Table Of ContentHiroyuki Osada Editor
Bioprobes
Biochemical Tools for Investigating Cell
Function
Second Edition
Bioprobes
Hiroyuki Osada
Editor
Bioprobes
Biochemical Tools for Investigating Cell
Function
Second Edition
Editor
HiroyukiOsada
ChemicalBiologyResearchGroup
RIKENCenterforSustainableResourceScience
Wako,Saitama
Japan
ISBN978-4-431-56527-7 ISBN978-4-431-56529-1 (eBook)
DOI10.1007/978-4-431-56529-1
LibraryofCongressControlNumber:2017936471
©SpringerJapanKK2000,2017
Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartof
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or
dissimilarmethodologynowknownorhereafterdeveloped.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publicationdoesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesareexempt
fromtherelevantprotectivelawsandregulationsandthereforefreeforgeneraluse.
Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthis
book are believed to be true and accurate at the date of publication. Neither the publisher nor the
authors or the editors give a warranty, express or implied, with respect to the material contained
hereinor for anyerrors oromissionsthat may havebeenmade. Thepublisher remainsneutralwith
regardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
Printedonacid-freepaper
ThisSpringerimprintispublishedbySpringerNature
TheregisteredcompanyisSpringerJapanKK
Theregisteredcompanyaddressis:ChiyodaFirstBldg.East,3-8-1Nishi-Kanda,Chiyoda-ku,Tokyo
101-0065,Japan
Preface
The first edition of this book Bioprobes was published in 2000. The term
“bioprobe” was introduced then as the name for a chemical tool, a probe, to
investigate biological functions. Eventually, the concept of the research inspired
by the bioprobe was similar tothat of chemical biology and it became familiar to
scientists.Theepoch-makingresearchresults,namely,thatFK506andcyclosporin
inhibit the activity of phosphatase calcineurin by forming the ternary complexes
FK506- FKBP12- calcineurin or cyclosporine-cyclophilin-calcineurin, opened a
newdoorinchemicalbiology.Itwastypicalresearchinbasicchemicalbiology.
ThenewtideofchemicalbiologyhasflowedfromtheInstituteofChemistryand
Cell Biology (ICCB) at Harvard Medical School and has initiated the building of
chemical biology-related centers in other parts of the world. One of the main
activities of the centers is the screening of small bioactive compounds from
chemical libraries. This tide merged into chemical genomics, which deals with
the systematic screening on a large scale from chemical libraries to discover lead
compoundsofdrugs.
Based on this background, the present volume describes bioprobes that are
mainly isolated from microorganisms, along with some synthetic compounds.
Most of the bioprobes are not developed for clinical use yet, but they are useful
aschemicalprobesaswellasleadcompoundsofdrugs.
Thebookconsistsofsixchapters.Afterabriefintroductionofthedevelopment
of bioprobes, the biological fields in which bioprobes affect the molecular targets
are surveyed. These reviews cover the broad, advancing areas of the cell cycle,
epigenetics, apoptosis–autophagy, and immunological responses. A final chapter
containsimportantbioprobesthatareusefulforinvestigatingbiologicalstudies.
InthissecondeditionofBioprobes,wedealwithsmallmoleculesisolatedfrom
marine organisms in addition to microbial metabolites. I hope this book will
v
vi Preface
contribute to the work of both natural product chemists and cell biologists, even-
tuallyresultingintheprogressofbioproberesearch.
Wako,Japan HiroyukiOsada
March25,2017
Contents
1 TrendsinBioprobeResearch. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
HiroyukiOsada
1.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 ScreeningofNewBioactiveCompounds. . . . . . . . . . . . . . . . . 2
1.3 TargetIdentificationofBioactiveCompounds. . . . . . . . . . . . . 4
1.4 TrendsandProspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2 CellProliferationandDifferentiation. . . . . . . . . . . . . . . . . . . . . . 11
NobumotoWatanabeandHiroyukiOsada
2.1 CellGrowthSignalingPathway. . . . . . . . . . . . . . . . . . . . . . . . 11
2.2 CellCycleRegulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.3 CellDifferentiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3 Epigenetics. . . . .. . . . .. . . . . .. . . . .. . . . . .. . . . . .. . . . .. . . . . 37
AkihiroItoandMinoruYoshida
3.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.2 HistoneAcetylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.3 HistoneMethylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.4 DNAMethylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.5 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4 ApoptosisandAutophagy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
EtsuTashiro,MitsuhiroKitagawa,andMasayaImoto
4.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.2 ApoptosisModulators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4.3 AutophagyModulators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
vii
viii Contents
5 AdaptiveandInnateImmuneSystems. . . . . . . . . . . . . . . . . . . . . . 115
TakaoKataoka
5.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
5.2 AntigenReceptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
5.3 Perforin/Granzymes. . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . 122
