0031-6997/93/4501-0087$03.00/0
`PHARMACOLOGICAL
`REVIEWS
`by The American
`Copyright
`© 1993
`
`Society
`
`for Pharmacology
`
`and
`
`Experimental
`
`Therapeutics
`
`Neutral
`
`Endopeptidase
`Experimental
`
`Inhibition,
`Structure,
`24.11:
`and
`Clinical
`Pharmacology
`
`Vol.
`Printed
`
`45, No.
`in U.S.A.
`
`1
`
`and
`
`BERNARD
`
`P.
`
`ROQUES,
`
`FLORENCE
`
`NOBLE,
`
`DAUGE,
`VALERIE
`ANN
`BEAUMONT
`
`MARIE-CLAUDE
`
`FOURNIE-ZALUSKI,
`
`AND
`
`Pharmacochimie
`1500
`
`Mol#{233}culaire et Structuro,ie
`Centre National
`de La Recherche
`
`(Unite
`266
`Scientifique,
`
`Medicale
`et de Ia Recherche
`de Ia Sante
`National
`Institut
`4 Avenue
`de
`I’Obseruatoire
`75270,
`Facult#{233} de Pharmo.cie,
`
`- Unite
`Pans
`
`de Recherche
`C#{233}dex06,
`
`Associ#{233}e D
`France
`
`Downloaded from
`
`88
`89
`90
`91
`93
`93
`93
`95
`95
`96
`96
`
`96
`97
`97
`98
`99
`100
`100
`
`a pharmacologically
`24.11
`purification
`and
`24.11
`mechanism
`action
`endopeptidase
`24.11
`inhibitors
`of neutral
`
`and
`neutral
`selective
`
`of
`
`multisubstrate-metabolizing
`relevant
`of neutral
`endopeptidase
`assay
`neutral
`endopeptidase
`of
`and
`aminopeptidase
`endopeptidase
`
`24.11
`N inhibitors
`24.11
`
`enzyme
`activity
`
`.
`
`24.11
`
`VI.
`
`VIII.
`
`24.11
`
`and
`
`aminopeptidase
`
`N and
`
`of
`
`24.11
`sequencing
`
`(common
`24.11
`
`acute
`
`lymphoblastic
`
`leukemia
`
`antigen)
`
`gene
`
`of neutral
`
`endopeptidase
`
`24.11
`
`in spinal
`
`cord
`
`and
`
`rats
`localization
`distribution
`
`of neutral
`of neutral
`
`endopeptidase
`endopeptidase
`
`in brain
`24.11
`24.11
`in the
`central
`
`tissues
`
`100
`. 101
`
`.
`
`.
`
`at Virginia Commonwealth Univ Tompkins McCaw Lib/Acq Srv on July 27, 2020
`
`103
`
`105
`105
`
`106
`107
`
`107
`109
`
`109
`
`112
`
`114
`115
`117
`. 118
`
`118
`
`Introduction
`I.
`endopeptidase
`II. Neutral
`endopeptidase
`III.
`Neutral
`Iv.
`specificity
`Substrate
`selective
`of
`V. Design
`of
`A. Development
`inhibitors
`1. Thiol
`inhibitors
`2. Carboxyl
`inhibitors
`3. Bidentate
`inhibitors
`4.
`Phosphorus-containing
`B. Aminopeptidase
`inhibitors
`peptidase
`dipeptidyl
`N and
`Development
`of mixed
`inhibitors
`of neutral
`endopeptidase
`neutral
`endopeptidase
`and
`angiotensin-converting
`enzyme
`VII. Molecular
`biology
`of neutral
`endopeptidase
`A. Complementary
`DNA
`cloning
`and
`B.
`Site-directed
`mutagenesis
`C. Human
`neutral
`endopeptidase
`of neutral
`Localization
`endopeptidase
`nervous
`A. Central
`system
`1. Distribution
`and
`ontogenesis
`meninges
`of humans
`and
`2. Lesion
`studies
`and
`cellular
`3. Cellular
`and
`subcellular
`nervous
`system
`In
`situ
`hybridization
`nervous
`system
`Localization
`of
`Pre-
`and
`postnatal
`tissues
`endopeptidase
`D. Neutral
`of neutral
`IX. Modulation
`the
`turnover
`at
`protein
`Pharmacological
`studies
`A.
`In vitro
`in vivo
`and
`24.11
`aminopeptidase
`Limited
`antinociceptive
`inhibitors
`Enhanced
`enzymes
`antinociception
`spinal
`Inhibitor-induced
`D.
`of
`selective
`and mixed
`E. Analgesic
`effects
`F.
`Interactions
`between
`the
`cholecystokinin
`Tolerance,
`dependence,
`and
`side
`G.
`effects
`endopeptidase
`24.11
`aminopeptidase
`
`4.
`
`of neutral
`
`endopeptidase
`
`24.11 messenger
`
`RNA
`
`in the
`
`central
`
`neutral
`
`endopeptidase
`ontogeny
`of neutral
`
`in peripheral
`24.11
`endopeptidase
`
`24.11
`endopeptidase
`cell
`surface
`
`fluids
`in physiological
`24.11
`expression
`
`and
`
`studies
`
`of neuropeptide
`
`degradation
`
`tissues
`24.11
`in central
`
`and
`
`peripheral
`
`possible
`
`role
`
`as
`
`regulator
`
`of
`
`by neutral
`
`endopeptidase
`
`and
`
`N
`effects
`
`of
`
`selective
`
`neutral
`
`endopeptidase
`
`24.11
`
`or
`
`aminopeptidase
`
`N
`
`analgesic
`
`responses
`
`induced
`
`by mixed
`
`inhibitors
`
`of enkephalin-degrading
`
`B.
`C.
`
`B.
`
`C.
`
`x.
`
`pain
`in chronic
`inhibitors
`and
`enkephalin
`systems
`and mixed
`selective
`
`of
`
`in the
`inhibitors
`
`of pain
`
`.
`
`.
`
`.
`
`control
`of neutral
`
`and
`
`N
`
`87
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1013, p. 001
`
`

`

`88
`
`ROQUES
`
`ET
`
`AL.
`
`on morphine
`inhibitors
`of peptidase
`Effect
`H.
