitors. In the current paper, virtual docking screening combined with drug likeness and ADMET properties predictions were jointly applied to evaluate potent 2-(1H-imidazol-2-yl) pyridines as V600E-BRAF kinase inhibitors. Results Most of the studied compounds showed better docking scores and favorable interactions with theiV600E-BRAF target. Among the screened compounds, the two most potent (14 and 30) with good rerank scores (−124.079 and − 122.290) emerged as the most effective, and potent V600E-BRAF kinase inhibitors which performed better than vemurafenib (−116.174), an approved V600E-BRAF kinase inhibitor. Thus, the docking studies exhibited that these compounds have shown competing inhibition of V600E-BRAF kinase with vemurafenib at the active site and revealed better pharmacological properties based on Lipinski’s and Veber’s drug-likeness rules for oral bioavailability and ADMET properties. Conclusion The docking result, drug-likeness rules, and ADMET parameters identified compounds (14 and 30) as the best hits against V600E-BRAF kinase with better pharmacological properties. This suggests that these compounds may be developed as potent V600E-BRAF inhibitors.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hu Lei ◽  
Han-Zhang Xu ◽  
Hui-Zhuang Shan ◽  
Meng Liu ◽  
Ying Lu ◽  
...  

AbstractIdentifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.


Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-36
Author(s):  
Mario Tiribelli ◽  
Roberto Latagliata ◽  
Massimo Breccia ◽  
Isabella Capodanno ◽  
Maria Cristina Miggiano ◽  
...  

Introduction : therapy of chronic phase (CP) chronic myeloid leukemia (CML) is based on tyrosine kinase inhibitors (TKIs) in virtually all patients. Three TKIs are approved for first-line therapy in Italy: imatinib and two second-generation (2G) TKIs, dasatinib and nilotinib. Choice of the front-line TKI is based on a combined evaluation of patient's and disease characteristics, age, risk, comorbidities and concomitant medications. Treating physician's preference and, in some cases, economic considerations, particularly after the advent of generic imatinib, may play a role in TKI selection. However, to date, few data are available on TKI use in a whole nation and on the possible drivers of treatment choice. Aim of the present work was to analyse the use of front-line TKI therapy in a large, unselected cohort of Italian CP-CML patients, correlating patient's features to drug choice. Methods: in the framework of the national Campus CML program, we retrospectively evaluated 1422 patients with CP-CML diagnosed from 2012 and 2019 in 21 haematologic Centres, mostly in academic and/or tertiary hospitals, widespread through the entire Italian territory and treated frontline with imatinib, dasatinib or nilotinib. Results: median age at diagnosis was 59.9 years [interquartile range (IQR) 47.1 - 71.7], with 317 (22.3%) patients under 45 years, 552 (38.8%) between 45 and 65 years and 553 (38.9%) older than 65 years; 821 (57.7%) patients were males. Among 1364 evaluable patients, CML risk according to Sokal score was low in 540 (39.6%), intermediate in 610 (44.7%) and high in 214 (15.7%) patients respectively; the number at low, intermediate or high risk according to the novel ELTS score among 1325 evaluable patients was 759 (57.3%), 402 (30.3%) and 164 (12.4%) respectively. Considering comorbidities, 1003 (70.6%) patients had at least one active disease at the time of CML diagnosis, the most common being hypertension (n=547, 38.5%), previous neoplasms (n=185, 13.0%), diabetes (n=150, 10.6%), chronic bronchopulmonary diseases (n=114, 8.0%), acute myocardial infarction (n=95, 6.7%), previous stroke (n=36, 2.5%) and other vascular diseases (n=98, 6.9%). Among 1335 evaluable patients, 813 (60.9%) were taking at least one concomitant medication, with 280 (21.0%) taking 3-5 drugs and 140 (10.5%) taking 6+ drugs at time of TKI start. As to the frontline therapy, 794 (55.8%) received imatinib and 628 (44.2%) were treated with 2G-TKIs, (226 dasatinib and 402 nilotinib) respectively. According to age, 2G-TKIs were chosen for majority of patients aged <45 (69.1%) while imatinib was used in 76.9% of patients over 65 (p<0.001). There was a predominance of imatinib use across all Sokal (51.1% in low, 61.3% in intermediate and 51.4% in high) and ELTS (50.3% in low, 60.4% in intermediate and 66.5%) risk categories. We observed a prevalent use of 2G-TKIs in patients presenting with higher WBC counts (55.1% if WBC >100,000/mm3 vs 38.2% if WBC <100,000/mm3; p<0.001), lower Hb (53.8% if Hb <10 g/dl vs 41.9 if Hb >10 g/dl; p=0.001) and bigger spleen (65.1% if spleen >5 cm vs 44.8% if spleen 1-5 cm vs 37.3% if spleen not palpable; p<0.001). There was a decreasing use of 2G-TKIs with higher number of concomitant drugs: 64.4% for 0, 47.7% for 1-2, 27.0% for 3-5 and 13.6% for >5 drugs, respectively (p<0.001). Concordantly, there was a significant higher use of imatinib in patients with hypertension (69.8%), diabetes (70.0%), COPD (73.7%), previous neoplasms (73.0%), AMI (86.3%) or stroke (97.2%) history (p<0.001 for all conditions). Lastly, we observed a wider use of imatinib (61.1%) in patients diagnosed in years 2018-19, compared to those of the period 2012-17 (53.7%; p=0.01). In multivariable analysis, factors correlated with imatinib use were age > 45 years, intermediate or high Sokal risk, presence of some comorbidities (2nd neoplasia and stroke) and number of concomitant medications. Conclusions: preliminary results of this observational study on almost 1500 patients show that around 55% of newly diagnosed Italian CP-CML patients receive imatinib as front-line therapy, and that the use of 2G-TKI is prevalent in the younger patients and in those with no concomitant clinical conditions. The counterintuitive finding of imatinib prevalence as frontline treatment in high risk patients might be explained by the older age of these patients. Introduction of the generic formulation in 2018 seems to have fostered the use of imatinib. Figure Disclosures Breccia: Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Abbvie: Consultancy; Bristol-Myers Squibb/Celgene: Consultancy, Honoraria. Cavazzini:Pfize: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Saglio:Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Ariad: Research Funding; Roche: Research Funding.


Sign in / Sign up

Export Citation Format

Share Document