Dipeptidyl Aminopeptidases: Basic Science and Clinical Applications

228 Pages·2006·3.308 MB·English

by  Inger Brandt, Anne-Marie Lambeir, Marie-Berthe Maes, Simon Scharpé, Ingrid De Meester (auth.), Uwe Lendeckel, Dirk Reinhold, Ute Bank (eds.)

Uwe Lendeckel
Dirk Reinhold
Ute Bank
Editors
Dipeptidyl Aminopeptidases
Basic Science and Clinical Applications
With77Figures
iii
UweLendeckel
DirkReinhold
UteBank
Otto-von-GuerickeUniversity
Magdeburg,Germany
[email protected]
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ISBN10:0-387-29058-3 e-ISBN0-387-32824-6
ISBN13:978-0387-29058-4
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TOPIC I
STRUCTURE AND FUNCTION
1
CHAPTER 1
PEPTIDE SUBSTRATES OF DIPEPTIDYL PEPTIDASES
Inger Brandt, Anne-Marie Lambeir, Marie-Berthe Maes, Simon Scharpe´
and Ingrid De Meester
LaboratoryofMedicalBiochemistry,DepartmentofPharmaceuticalSciences,
UniversityofAntwerp,Universiteitsplein1,B-2610Antwerp,Belgium
1. INTRODUCTION
DPP IV has been attributed a large array of functions, some of which are mediated by
itsexopeptidaseactivity.AlthoughitonlyremovestwoaminoacidresiduesattheN-terminus
ofthepeptide,thiscleavagecaninactivateormodifytheactivityofregulatorypeptides,pep-
tidehormones,chemokinesandneuropeptides.SeveralexcellentDPPIVsubstrateswithhigh
specificityconstantswereidentifiedbytheinvitrokineticstudyofthetruncationofbioactive
peptidesbyDPPIV.Invivostudiese.g.withDPPIVnegativeanimalsandinvivoinhibition
experimentscouldenlightenusonthephysiologicalrelevanceofthesetruncations.
TheDPPIVmediatedtruncationofbioactivepeptideshasbeenreviewedonseveralocca-
sions(Mentleinetal1999,Lambeiretal2003,DeMeesteretal2000and2003).Herewewant
(1)topresentsomedataonanovelDPPIVsubstrate,(2)toreviewsomeoftherecentliterature
onknownsubstratesand(3)todiscussthepossibletruncationofknownDPPIVsubstratesby
relateddipeptidylpeptidases.
2. INVITROTRUNCATIONOFBIOACTIVEPEPTIDESBYDPPIV
2.1. OverviewofSpecificityConstants
Figure 1 represents the specificity constants k /K of the cleavage of natural peptides
cat m
by DPP IV (Lambeir et al 2001 and 2002). All data were obtained using the same batch of
human natural DPP IV, purified from seminal plasma. The experiments were performed in
identicalcircumstancesofincubationandanalysis,exceptforthedataonhemorphin-7which
wereobtainedfromCohenetal(2004).Thefullnamesofthepeptidesaregivenfurtherinthis
paper.
From the in vitro studies several conclusions can be drawn. Not only the amino acid
sequencesurroundingthescissilebond,butalsothespecificstructuralfeaturesofthesubstrate
3
4 INGERBRANDTetal
LD78beta
PACAP27
GLP-2 LD78beta
PACAP38 PACAP27
RANTES
GLP-2
GIP
PACAP38
Eotaxin
RANTES
SP(3-11)
GIP
SP(1-11)
Eotaxin
Hemorphin-7
GLP-1 SP(3-11)
BNP SP(1-11)
Mig Hemorphin-7
MDC (3-69) GLP-1
IP-10 0 0,1 0,2
NPY
I-TAC
GRP
MDC(1-69)
SDF-1alfa
0 1 2 3 4 5
k /K (106M−1s−1)
cat m
Figure1. Thespecificityconstantsk /K ofthecleavageofnaturalpeptidesbyDPPIV.
cat m
areofimportance(Ku¨hn-Wacheetal2003).Thereseemstobenolinkbetweenpeptidelength
up to 125 amino acids and the kinetic constants. A free and flexible N-terminus is required
(Lambeiretal2003).