`endopeptidase
`of neutral
`I. Behavioral
`effects
`N inhibitors
`24.11/aminopeptidase
`of enkephalin-degrading
`effects
`J. Gastrointestinal
`agents
`use
`as
`antidiarrheal
`in airways
`endopeptidase
`Role
`of neutral
`24.11-induced
`Inhibition
`of neutral
`endopeptidase
`and
`clinical
`implications
`pharmacological
`Possible
`the
`common
`acute
`lymphoblastic
`of
`24.11
`on
`lymphoblastic
`cells
`endopeptidase
`XI. Conclusions
`perspectives
`XII.
`References
`
`withdrawal
`24.1 1 and
`
`neutral
`
`endopeptidase
`
`24.11
`
`enzyme
`
`inhibitors
`
`and
`
`their
`
`clinical
`
`inactivation
`
`of atrial
`
`natriuretic
`
`peptide:
`
`leukemia
`
`antigen
`
`identified
`
`as neutral
`
`121
`
`121
`
`125
`126
`
`127
`
`131
`132
`133
`
`K.
`L.
`
`M.
`
`roles
`
`and
`
`I.
`
`Introduction
`
`in membrane-bound
`interest
`the
`1970s
`following
`end
`of
`of ACE*
`(peptididyl-dipeptidase,
`enzyme
`implicated
`in
`the
`from
`angiotensin
`I, produced
`(Ondetti
`et
`1977)
`and
`al.,
`of
`another
`membrane-bound
`involved
`in
`inactivation
`in
`enkephalins
`brain,
`induced
`al.,
`et
`(Roques
`1980).
`Given
`this
`activity,
`latter
`enzyme
`(Malfroy
`et
`al.,
`1978b)
`to
`identical
`be
`well-characterized
`present
`in
`the
`of
`kidney
`
`the
`
`II
`
`the
`the
`
`the
`
`Considerable
`emerged
`at
`that
`inhibition
`3.4.15.1),
`the
`angiotensin
`effects
`tensive
`inhibition
`that
`lopeptidase,
`peptide
`sponses
`metabolizing
`“enkephalinase”
`shown
`was
`later
`an
`already
`3.4.24d
`1),
`tidase
`known
`to
`be
`the
`proximal
`tubules
`1974a,b).
`of metabolizing
`As
`studies
`that,
`as was
`came
`apparent
`peptidases
`substrates,
`these
`the
`concept
`of “one
`peptidase:
`be
`realized
`(Schwartz,
`1983;
`
`the
`
`peptidases
`discovery
`E.C.
`of
`
`formation
`antihyper-
`3 years
`later
`Zn metal-
`the
`opioid
`of
`analgesic
`re-
`enkephalin-
`designated
`some
`time
`NEP
`(E.C.
`Zn metallopep-
`border
`cells
`and
`Kenny,
`
`its
`was
`and
`
`with
`
`brush
`(Kerr
`
`of
`
`progressed,
`enzymes
`for
`their
`the
`case
`distributed,
`were
`widely
`not
`one
`substrate”
`was
`Kenny,
`1986).
`instance,
`
`it be-
`peptide
`and
`to
`
`For
`
`angiotenSin-conVerting
`
`ACE,
`ANP,
`leukemia
`
`neutral
`acute
`dipep-
`
`system;
`
`peptide;
`
`rat;
`
`TLN,
`tegmental
`natriuretic
`lymphoblastic
`
`thermo-
`area;
`factor;
`leu-
`
`*5 Abbreviations:
`NEP,
`enzyme;
`endopeptidase;
`common
`CALLA,
`peptide;
`natniuretic
`atnial
`antigen;
`APN,
`lymphoblastic
`N; DAP,
`aminopeptidase
`SP,
`P;
`tidylaminopeptidase;
`i.c.v.,
`intracerebroventricular;
`substance
`CCK,
`FTI,
`cholecystokinin;
`N-([fluoresceinyl)-N’-[1-(6-(3-mercapto-
`2.benzyl.1-oxopropyl)-amino-1-hexylj-thiocarbamide;
`VTA,
`lysin(cid:1)CNS,
`central
`nervous
`Ventral
`CGRP,
`atrial
`ANF,
`gene-related
`calcitonin
`acute
`ALL,
`SHR,
`hypertensive
`spontaneously
`HACBO-Gly,
`N-[(2RS)-4-(hydroxyamino)-1,4-dioxo-2-(phen-
`kemia;
`DAMGO,
`Try-D.Ala-Gly-(Me)Phe-Gly.ol;
`ylmethyl)butyhj-glycine;
`DPDPE,
`Tyr-D.P#{233}n-Gly-Phe-D.P(cid:1)n;
`DSTBULET,
`Tyr-D.Ser(O-ter-
`BUBU,
`tiobutyl)-Gly-Phe-Leu-Thr;
`Tyr-D.Ser(O-tertiobutyl)Ghy-
`DTLET,
`Phe-Leu-Thr(O-tertiobutyl);
`Tyr-D.Thr-Gly-Phe-Leu-
`BDNL,
`Thr-,
`Boc-Tyr(SO3H)Nle-Gly-Trp-Nle-Asp-PheNH2;
`N-[(R,S)-2-benzyl.3[(S)(2-amino-4-methylthio)butyldithioj-1-oxopro-
`[‘ThIIRB 104,
`pylj-L-phenylalanine;
`2-(cid:1)(3-[’25I]iodo-4-hydroxy)-phenyh-
`methyhl-4-N-[3-hydroxyamino-3-oxo-1-(phenyl
`methyl)propyljamino-
`BC
`264,
`Boc-Tyr(SO3H)-gNhe-mGhy-Trp-(NMe)
`4-oxobutanoic
`acid
`RB 38A,
`N-[3(R)hydroxyaminocarbonyl)-2-benzyl-
`Nle-Asp-PheNH2;
`1-oxopropyl)-L-phenyhalanine.
`
`RB
`
`101,
`
`et
`
`was
`NEP
`circulating
`phenson
`Olins
`one
`kemia
`nated
`al.,
`
`of
`
`et
`
`participate
`(Koehn
`Sonnenberg
`also
`NEP
`was
`in
`immunology
`markers
`and
`et
`a!.,
`1975;
`
`the
`clearing
`in
`Ste-
`a!.,
`1987;
`1988;
`et
`al.,
`to be
`shown
`leu-
`and
`desig-
`was
`Letarte
`
`et
`
`to
`found
`recently
`from
`plasma
`ANP
`and
`Kenny,
`1987a;
`al.,
`1989). Moreover,
`used
`the many
`proteins
`research
`as
`lymphocyte
`CALLA
`(CD1O)
`(Greaves
`1988).