5.4 FasL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
5.5 TLRFamily. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
5.6 NLRFamily. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
5.7 RLRFamily. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.8 CLRFamily. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
5.9 TNFReceptorFamily. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6 BioprobesataGlance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
HiroyukiKoshinoandTakeoUsui
AuthorsandCollaborators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Chapter 1
Trends in Bioprobe Research
HiroyukiOsada
Keywords Screening(cid:129)PPI(cid:129)TargetIdentification(cid:129)CellMorphology(cid:129)Profiling(cid:129)
Proteome(cid:129)AffinityBeads(cid:129)DARTS(cid:129)CETSA
1.1 Introduction
Theterm“bioprobe”wasusedforthefirsttimeinthepreviousversionofthisbook
[1]. Bioprobes were defined as small molecules which are useful not only for
biochemical research but also for the source of drug candidates with diverse
activities. Historically, microbial metabolites have been quite useful not only as
medicines but also as bioprobes. As a typical example, penicillin was originally
discoveredasatherapeuticagentagainstbacterialinfectiousdiseases.Afterwards,
researchonthemodeofactionofpenicillingaveustheinsightonthestructureand
the biosynthesizing mechanism of the bacterial cell wall [2, 3]. Another well-
known example is that the reverse transcriptase was discovered by the aid of
daunomycin and actinomycin D. In the twentieth century, the central dogma of
molecularbiologysuggestedthatthegeneticinformationflowsfromDNAtoRNA,
and then finally to protein. However, some tumor viruses transcribe DNA from
RNA by reverse transcriptase which is RNA-dependent DNA polymerase
[4, 5]. Specific inhibitors of DNA and RNA synthesis were used to prove the
templateofthepolymerase.
Based on the above mentioned background, we especially focused on the
microbial metabolites in the first version of Bioprobes and attempted to merge
theinterestsoforganicchemistswhoareinterestedintheisolationofinhibitors,and
molecularbiologistswhoareinterestedinthebiologicalfunctionofenzymes,into
the same stream. The attempt was fulfilled as chemical biology. There are many
success stories to prove the usefulness of microbial metabolites [6, 7]. The most
recent glorious exampleis the development ofthe antiparasitic agentsavermectin
and ivermectin by Omura and Campbell [8]. Unfortunately, pharmaceutical
H.Osada(*)
ChemicalBiologyResearchGroup,RIKENCenterforSustainableResourceScience,
2-1Hirosawa,Wako,Saitama351-0198,Japan
e-mail:[email protected]
©SpringerJapanKK2017 1
H.Osada(ed.),Bioprobes,DOI10.1007/978-4-431-56529-1_1
2 H.Osada
companies gave up natural products screening and shifted to high-throughput
screening using small molecules synthesized by combinatorial chemistry. How-
ever,theNobelPrize2015recalledtheusefulnessofthenaturalproducts.
1.2 Screening of New Bioactive Compounds
1.2.1 Cell-Based Screening
To screen for bioactive small molecules, it is essential to establish a simple rapid
bioassay system. Screening systems are roughly categorized into enzyme-, cell-,
andanimal-basedassays,eachwithitsownprosandcons.Whenconstructinganew
bioassay system, we make a point of verifying whether it meets the 4S (simple,
speedy,sensitive,andspecific)and1D(distinctive)criteria[9].Inanotherwords,a
good screening system provides an easy-to-follow assay protocol, a quick turn-
around time for assay results, high sensitivity, high specificity, and easy interpre-
tation of assay results. Regarding these criteria, enzyme-based assay is easy to
transformtoahigh-throughputscreeningsystem.Conversely,cell-basedassaysare
generallytimeconsumingandlaborious,therefore,cell-basedscreeningisdifficult
to transform to high-throughput screening. However, phenotypic screening by
usingcellsissuitablefornaturalproductsscreening[10].
Recently, high-content screening using mammalian cells is becoming popular
because automatic microscope systems such as IN Cell Analyzer (GE Healthcare
Life Science), Cell Voyager (Yokogawa Electric Corporation), Cell Insight
(Thermo Fisher Science), Opera Phenix (Perkin Elmer), etc., are commercially
available. With these visualization tools, it is now possible to analyze qualitative
and quantitative cellular phenotypes such as morphological changes in a high-
throughputmanner.
Moreinterestingly,wefoundthatmammaliancancercellsalsoshowedspecific
morphological changes in response to the mode of action of a drug. Thus, we
constructedaphenotypicscreeningsystembasedonthehigh-contentcellmorpho-
logicaldatabase“MorphoBase”[11].Afterseveralexperimentsusingvariouscell
lines,wedecidedtousesrcts-NRK[12]andHeLacellsfortheassaysystem.Both
cell lines exhibited a variety of changes in shape in response to various small
molecules. To eliminate bias of individuals during observation, we performed
automatedobservationsusingINCellAnalyzer.Specifically,wedesignedahigh-
content imaging algorithm that segments an individual cell into three fragments
(cell, nucleus, and granule) and quantified 12 morphological parameters. This
system enables us to classify the mode of action of typical anticancer drugs and
profile various molecular targets of bioactive compounds and even crude extracts
[13,14].