2.2. BrainNatriureticPeptide32(BNP-32)isCleavedEfficientlybyDPPIV
The natriuretic peptide family consists of four members which all have a characteristic
17aminoacidresidueringformedbyanintramoleculardisulfidebridgebetweentwocysteine
residues. The heart secretes two cardiac natriuretic peptides, atrial natriuretic peptide (ANP)
andbrainnatriureticpeptide(BNP),whichareinvolvedintheregulationofextracellularfluid
andbloodpressurehomeostasis.
Beforetheyarereleasedintothecirculationtheyeachexistasahighmolecularweightpro-
hormone(Vanderheydenetal2004).BNPprohormone,proBNP,consistsof108aminoacidsand
isproducedmainlybythecardiacmyocytes.ThebiologicallyactiveBNP-32isseparatedfrom
theN-terminalpartoftheprohormone,calledNT-proBNP.ThephysiologicaleffectsofBNP-32
are caused by its binding to the natriuretic peptide receptor type A that is coupled to cGMP.
Theeffectsthusproducedincludediuresis,vasodilatation,inhibitionofreninandaldosterone
productionandofcardiacandvascularmyocytegrowth(Hall2004).Measurementsofblood
BNP-32 proved useful in identifying patients with left ventricular dysfunction. In addition,
nesiritide,arecombinantformofhumanBNP,hasbeenapprovedforthetreatmentofacutely
decompensatedheartfailure(Munagalaetal2004).
PEPTIDESUBSTRATESOFDIPEPTIDYLPEPTIDASES 5
BNP-32 turned out to be a very good substrate for DPP IV having a higher specificity
constant (k /K 0.37∗106 M−1s−1) than several known physiological substrates of the en-
cat m
zyme.Shimizuetalreportedthereleaseof2amino-acids(Ser-Pro)fromtheaminoterminus
ofBNP(1-32)uponincubationinwholeblood.Intheirassaysytem,theimmunoreactivityof
BNP was preserved. No other fragments of BNP were observed after 24-h incubation. The
authors did not identify the protease responsible for the observed cleavage. (Shimizu et al
2002)WhetherthecleavageofBNP-32byDPPIVhasphysiologicalimplicationsremainsto
beelucidated.
3. INVIVORELEVANCEOFPEPTIDETRUNCATIONBYDPPIV
InvitroalargenumberofsubstratesarecleavedbyDPPIV.Todateonlyafewofthem
havebeenproventobeendogenoussubstratesuponthefollowingcriteria:(1)cleavageoccurs
invitroatthepenultimateresidue,and(2)cleavageproductsareobservedinvivobutareabsent
inthepresenceofaselectiveinhibitororinDPPIV−/−mice(Weber2004).Evenifinvivo
truncationbyDPPIVcanbedemonstrated,therelevanceoftheDPPIVcleavageandeventually
itsinhibition,shouldbestudiedthoroughlybeforeconclusionsaredrawn(Mentleinetal2003).
Forsomeknown(e.g.substanceP(Ahmadetal1992))orputative(e.g.procalcitonin(Wrenger
etal2002))invivosubstrates,nonewdatabecameavailablesincethepublicationofourmost
recentreview(Lambeiretal2003).
3.1. ThePancreaticPolypeptideFamily
3.1.1. NeuropeptideY
NeuropeptideY(NPY)displaysalargearrayoffunctions;itisinvolvedinthecontrolof
energy homeostasis, blood pressure, angiogenesis, immune responses and behavioural stress
responses.Ithasatleast5receptors,Y1,Y2,Y4,Y5andY6(forrecentreviewseeGroneberg
etal2004).
The N-terminally truncated form of NPY(3-36), the NPY fragment created by DPP IV,
hasconsiderablyreducedactivityfortheY1receptor,butremainsanagonistfortheY2and
Y5receptorsubtypes.Invitrostimulationofhumanumbilicalendothelialcellsbyacocktail
ofinflammatorycytokines(LPS,TNF-αandIFN-γnotonlyincreasedtheNPYandDPPIV
synthesis,butalsochangedtheY2andY5receptorexpressions.TheNPYsystemthusseems
veryflexibleduringtheprocessofaninflammatoryresponse(Silvaetal2003).
TheNPYY1receptormediatesanxiolytic-likeeffectsofNPY.Karletal(2003)admin-
istered intracerebroventricularly different doses of NPY in wild-type F344 rats and mutants
lackingendogenousDPPIV-likeactivity.IntheDPPIV-negativesubstrainsNPYadministra-
tionwasmorepotentinexertinganxiolytic-likeandsedative-likeeffects.