`nervous
`in
`both
`that,
`appears
`now
`It
`extracellularly
`degraded
`are
`peptides
`tissue,
`with
`enzymes
`of
`number
`limited
`a
`probably
`extracellular
`these
`of
`Most
`specificities.
`broad
`i.e.,
`integral
`ectoenzymes,
`are
`enzymes
`degrading
`sites
`facing
`active
`have
`their
`that
`proteins
`brane
`Zn metallopeptidases
`and many
`space,
`extracellular
`rela-
`Despite
`their
`al.,
`1987; Maroux,
`et
`(Turner
`in
`vivo
`specificity
`a
`specificities,
`broad
`tively
`both
`the
`distribution
`governed
`achieved,
`to be
`seems
`potential
`substrates.
`of
`and
`that
`of
`a given
`peptidase
`cord,
`the
`enkephalins,
`spinal
`and
`the
`Thus,
`in the
`brain
`with
`opiates,
`interact
`other
`classical
`like morphine
`and
`and
`recently
`cloned
`the
`sites
`such
`as
`different
`binding
`Evans
`et
`al.,
`1992;
`sequenced
`#{244}-opioidreceptor
`(Kieffer
`of pharmacological
`al.,
`1992)
`to produce
`a multiplicity
`et
`the
`and
`euphoria.
`Increasing
`responses,
`such
`as
`analgesia
`their
`of
`levels
`endogenous
`opioid
`peptides
`by
`inhibiting
`inactivating
`enzymes
`was,
`therefore,
`proposed
`as a means
`of eliminating
`or minimizing
`the
`serious
`drawbacks
`(res-
`piratory
`depression,
`constipation,
`tolerance,
`physical,
`psychic
`and
`dependence)
`induced
`chronically
`admin-
`by
`opiates
`istered
`(Roques
`al.,
`These
`effects
`et
`1980).
`are
`thought
`to be
`caused
`by an
`overstimulation
`of opioid
`receptors
`all
`brain
`areas
`in which
`the
`enkephalins
`in
`might
`tonically
`or phasically
`released.
`A similar
`either
`the
`approach
`is now being
`followed
`to potentiate
`diuretic,
`natriuretic,
`and
`vasorelaxant
`effects
`of circulating
`ANP
`new
`with
`the
`aim of obtaining
`antihypertensive
`agents
`(reviewed
`by Roques
`and Beaumont,
`1990).
`The
`literature
`concerning
`these
`subjects
`the
`voluminous
`and
`includes
`reviews
`of
`inhibitors
`for
`Zn metallopeptidases
`ment
`of
`
`and
`
`is
`
`peripheral
`by what
`relatively
`peptide-
`mem-
`the
`
`are
`1987).
`certain
`by
`its
`
`side
`
`be
`
`recent
`various
`
`is
`
`already
`develop-
`(Ro-
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1013, p. 002
`
`

`

`NEUTRAL
`
`ENDOPEPTIDASE
`
`24.11
`
`89
`
`1984; Roques,
`and Cushman,
`Ondetti
`1982a,b;
`et al.,
`ques
`Chipkin,
`1986;
`Fourni#{233}-Zaluski,
`1986;
`and
`Roques
`1985;
`and
`their
`phar-
`and Wyvratt,
`1987;
`Rich,
`1990)
`Thorsett
`effects
`(Roques
`and
`Fourni#{233}-Zaluski,
`1985,
`macological
`et
`al.,
`1985;
`Roques,
`1988a,
`1991;
`Dick-
`1986;
`Schwartz
`Needelman
`et
`al.,
`1989;
`Le Bien
`and Mc-
`enson,
`1986;
`Cormack,
`1989;
`Noble
`al.,
`1992d).
`The
`main
`results
`et
`be
`will
`recalled
`when
`necessary,
`reported
`these
`papers
`in
`but
`the
`present
`review
`is mainly
`devoted
`to the molecular
`the
`biology
`and
`structural
`characterization
`of NEP
`and
`potential
`use
`ofthese
`results
`for designing
`either
`selective
`or mixed
`inhibitors
`of NEP
`and
`APN,
`another
`Zn me-
`talloenzyme
`involved
`in enkephalin
`metabolism,
`or NEP
`at
`and
`ACE.
`We
`have
`also
`looked
`the
`distribution
`tissues.
`role
`NEP
`and
`discussed
`its
`possible
`in
`various
`survey
`an
`Finally,
`have
`provide
`extensive
`to
`we
`and
`of
`the
`in
`actions
`the
`peptidase
`inhibitors
`vivo
`achieve
`used
`show
`how these
`molecules
`can
`be
`(a)
`to
`better
`understanding
`of
`physiological
`functions
`in therapeutics.
`peptidergic
`systems
`
`tried
`
`the
`(b)
`
`and
`
`of
`
`of
`to
`a
`of
`
`II. Neutral
`Pharmacologically
`
`Endopeptidase
`Relevant
`
`metabolizing
`
`A
`
`24.11:
`Multisubstrate-
`Enzyme
`
`the
`they
`a!.,
`ACE
`al.,
`to
`
`that
`
`aL,
`
`of
`
`rapidly
`were
`enkephalins
`observation
`The
`that
`suggested
`homogenates
`tissue
`by
`brain
`graded
`inactivation
`in the
`physiological
`could
`be involved
`tidases
`accounted
`endogenous
`opioid
`peptides.
`This
`of
`these
`only with
`and
`transient
`analgesia
`obtained
`the weak
`doses
`(0.1 mg)
`of
`i.c.v.
`administered
`Met-enkephalin
`mice
`(Belluzi
`et
`al.,
`1976)
`and
`higher
`potency
`the
`enkephalin
`analogues
`protected
`peptidase
`macti-
`from
`by
`vation
`by modifications
`such
`replacement
`of Gly2
`as
`a!.,
`an
`amino
`acid
`of
`the
`dextro
`configuration
`(Pert
`et
`1976),
`amidation,
`or
`reduction
`ofthe
`free
`carboxyl
`group,
`of
`N-methy!ation
`the G!?-Phe4
`bond,
`or
`replacement
`Phe4
`by
`its D-isomer
`(Fourni#{233}-Zaluski
`et
`al.,
`1979).
`cause
`the
`enkephalins
`are
`unable
`enter
`cells,
`to
`of
`detection
`the metabolites
`Tyr,
`Tyr-Gly-Gly,
`and
`Gly
`following
`i.c.v.
