Competitive affinity-based proteome profiling and imaging to reveal potential cellular targets of betulinic acid.

Betulinic acid (BA), a pentacyclic triterpenoid natural product, has received considerable attention owing to the broad spectrum of biological activities such as anti-HIV, anti-inflammatory, and especially, antitumor. Despite numerous studies on the antitumor mechanisms over the past decade, the cellular targets in cancer cells remain obscure. Here, we developed two photoprobes, BA-1/BA-2, by attaching terminal alkyne-containing "minimalist" tetrazole/diazirine photo-crosslinkers (L3 and L7) at the C-3 and C-28 positions of BA, respectively, based on well-established structure-activity relationships. The potential cellular targets were identified via competitive proteome profiling and bioimaging approaches.

[1]  S. Sze,et al.  Design and synthesis of minimalist terminal alkyne-containing diazirine photo-crosslinkers and their incorporation into kinase inhibitors for cell- and tissue-based proteome profiling. , 2013, Angewandte Chemie.

[2]  W. Gao,et al.  Synthesis of heterocycle-modified betulinic acid derivatives as antitumor agents. , 2015, European journal of medicinal chemistry.

[3]  Anna K. Schrey,et al.  Comprehensive identification of staurosporine-binding kinases in the hepatocyte cell line HepG2 using Capture Compound Mass Spectrometry (CCMS). , 2010, Journal of proteome research.

[4]  Lei Wang,et al.  Betulinic Acid and its Derivatives as Potential Antitumor Agents , 2015, Medicinal research reviews.

[5]  G. Kroemer,et al.  Targeting mitochondrial apoptosis by betulinic acid in human cancers. , 2009, Drug discovery today.

[6]  Li Huang,et al.  Synthesis and anti-HIV activity of bi-functional betulinic acid derivatives. , 2006, Bioorganic & medicinal chemistry.

[7]  Anna K. Schrey,et al.  Dasatinib, imatinib and staurosporine capture compounds - Complementary tools for the profiling of kinases by Capture Compound Mass Spectrometry (CCMS). , 2011, Journal of proteomics.

[8]  Mingyao Liu,et al.  Synthesis and biological evaluation of heterocyclic ring-fused betulinic acid derivatives as novel inhibitors of osteoclast differentiation and bone resorption. , 2012, Journal of medicinal chemistry.

[9]  S. Yao,et al.  Tetrazole Photoclick Chemistry: Reinvestigating Its Suitability as a Bioorthogonal Reaction and Potential Applications. , 2016, Angewandte Chemie.

[10]  S. Yao,et al.  "Minimalist" cyclopropene-containing photo-cross-linkers suitable for live-cell imaging and affinity-based protein labeling. , 2014, Journal of the American Chemical Society.

[11]  H. Waldmann,et al.  Small-Molecule Target Engagement in Cells. , 2016, Cell chemical biology.

[12]  Xiaoguang Lei,et al.  Chemoproteomic Profiling of Bile Acid Interacting Proteins , 2017, ACS central science.

[13]  S. Shukla,et al.  Betulinic acid suppresses constitutive and TNFα‐induced NF‐κB activation and induces apoptosis in human prostate carcinoma PC‐3 cells , 2008, Molecular carcinogenesis.

[14]  David J. Lapinsky,et al.  Recent developments and applications of clickable photoprobes in medicinal chemistry and chemical biology. , 2015, Future medicinal chemistry.

[15]  P. Clemons,et al.  Target identification and mechanism of action in chemical biology and drug discovery. , 2013, Nature chemical biology.

[16]  Stephen R. Johnson,et al.  Ligand and Target Discovery by Fragment-Based Screening in Human Cells , 2017, Cell.

[17]  Bernhard Kuster,et al.  Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors , 2007, Nature Biotechnology.

[18]  G. Superti-Furga,et al.  Target profiling of small molecules by chemical proteomics. , 2009, Nature chemical biology.

[19]  Zhengqiu Li,et al.  Developments of bioorthogonal handle-containing photo-crosslinkers for photoaffinity labeling. , 2017, MedChemComm.

[20]  J. Slupsky,et al.  Acetyl-boswellic acids are novel catalytic inhibitors of human topoisomerases I and IIalpha. , 2000, Molecular pharmacology.

[21]  Wayne N. Frankel,et al.  The harlequin mouse mutation downregulates apoptosis-inducing factor , 2002, Nature.

[22]  J. Kusz,et al.  Synthesis, structure and cytotoxic activity of acetylenic derivatives of betulonic and betulinic acids , 2016 .

[23]  K. Lim,et al.  Puromycin Analogues Capable of Multiplexed Imaging and Profiling of Protein Synthesis and Dynamics in Live Cells and Neurons. , 2016, Angewandte Chemie.

[24]  M. Bentley,et al.  Betulin and lupeol in bark from four white-barked birches , 1988 .

[25]  Jasmina Marjanovic,et al.  2-Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification , 2016, Journal of the American Chemical Society.

[26]  S. Son,et al.  Identification of malate dehydrogenase 2 as a target protein of the HIF-1 inhibitor LW6 using chemical probes. , 2013, Angewandte Chemie.

[27]  Yuguo Zheng,et al.  Target identification of biologically active small molecules via in situ methods. , 2013, Current opinion in chemical biology.

[28]  D. Ströhl,et al.  Betulinic acid derived hydroxamates and betulin derived carbamates are interesting scaffolds for the synthesis of novel cytotoxic compounds. , 2015, European journal of medicinal chemistry.

[29]  R. Csuk.  Betulinic acid and its derivatives: a patent review (2008 – 2013) , 2014, Expert opinion on therapeutic patents.

[30]  Theonie Anastassiadis,et al.  Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity , 2011, Nature biotechnology.

[31]  Herbert Waldmann,et al.  Target identification for small bioactive molecules: finding the needle in the haystack. , 2013, Angewandte Chemie.

[32]  W. Lee,et al.  Human ACAT-1 and ACAT-2 inhibitory activities of pentacyclic triterpenes from the leaves of Lycopus lucidus TURCZ. , 2006, Biological & pharmaceutical bulletin.