Theabovementionedandotherrecentstudies(Lietal2005,chapter14inthisvolume;
Kitlinskaetal2003)provideevidencethatNPY-mediatedeffectsintheperipheryaswellasin
thecentralnervoussystemaremodulatedbyDPPIV-likeenzymaticactivity.Whetherlong-term
inhibitionofDPPIVleadstoincreasedNPYactivityintreatedpatients,istobeawaited.
Atthemoment,noNPY-basedmoleculesareinclinicaluse,sothehumanNPYsystem
remainslargelyelusive(ChronwallandZukowska,2004).Anumberofrecentstudiesanalyzing
NPYandYreceptoroverexpressingandknockoutanimalmodelsmaygivesomehintsonthe
specificfunctionsofsignallingviathedifferentreceptors(Linetal2004).
6 INGERBRANDTetal
3.1.2. PeptideYY(PYY)
Another member of the pancreatic polypeptide family is the gut hormone peptide YY
for which the L-cells of the gastro-intestinal tract are the major source. PYY is produced by
endocrinecellsofthesmallintestineandthecolon.Postprandiallyitisreleasedasapeptide
hormone.Itcirculatesinthebloodandinhibitsseveralgastro-intestinalfunctions,forexample
gastricacidrelease(Mentlein1999).TherearetwoendogenousformsofpeptideYY,PYY(1-36)
andPYY(3-36),thelatterisproducedbyDPPIV.TheliberationoftheN-terminaldipeptidefrom
peptideYY,convertsitfromavasoconstrictiveandinsulinotrophicfactorintoapotentsecretion
inhibitor (Keire et al 2002). In humans, peripheral infusion of PYY(3-36) at a dose which
producednormalpostprandialconcentrationssignificantlydecreasedappetiteandreducedfood
intake(BatterhamandBloom2003).Theseresultsindicatethattheregulationoffoodintake
doesnotoccurviatheY1receptor.
3.2. OpioidPeptides
3.2.1. Hemorphins
Thehemorphinsareafamilyofsmallbioactivepeptidesderivedfromhemoglobinhydrol-
ysis throughayetunknown mechanism. Inadditionto theiropioid likeactivityanumberof
otherbiologicalactivitiesofhemorphinswereobserved,includinginteractionwiththerenin-
angiotensinsystem.Althoughthepresenceinphysiologicalorpathologicalconditionsaswell
aspotentialactivitiesofhemorphinshavebeenextensivelystudied,onlyfewreportsarerelated
totheirmetabolism.CathepsinDhasbeenhypothesizedtobeinvolvedinthefirststepoftheir
generation,withthereleaseofLVV-Hemorphin-7(LVVH7;sequence:LVVYPWTQRF)and
VVH7(Fruitier-Arnaudinetal2003a).LVVH7incubatedwithmicrosomalfractiongaverise
toHemorphin-7,whichcouldbedegradedbytheDPPIVpresentinthisfraction.Asubsequent
kineticstudywithpurifiedDPPIVpermittedtoconfirmthatH7isverygoodsubstrateforthe
enzyme(Cohenetal2004).
In vivo relevance for a role for DPP IV in the hemorphin-7 metabolism has not been
reportedyet.Intriguingly,significantlylowerlevelsofserumhemorphin-7peptidesarefound
indiabeticpatients.Itishypothesizedthattheinvivoformationofhemorphinsresultingfrom
hemoglobinhydrolysisisalteredbyglycosylation(Fruitier-Arnaudinetal2003b).Giventhe
manyeffectsofhemorphinsinvivo,onecanwonder1)whetherthesignificantlyalteredlevels
indiabeticscontributetothesymptomatologyand2)whetherinhibitionofDPPIV‘rescues’
partofthehemorphinactivities.
3.2.2. Endomorphins
Endomorphin-1(EN1)isanendogenousmammalianopioidagonistwithhighaffinityand
selectivityforµ-opioidreceptors.Duringtestsinabuccaldeliverysystem,EN1wasshownto
beveryunstable.DiprotinA,adipeptidylpeptidaseinhibitorprovidedsignificantinhibitionof
itsdegradation(Birdetal2001).