`administration
`of
`Leu-enkephalin
`suggested
`that
`the
`peptide
`was
`by
`aminopepti-
`degraded
`dase,
`DAP,
`and
`peptidyl-dipeptidase
`activities
`located
`therefore,
`the
`cell
`surface
`and
`acting,
`as
`ectoenzymes
`(Hambrook
`et
`a!.,
`1976;
`Dupont
`et
`Craves
`1977;
`al.,
`1978).
`low affinity-binding
`a relatively
`The
`existence
`[3H]Leu-enkephalin
`90 nM)
`for
`approximately
`(KD
`from
`the
`receptor-binding
`different
`membranes
`striatal
`was
`subsequently
`attributed
`1978a)
`a!.,
`(Malfroy
`et
`site
`des-
`of a Tyr-Gly-Gly-releasing
`enzyme
`to the
`existence
`its
`and
`al.,
`enkephalinase
`(Malfroy
`et
`1978b),
`ignated
`(1978).
`was
`presence
`in brain
`confirmed
`by Sullivan
`et al.
`was
`not
`In
`addition,
`it was
`shown
`that
`the
`tripeptide
`produced
`by the
`sequential
`action
`of a carboxypeptidase
`The
`(Guyon
`et
`a!.,
`1979).
`Km of
`the
`enkephalins
`enkephalinase
`was
`subsequently
`shown
`to be
`higher
`
`de-
`pep-
`
`for
`high
`in
`of
`
`of
`Be-
`the
`Tyr-
`
`at
`
`et
`
`site
`in rat
`
`for
`(ap-
`
`20 (cid:1)tM)
`1979)
`a!.,
`et
`The
`90 nM).
`in
`enkephalin
`naloxone-reversible
`by
`the
`synthetic
`(Roques
`et
`a!.,
`levels
`in
`enzyme
`et
`a!.,
`1978b)
`particularly
`quantitative
`
`also
`
`cleave
`a!.,
`
`of
`
`et
`
`in
`and
`
`et
`
`common
`
`1979;
`and Altstein,
`(Vogel
`previously
`estimated
`than
`physiological
`relevance
`metabolism
`was
`firmly
`antinociceptive
`inhibitor,
`enkephalinase
`the
`reported
`1980).
`However,
`morphine
`treatment
`after
`chronic
`confirmed
`(Barchas
`was
`not
`when
`enzyme
`levels
`were
`autoradiography
`(De!ay-Goyet
`
`Fourni#{233}-
`(approxi-
`of enkepha-
`established
`properties
`thior-
`in-
`
`meas-
`
`et
`
`et
`
`of
`
`et
`and
`et
`
`an
`
`of
`as
`
`proximately
`Zaluski
`mately
`linase
`the
`by
`elicited
`phan
`crease
`(Ma!froy
`a!.,
`1981),
`ured
`using
`a!.,
`1989a).
`bond
`the Gly3-Phe4
`to
`found
`ACE
`was
`although
`1978);
`however,
`(Erd#{246}set
`enkephalins
`(Swerts
`to
`be
`identical
`suggested
`initially
`were
`1979),
`enkephalinase
`and Marks,
`Benuck
`1979a;
`distinct
`enzymes
`(Swerts
`to
`be
`two
`shown
`were
`The
`enkephalins
`were
`found
`1979).
`Buckett,
`1979b;
`ACE
`(Km approximately
`i0(cid:1)
`for
`low
`affinity
`a
`have
`a major
`role
`for
`this
`enzyme
`in the
`excluding
`thereby
`M),
`these
`peptides.
`However,
`the
`ex-
`in vivo metabolism
`Met-enkephalin-Arg(cid:1)-Phe7,
`could
`tended
`opioid
`peptide,
`Met-enkepha-
`less
`potent
`peptide
`be
`transformed
`to the
`the
`explain
`might
`al.,
`1982).
`This
`lin
`by ACE
`(Benuck
`with
`treated
`elevation
`patients
`often
`reported
`mood
`in
`for
`conforma-
`ACE
`inhibitors.
`Interestingly,
`probably
`dynorphin
`1-13
`tional
`reasons,
`fl-endorphin,
`as we!!
`as
`ACE,
`to a
`and,
`and
`1-17,
`appear
`to be
`resistant
`to NEP,
`The
`coidentity
`APN
`(Turner
`et
`a!.,
`1987).
`lesser
`extent,
`established
`when
`of enkephalinase
`and NEP
`was
`clearly
`the
`two
`enzymes
`were
`found
`be
`immunologically
`in-
`to
`distinguishable
`(Relton
`et
`a!.,
`1983; Matsas
`et
`a!.,
`1983).
`Because
`their
`rather
`homogeneous
`distribution
`ap-
`of
`that
`of
`enkephalins
`or
`opioid
`re-
`parently
`unrelated
`to
`a physiological
`role
`for
`aminopeptidases
`as
`en-
`ceptors,
`kephalin-metabolizing
`enzymes
`was
`initially
`questioned
`Nevertheless,
`two
`aminopepti-
`1981).
`(Schwartz
`et
`a!.,
`sensitivity
`to the
`natural
`inhibi-
`their
`dases,
`differing
`bestatin,
`were
`purified
`(Hersh,
`1981;
`puromycin
`tors
`both
`were
`capable
`of cutting
`the
`Tyr’-
`1983);
`et
`al.,
`Hui,
`of enkephalins.
`Bestatin,
`but
`not
`puromycin,
`bond
`Gly2
`to
`potentiate
`the
`analgesic
`effect
`of
`i.c.v.
`shown
`was
`a!.,
`Met-enkephalin
`(Carenzi
`et
`1981),
`suggest-
`injected
`the
`puromycin-sensitive
`aminopeptidase
`was
`not
`that
`ing
`of
`the
`relevant
`enzyme.
`The
`identity
`the mem-
`brane-bound
`bestatin-sensitive
`aminopeptidase
`and
`APN
`was
`established
`titration
`with
`anti-
`finally
`by
`body
`directed
`toward
`the
`enzyme
`(Matsas
`et
`al.,
`kidney
`1985; Gros
`al.,
`1985).
`The
`preferential
`involvement
`of
`the
`APN
`in
`physiological
`inactivation
`of
`enkephalins
`was
`demonstrated
`by the
`increased
`analgesic
`potency
`inhibitors
`more
`selective
`for APN
`than
`bestatin,
`such
`et
`al.,
`1985a).
`kelatorphan
`(Waksman
`a membrane-bound
`In
`addition
`to these
`two
`enzymes,
`DAP
`was
`identified
`in rat
`brain
`(Gorenstein
`and
`Snyder,
`1979)
`and
`purified
`from pig
`kidney
`(Ch#{233}rotet a!.,
`1986a).
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1013, p. 003
`
`

`

`90
`
`ROQUES
`
`ET
`
`AL.
`
`Selected
`
`mixed
`
`enzyme
`
`TABLE
`inhibitors
`
`1
`of NEP/APN
`
`or NEP/ACE
`
`L7CdIRLIbIUNIPTM!NSrND1ACE
`
`#{149}O0
`II
`HN-C-CH,
`
`CH,
`I
`-CH-CONH-CH.C00
`
`CH,
`I
`
`K#{233}latorphan
`
`K1 (nM)
`
`INEP
`
`1.8
`
`APNI
`
`380
`
`#{149}?(cid:1):j(cid:1) (cid:1):H(cid:1)d(cid:1)
`
`HN-C-CH2-CH-CONH.CH-C00
`
`.00
`
`I
`
`HN
`
`II
`-C.CH2
`
`CH4
`
`I
`-CH
`
`- NHCO-C}12
`
`CH
`
`I
`-CH
`
`RB
`
`38A
`
`0.9
`
`120
`
`-C00
`
`JFH 19
`
`0.2
`
`2.000
`
`CH2(cid:1)
`
`()
`
`HN
`
`- C - CH2
`
`- CH
`
`- CONH
`
`-CH-CH
`
`- COO
`
`-
`
`XJ9
`
`3.8
`
`SCH,
`
`(CH,),
`
`H,N-CH.CH,
`
`-S
`
`PC 18
`
`74
`
`8
`
`CH,4’
`
`CH,4
`
`S-CH2-CH-CONH-CH-C00
`
`ST43
`
`1.5
`
`>10.000
`
`>10.000
`
`CH,
`
`(cid:1)CH
`
`CH,
`
`- CH,
`
`CH,’I’
`
`S-CH2-CH-CONH-CH-COO
`
`CH,4
`
`OCH,O
`
`S
`
`-CH,-CH-CONH-CH-C00
`
`ES34
`
`ES 37
`
`INEP
`
`ACE
`
`4.5
`
`55
`
`5.2
`
`14
`
`CH,
`
`s
`
`CH,
`
`(cid:1)CH,
`
`CH,cD . OH
`
`CH3
`
`-CH-CH-CONH
`
`-CH-CONH-CH-C00
`
`PC 57
`
`1.4
`
`0.2
`
`specific
`using
`demonstrated
`been
`it has
`However,
`involved
`critically
`is not
`enzyme
`this
`that
`itors
`1986b).
`(Ch#{233}rotet
`a!.,
`metabolism
`kephalin
`degradation
`and APN
`in enkephalin
`of NEP
`role
`The
`of numerous
`use
`confirmed
`by
`the
`now been
`amply
`has
`to
`or
`able
`specific
`for
`one
`enzyme
`inhibitors,
`either
`IX).
`A clear
`tables
`1 and
`2 and
`section
`both
`inhibit
`(see
`of other
`participation
`of NEP
`in the
`in vivo
`inactivation
`However,
`as
`neuropeptides
`is
`still
`not well
`established.
`participate
`could
`discussed
`following
`section,
`NEP
`in the
`in
`of SP
`actions
`the
`in the
`of
`physiological
`interruption
`and
`1987a)
`a!.,
`the
`rat
`nigra
`(Mauborgne
`substantia
`(Chec!er
`defined
`not well
`neurotensin
`structures
`as
`yet
`in
`1983;
`involvement
`et
`al.,
`Coquerel
`a!.,
`1986).
`The
`in the
`NEP
`physiological
`inactivation
`of other
`neuropep-
`will
`tides
`have
`to
`be
`confirmed
`the
`use
`of
`specific
`antagonists
`of
`neuropeptides
`of
`reversing
`both
`the
`pharmacological
`response
`by the
`endogenous
`
`et
`
`of
`
`et
`
`by
`capable
`induced
`
`inhib-
`in
`en-
`
`at
`
`that
`and
`effector
`end
`the
`cussed
`for ANP;
`fulfilled
`messenger
`peptide
`This
`does
`NEP.
`in other
`peptidase
`be
`characterized.
`
`inhibitor.
`an NEP
`by
`induced
`requirements
`review,
`these
`of
`this
`peptide
`is,
`therefore,
`the
`this
`action
`clearly
`regulated
`whose
`not
`exclude
`participation
`the
`peptidergic
`pathways
`which
`
`As
`
`dis-
`are
`second
`
`by
`the
`to
`
`of
`remain
`
`is
`
`HI. Neutral
`and
`Assay
`
`Endopeptidase
`of Neutral
`Activity
`
`1 1 Purification
`24.
`24.11
`Endopeptidase
`
`has
`NEP
`(Kerr
`kidney
`1981),
`al.,
`(Fulcher
`philic
`tein.
`
`et
`solvents
`Immunoaffinity
`
`such
`tissues,
`various
`from
`purified
`been
`(Almenoff
`pituitary
`1974b),
`and Kenny,
`and
`intestine
`1983),
`et
`al.,
`brain
`(Relton
`detergents
`or
`lipo-
`various
`a!.,
`1983),
`using
`this
`membrane-bound
`pro-
`to
`solubilize
`chromatography
`with
`polyclonal
`
`as
`et
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1013, p. 004
`
`(cid:1)
`

`

`NEUTRAL
`
`ENDOPEPTIDASE
`
`24.11
`
`91
`
`monoclona!
`especially,
`(Fulcher
`facilitated
`has
`greatly
`bodies
`quantities
`of pure
`enzyme
`rapid
`studies.
`functional
`that
`developed
`been
`assays
`have
`Numerous
`selectivity
`of NEP.
`substrate
`on
`based
`are
`the
`tripeptide
`frag-
`measuring
`methods
`Current
`[3H]Tyr-D-Ala-G!y
`formed
`by
`or
`[3H]Tyr-G!y-Gly
`ments
`bond
`of
`[3H]Leu-enkephalin
`or
`of
`the Gly-Phe
`cleavage
`respectively
`(Vogel
`and Alt-
`[3H]D-Ala2-Leu-enkephalin,
`1982).
`[3H]Leu-enkepha!in
`is
`al.,
`stein,
`1977;
`Llorens
`study
`APN
`activity.
`Internally
`also
`currently
`used
`as
`substrates
`such
`the
`NEP
`quenched
`fluorogenic
`available,
`dansyl-D-Ala-Gly-
`selective,
`commercially
`continuous
`recording
`of NEP
`Phe(pNO2)-G!y
`allows
`1984).