Anotherendogenousligandfortheµ-opioidreceptor,endomorphin-2,wasincubatedwith
mouse brain synaptic membranes in order to study its metabolism. The degradation of the
substrate was inhibited by the use of DPP IV inhibitors, such as diprotin A and B. Further-
more,purifiedDPPIVhydrolyzedendomorphin-2.Thesefindingsaddfurtherevidenceforthe
PEPTIDESUBSTRATESOFDIPEPTIDYLPEPTIDASES 7
proposedroleofDPPIVinthemetabolismofendomorphin-2.Theintracerebroventricularly
injectionofdiprotinAtogetherwithendomorphin-2inmiceenhancedendomorphin-2-induced
antinociception(Sakuradaetal2003),suggestingarolefordiprotinA-sensitiveDPPactivity.
Repetitionofthiskindofexperimentswithhighlyselective,potentinhibitorsofthedifferent
members of the dipeptidyl peptidase family would greatly enhance our understanding of the
centralmetabolismoftheendomorphins.
3.3. PACAP/GlucagonFamilyofPeptides
3.3.1. PituitaryAdenylateCyclaseActivatingPolypeptide(PACAP)
PACAPhasbeenattributedanumberoffunctions,suchasglucosecontrol,lipidmetabolism
andadaptivethermogenesis.AnintactN-terminusisrequiredforitsbioactivity.PACAPisa
38aminoacidpeptidewithaC-terminallytruncated27-residuesplicingvariantPACAP27.In
vitroadifferentialprocessingofPACAP27andPACAP38hasbeenobserved.PACAP38has
a higher affinity for DPP IV and a higher turnover rate (Lambeir et al 2001). Using tandem
mass spectrometry Zhu et al (2003) measured the intact PACAP(1-38) and the metabolite
PACAP(3-38) in plasma of wild type and DPP IV deficient C57Bl/6 mice after intravenous
dosingwithsupraphysiologicallevelsoftheneuropeptide.Theyfoundthatinwildtypemice
PACAP38wasrapidlydegradedwithconcomitantformationoftheinactivePACAP(3-38).This
metabolitewasvirtuallyabsentinDPPIV−/−miceandtheclearanceofintactPACAP38from
thecirculationwassignificantlylowerintheseanimals.Inasimilarexperimentmonitoringthe
catabolismofexogenousPACAP27inwildtypemice,therewasnoevidencefortheformation
ofthecorrespondingPACAP(3-27)metabolite.RecentlyAhre´nandHughes(2005)wereable
toshowthatinmiceDPPIVinhibitionbyValine-pyrrolidideaugmentedtheinsulinresponses
tointravenousadministrationofPACAP38.Fromtheseresultstheyconcludedthatprevention
ofinactivationofthispeptideaugmentsisletfunction.
3.3.2. VasoactiveIntestinalPeptide(VIP)
VIPisaprominentneuropeptidewithawidespectrumofbiologicalactivitiesinmammals,
suchassuppressionofinflammationandperceptionofpain.Itisreleasedafterstimulationof
theparasympatheticnervesinthepancreasandincreasesinsulinandglucagonsecretionina
glucose-dependentmanner.Asresidues1and5areinvolvedinVIPbindingandresidue3is
involved in VIP binding and VIP structure (Nicole et al 2000), the sequential truncation of
thispeptidebyDPPIVislikelytohaveanimpactonitsbioactivity.Sofarthereisnoinvivo
evidenceforrelevanceofDPPIVmediatedtruncationofVIP.
3.3.3. Glucagon-LikePeptide1(GLP-1)andGlucose-DependentInsulinotropicPeptide
(GastricInhibitoryPeptide,GIP)
TheincretinsGIPandGLP-1aregut-derivedfactorsthatpotentiatepostprandialinsulin
secretion and glucose clearance; they are released in response to the presence of food in the
intestinal lumen. Both are subjected to DPP IV mediated N-terminal cleavage which causes
acompletelossofinsulinotropicactionofthesehormonesastheseresiduesareimportantin
receptoractivation.GLP-1andGIParethebeststudiedinvivosubstratesofDPPIVandthey
aresubjectofanumberofexcellentrecentreviews(Ahren2003;Drucker2003;Kieffer2004).