`tivity
`(Florentin
`et
`a!.,
`two-step
`a
`using
`NEP
`activity
`can
`also
`be
`and APN.
`The
`reaction
`catalyzed
`sequentially
`is
`cleaved
`by
`substrate
`benzyl-Gly-Arg-Arg-Leu-2NA
`addition
`of
`leading,
`NEP
`the
`Arg-Leu
`bond,
`at
`removal
`of
`by
`APN,
`to the
`formation
`of 2-naphtylamine
`can
`the
`N-terminal
`amino
`acid,
`Lou.
`2-Naphtylamine
`(Al-
`then
`be quantified
`by fluorescence
`after
`diazotation
`iden-
`menoff
`and Orlowski,
`1984).
`Owing
`to the
`common
`in
`the
`and
`tity
`of NEP
`and
`CALLA
`to
`clinical
`interest
`sur-
`this
`characterizing
`and
`quantifying
`protein
`on
`the
`face
`of
`various
`cells,
`new methods
`have
`been
`recently
`proposed.
`A highly
`sensitive
`enzyme-linked
`immunosor-
`bent
`assay
`method
`for NEP
`applicable
`to material
`has
`porcine
`and
`human
`origin
`been
`developed
`(Howell
`et
`al.,
`1991).
`The
`limits
`of using
`immunological
`methods
`to
`quantify
`NEP
`are
`their
`species
`specificity
`and
`their
`generally
`lower
`sensitivity
`compared
`to enzymatic
`meth-
`Thus,
`ods
`al.,
`1989; Milhiet,
`1992).
`nu-
`(Beaumont
`et
`merous
`lymphoid
`cells,
`defined
`as CALLA
`negative
`using
`detection
`by
`flow cytometry,
`were
`shown
`immunological
`to
`have
`significant
`levels
`of NEP
`activity
`at
`the
`low but
`surface
`[3H]D-Ala2-Leu-enkephalin
`was
`used
`as
`when
`substrate.
`However,
`due
`to
`the
`low specificity
`of NEP,
`enzymatic
`methods
`require
`control
`with
`selective
`inhib-
`itors.
`For
`this
`reason,
`highly
`sensitive
`methods
`based
`on
`the
`use
`of
`highly
`selective
`fluorescent
`(Milhiet
`et
`a!.,
`1992b)
`or
`radioiodinated
`(Fourni#{233}-Zaluski
`et
`al.,
`1992c)
`NEP
`inhibitors
`have
`recently
`developed.
`The major
`been
`these
`over monoclonal
`antibod-
`compounds
`advantage
`of
`that
`ies
`is
`they
`NEP
`independently
`of
`recognize
`and
`species
`the
`degree
`of glycosylation
`of
`the
`enzyme.
`study
`recent
`has
`shown,
`for
`example,
`that,
`of 20 human
`monoclonal
`antibodies
`tested,
`only
`four
`cross-reacted
`(H#{233}l#{232}neet a!.,
`cell
`with
`a rat
`line
`
`
`1992).
`The
`fluorescent
`thiorphan
`derivative
`NEP)
`has
`been
`used
`to
`detect
`NEP
`to
`follow
`its
`intracellular
`traffic
`using
`et
`a!.,
`1993),
`to quantify
`(Milhiet
`enzyme
`on various
`or
`rodent
`etry
`(Milhiet
`et
`However,
`
`the
`
`cellular
`is based
`enzymatic
`
`of
`
`characterizing
`directly
`ing method
`and
`the
`the
`origin
`NEP,
`whatever
`the metallopeptidase,
`of
`localization
`preserves
`some
`of
`its
`that
`NEP
`dodecyl
`sulfate-polyacrylamide
`sodium
`(Sullivan
`Johnson,
`and
`sis
`to
`the
`content
`high
`due
`Thus,
`highly
`the
`dase.
`[‘25I]RB1O4
`inhibitor
`as 2 ng of
`little
`tify
`as
`gel
`electrophoresis
`after
`Soleilhac
`et a!.,
`1992).
`
`presence
`subeellular
`on
`the
`activity
`electrophore-
`gel
`a property
`probably
`1989),
`bonds
`in the
`pepti-
`of disulfide
`0.03
`nM)
`and
`specific
`potent
`(KD =
`to visualize
`and
`quan-
`can
`be used
`in crude
`tissue
`extracts
`the
`enzyme
`(Fourni#{233}-Zaluski
`et
`1992c;
`
`or
`
`of
`
`fact
`after
`
`a!.,
`
`Iv.
`
`Substrate
`Action
`
`Specificity
`of Neutral
`
`and
`Endopeptidase
`
`Mechanism
`
`of
`
`24.11
`
`such
`
`the
`involves
`bond
`
`of
`
`by
`of
`
`carbon
`subsequent
`them
`corre-
`of exopepti-
`Crystal-
`carboxyl
`hydrolysis
`com-
`scissile
`the
`of
`the
`acids
`molecule
`a!.,
`1984;
`
`or
`
`of
`
`lographic
`
`studies
`
`through
`the
`
`of
`and
`
`to
`
`atom
`
`inhibited
`Zn metallopeptidase
`a glycosylated
`is
`NEP
`as
`fi mer-
`reagents,
`metal-chelating
`nonspecific
`by
`acid
`(Kerr
`or
`ethylenediaminetetraacetic
`captoethanol
`a
`form
`1974a,b).
`The
`Zn metallopeptidases
`and
`Kenny,
`to
`addition
`of
`enzymes
`which
`include,
`in
`large
`group
`colla-
`carboxypeptidases
`A, B,
`and
`E, ACE,
`NEP,
`APN,
`the
`bacterial
`endopeptidase
`TLN
`(reviewed
`genases,
`and
`by Vallee
`and Auld,
`1990).
`car-
`analyses
`As
`shown
`from
`the
`crystallographic
`and
`TLN
`(Kester
`and
`A (Lipscomb,
`1980)
`boxypeptidase
`have
`Matthews,
`1977a),
`all
`of
`Zn metalloproteases
`the
`respective
`similarities
`in
`their
`active
`sites
`and
`in
`their
`1977b;
`mechanisms
`action
`(Kester
`and
`Matthews,
`of
`hydrol-
`reviewed
`by Matthews,
`1988).
`Schematically,
`(a)
`the
`bond
`by these
`enzymes
`ysis
`of
`a peptide
`to the
`Zn
`coordination
`the
`oxygen
`of
`the
`scissile
`of
`attack
`by a
`atom;
`a glutamate-promoted
`nucleophilic
`(b)
`water
`molecule
`on
`the
`carbonyl
`carbon
`polarized
`the
`ion;
`Zn
`and
`the
`protonation
`of
`the
`nitrogen
`the
`(c)
`peptide
`bond
`be
`cleaved,
`leading
`to
`a breakdown
`of
`to
`the
`weakened
`linkage
`between
`the
`tetrahedral
`and
`the
`protonated
`nitrogen
`atoms,
`with
`release
`the
`two
`peptide
`fragments,
`one
`of
`sponding
`to
`a single
`amino
`acid
`in the
`case
`dases
`(aminopeptidases
`or
`carboxypeptidases).
`with
`complexed
`of TLN
`that
`have
`suggested
`inhibitors
`hydroxamate
`the
`formation
`of a pentacoordinate
`occurs
`metal,
`including
`the
`oxygen
`of
`plex
`three
`Zn
`coordinating
`amino
`the
`bond
`the
`water
`without
`displacement
`of
`peptidase,
`bound
`the
`Zn
`(Hangauer
`et
`initially
`1988).
`Matthews,
`specificity
`The
`ensured
`tially
`their
`between
`the
`of
`chains
`substrate.
`of
`the
`well-positioned
`ceptor
`groups
`dues
`the
`
`of
`by Van
`
`essen-
`is
`Zn meta!lopeptidases
`the
`interactions
`der Waals
`and
`ionic
`the
`lateral
`Si’
`and
`subsites
`and
`52’
`52,
`51,
`P2’ moieties
`corresponding
`P2, P1, P1’
`and
`by several
`Specificity
`is also
`determined
`and
`ac-
`donor
`hydrogen
`bonds
`between
`polar
`resi-
`the
`the
`bound
`molecule
`and
`of
`peptidases,
`such
`as As&’2
`and Arg(cid:1)#{176}3in TLN.
`
`of
`
`a!.,
`
`et
`(Aubry
`preparation
`and
`
`and,
`1983)
`1987)
`et
`al.,
`large
`of
`structural
`sensitive
`the
`
`wide
`include
`
`et
`to
`
`detected
`by NEP
`
`after
`
`anti-
`the
`for
`
`ac-
`
`of
`
`the
`A
`
`on
`
`10 nM on
`(IC50
`=
`surface,
`cell
`the
`photomicroscopy
`the
`of
`the
`presence
`cells
`by flow cytom-
`the most
`interest-
`
`and
`human
`1992b).
`
`aL,
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1013, p. 005
`
`

`

`92
`
`ROQUES
`
`ET
`
`AL.
`
`in
`
`a
`
`a
`
`re-
`
`of
`the
`
`and
`
`than
`
`the
`
`Si’
`
`and
`
`detail
`in greater
`is discussed
`of NEP
`structure
`The
`section
`this
`for
`the
`purposes
`of
`section,
`but
`following
`the
`that
`the
`enzyme
`preferentially
`to
`note
`important
`is
`it
`resi-
`amino
`side
`of hydrophobic
`on
`the
`peptides
`cleaves
`of
`suggesting
`the
`occurrence
`Met),
`(Phe,
`Leu,
`dues
`(Kerr
`similar
`to that
`found
`in TLN
`lipophilic
`subsite
`these
`of
`A more
`detailed
`comparison
`and Kenny,
`1974b).
`in the
`P2
`that
`the
`presence
`of a Phe
`enzymes
`shown
`has
`the
`specificity
`and
`the
`turnover
`rate
`position
`optimizes
`the
`provided
`that
`S1 subsite
`is occupied
`constant
`of NEP,
`residue,
`a restriction
`that
`does
`not
`occur
`by
`a Gly
`or Ala
`and Morihara,
`1986;
`Pozsgay
`et a!.,
`1986).
`in TLN
`(Hersh
`we
`consider
`design
`of better
`adapted
`the
`However,
`when
`results
`of
`enzymatic
`studies
`suggesting
`inhibitors,
`the
`of putative
`well-defined
`subsites
`in metal-
`the
`presence
`used
`must
`be
`cautiously.
`The
`rate
`of cleav-
`lopeptidases
`is critically
`dependent
`on
`the
`dissocia-
`age
`of
`substrates
`two
`metabolites
`formed,
`which
`is
`the
`tion
`rate
`of
`the
`solvatation
`and
`energy-consuming
`their
`of
`function
`processes
`associated
`with
`their
`transconformational
`active
`site. Most
`these
`param-
`of
`enzyme’s
`lease
`from the
`on
`the
`binding
`affinity
`inhib-
`of
`influence
`have
`no
`eters
`by
`ionic
`Van
`der Waals
`dominated
`itors
`which
`is
`(or
`antagonists
`in
`the
`case
`Inhibitors
`interactions.
`better
`adapted
`to scrutinize
`therefore,
`are,
`receptors)
`of putative
`subsites
`in proteins.
`the
`nature
`and
`presence
`and
`selectivity
`can
`cleave
`various
`a broad
`has
`NEP
`such
`peptides
`as endothelin
`(Fagny
`or
`cyclic
`linear
`short
`or
`the ANP
`(Stephenson
`and Kenny,
`1987a),
`1991)
`et a!.,
`polypeptides
`of
`intermediate
`or
`long
`length,
`as
`as well
`insulin-B
`(approximately
`3000
`daltons)
`chain
`such
`as
`and
`Kenny,
`and
`interleukin-ce1
`(17,000
`1974b)
`(Kerr
`(Pierart
`although
`it
`sometimes
`acts
`et al.,
`1988),
`daltons)
`efficiently
`as
`a dipeptidylcarboxypeptidase
`as
`more
`endopeptidase.
`The
`Km of Leu-enkephalin-amide,
`a true
`of Leu-
`for
`example,
`is about
`20 times
`higher
`than
`that
`due
`to
`enkephalin.
`This
`finding
`was
`interpreted
`as being
`COOH-
`a
`favorable
`ionic
`interaction
`between
`free
`a well-
`and
`of
`terminal
`carboxyl
`group
`Leu-enkephalin
`active
`acid
`charged
`positioned,
`positively
`amino
`the
`in
`NEP
`1981a),
`et
`al.,
`site
`(Fourni#{233}-Zaluski
`1979;
`leading
`to a
`structurally
`to be
`considered
`as more
`closely
`related
`and
`than
`to an endopeptidase
`(Malfroy
`carboxypeptidase
`How-
`Schwartz,
`1985;
`Hersh
`and Morihara,
`1986).