8 INGERBRANDTetal
Theinvivostabilizationoftheseincretinsisnowwidelyacceptedasanewapproachin
antidiabetictreatment.InadditiontotheuseofDPPIVinhibitors,stableincretinanalogueshave
beendevelopedtotreatdiabetes.Leeetal(2005)developedaspecificDPPIVresistantmono-
polyethyleneglycol-conjugatedGLP-1preparationwithagreatlyimprovedpharmacological
profile. In a recent study by Green et al (2004a) alanine residues in both GLP-1 (Ala8) and
GIP (Ala2) were substituted for a larger amino acid structure, 2-aminobutyric acid (Abu).
(Abu8)GLP-1 turned out to be profoundly resistant to DPP IV degradation, but surprisingly
(Abu2)GIPonlyhadamoderatelyprolongedhalf-lifecomparedtonativeGIP.Thesamegroup
ofresearchersalsocombinedthisGLP-1modificationwiththeattachmentofpalmitoylmoieties
to its epsilon-amino group in the side chain of the Lys26 residu. They indicate a potential
usefulnessoffattyacidslinkedanaloguesofGLP-1,butindicatetheimportanceofthechain
lengthforpeptidekineticsandbioavailability(Greenetal2004b).TheN-terminalmodification
ofHis7inGLP-1alsogeneratesDPPIVstableanalogues(Greenetal2004c).Islametal(2005)
transfectedratinsulomacellswithaplasmidencodingamutatedformofGLP-1(GLP-1-Gly8).
The cells produced and stored GLP-1-Gly8, moreover they displayed an enhanced glucose
responsiveness.TheresultsobtainedwithstabilizedGLPderivativesaswellaswithDPPIV
inhibitors(HolstandDeacon2004;Ahrenetal2004)allsupportacrucialroleforDPPIVin
theinvivometabolismofthisincretin.
3.3.4. Glucagon-LikePeptide2(GLP-2)
Nexttoitsimportantroleinintestinalmucosaregenerationandpermeability,GLP-2aug-
ments intestinal hexose transport and reduces gastric acid secretion and gastric emptying. In
invitroexperimentsDPPIV-mediatedtruncationofGLP-2turnedouttobealmosttentimes
slowerthanthatofGLP-1(Lambeiretal2002).InanelegantstudyonGLP-2degradationin
humans,Hartmannetal.2000identifiedGLP-2(3-33)astheonlymetaboliteformed.However,
1hraftersubcutaneousinjection,still69%oftheGLP-2wasintact,confirmingasignificantly
slower cleavage than GLP-1. Nothwithstanding this relatively slow truncation, preclinical in
vivostudiesonthetherapeuticpotentialofGLP-2,useDPPIV-resistantanaloguesofthepep-
tidee.g.Teduglutide,(Gly2)GLP-2(Boothetal2004).Severalinvivoexperimentshavebeen
conductedinordertotestthehypothesisthatthedegradingactivityofDPPIVwouldbelim-
iting for the effects of GLP-2. Hartmann et al (2000) found that the GLP-2 degradation in
Wistar rats could be reduced by Valine-Pyrrolidide and that DPP IV inhibition enhanced the
insulinotropiceffectofGLP-2inbothratsandandC57BLmice.AdministrationofratGLP-2
to DPP IV deficient rats was found to be associated with markedly increased bioactivity of
rat GLP-2 resulting in an increase in small bowl weight (Drucker et al 1997). Tavares et al
(2000)determinedtheimportanceofDPPIVintheregulationofcirculatingGLP-2levelsin
vivobyinjectingGLP-2ortheDPPIV-resistantanalogue[Gly2]GLP-2innormalandinDPP
IV-negativerats.TheyfoundthatnormalratsshowedasteadydegradationofGLP-2(1-33)to
GLP-2(3-33) over time. In contrast, little or no conversion of GLP-2(1-33) was observed in
DPP IV negative rats. [Gly2]GLP-2 also remained intact in wild type rats. GLP-2 is known
to enhance the repair of damaged intestinal tissue, however Geier et al (2005) demonstrated
that DPP IV(−/−) mice did not display an increased resistance to or an enhanced recov-
eryfromdextransulphatesodium-inducedcolitiscomparedtoDPPIV(+/+).Astheyfound
thatDPPIV(−/−)micestillpossessedsignificantplasmalevelsofDPPIV-likeactivity,they
hypothesized that other DPP IV family members might also be involved in the cleavage of
GLP-2.