`1982,
`endo-
`ever,
`the
`also
`behaves
`as
`a very
`efficient
`enzyme
`such
`peptidase,
`various
`linear
`peptides
`in
`vitro,
`cleaving
`Ste-
`as SP
`(Matsas
`a!.,
`19Mb;
`1983,
`neurokinins
`et
`phenson
`198Th),
`and
`CCKS
`(Matsas
`and
`Kenny,
`gastrin
`et
`a!.,
`et
`al.,
`Durieux
`et
`al.,
`1986;
`1984a;
`Durieux
`1985;
`et al.,
`1985),
`neurotensin
`(Checler
`et a!.,
`1983),
`and
`Zuzel
`Met-enkephalin-Arg-Phe.
`chemotactic
`peptide
`for-
`The
`was
`myl-Met-Leu-Phe
`shown
`to
`be
`hydrolyzed
`by
`also
`NEP.
`The
`colocalization
`of NEP
`immunoreactivity
`with
`senile
`plaques
`in
`brain
`of
`patients
`suffering
`Alz-
`from
`heimer
`disease
`warrants
`an
`investigation
`a possible
`role
`of NEP
`f3-amyloid
`protein
`degradation
`(Sato
`
`in
`
`of
`
`et
`
`recently
`(fragment
`
`shown
`1-40)
`
`that
`
`at
`
`vitro
`in
`several
`
`NEP
`sites
`
`at
`
`is
`
`to
`
`in
`
`ANP
`and
`
`in
`
`the
`
`have
`1991). We
`al.,
`cleaves
`the
`(cid:1)3-amy1oid
`(unpublished
`results).
`subsite,
`catalytic
`the
`especially
`The
`active
`site
`of NEP,
`1983),
`(Fourni#{233}-Zaluski
`et al.,
`been
`shown
`to be
`large
`has
`of
`the
`enzyme
`to
`cut
`the Cys-
`accounting
`for
`the
`ability
`a site within
`the
`ring
`formed
`Phe
`bond
`of ANP,
`i.e.,
`present
`this
`peptide,
`by
`the
`two
`half-cystines
`(Stephen-
`SLRRSSOFGGRMDRIGAQSGLG(cid:1)CNSFRY
`notice
`that
`important
`and
`Kenny,
`1987a).
`It
`son
`the
`the
`same
`at
`and
`TLN
`cleaves
`enkephalins
`NEP
`B. P. Roques,
`Soroca
`(E. Lucas
`bonds
`as
`does
`results),
`the
`close
`corre-
`again
`unpublished
`emphasizing
`in the
`active
`enzymes.
`spondence
`sites
`of both
`to
`the
`In
`addition
`enkephalins
`and Met-enkephalin-
`shows
`opioid
`Arg-Phe,
`NEP
`little
`activity
`toward
`other
`these
`of
`by
`the
`peptides.
`All
`peptides
`are
`characterized
`sequence
`but
`the
`N-terminal
`of Met
`or Leu-enkephalin,
`of NEP
`bonds
`is
`efficiency
`cleaving
`their
`G1y3-Phe4
`sensitive
`amino
`acid
`exquisitely
`to
`the
`length
`of
`the
`added
`at
`sequence
`COOH
`terminus
`(Turner
`al.,
`fi-
`Thus,
`dynorphin
`1987).
`1-9,
`dynorphin
`1-13,
`neoendorphin,
`and
`(cid:1)3-endorphin
`are
`substrates,
`poor
`gesting
`a conformationally
`access
`hindered
`This
`site.
`active
`bonds
`enzyme-sensitive
`that
`indicates
`it
`important
`“opioid”
`induced
`by NEP
`inhibitors
`acting
`itors,
`NEP
`to the
`protection
`two
`and
`perhaps
`partially
`that
`Met-enkephalin-Arg-
`Phe.
`colocalized,
`found
`often
`is
`with
`NEP,
`Like
`specificity
`a broad
`has
`also
`the membrane-bound
`hydrophobic
`although
`1986),
`Barrett,
`(McDonald
`and
`NH2-terminal
`in
`the
`aromatic,
`residues,
`preferentially
`addition,
`the’
`In
`removed.
`rapidly
`position
`are more
`also
`seem to prefer
`hydrophobic
`of APN
`and
`subsites
`52’
`al.,
`1988; Xie
`et
`a!.,
`1989a,b).
`et
`residues
`(Hernandez
`of NEP
`in the
`degradation
`The
`possible
`involvement
`preparation
`contaminated
`of a peptide
`a crude
`tissue
`in
`using
`demonstrated
`by
`by
`other
`peptidases
`usually
`is
`inhibi-
`as
`selective
`either
`thiorphan
`or phosphoramidon
`a peptide
`degradation
`of
`tors.
`Nevertheless,
`the
`in vitro
`even
`by
`enzyme,
`using
`a tissue
`in which
`both
`mole-
`cules
`are
`localized,
`does
`not
`necessarily
`mean
`the
`that
`peptidase
`is responsible
`for
`its metabolism
`in vivo.
`Thior-
`for
`phan,
`example,
`little
`effect
`on
`degradation
`has
`al.,
`striatal
`rat
`CCKS
`(Butcher
`et
`or
`spinal
`et
`and
`Chipkin,
`1989; Mauborgne
`al.,
`1991)
`(Yaksh
`tissues.
`of
`spite
`the
`presence
`of NEP
`in these
`However,
`SP
`in
`other
`brain
`regions,
`such
`as
`the
`substantia
`nigra,
`al.,
`might
`well
`degraded
`by NEP
`(Mauborgne
`et
`1987a).
`Owing
`to the wide
`distribution
`of NEP
`in periph-
`era!
`tissues,
`is probable
`that
`other
`putative
`physiolog-
`ical
`substrates
`are
`yet
`to be
`characterized.
`
`related
`the
`to
`because
`effects
`by mixed
`are mainly
`enkephalins
`heptapeptide,
`
`due
`
`observation
`pharmacological
`probably
`and
`APN,
`and
`endogenous
`of
`the
`extended
`
`et
`and
`
`a-
`
`sug-
`of
`the
`is
`an
`the
`inhib-
`both
`the
`to
`
`of
`
`on
`
`it
`
`which
`APN
`
`an
`
`be
`
`it
`
`the
`1989)
`
`of
`SP
`in
`
`BIOCON PHARMA LTD (IPR2020-01263) Ex. 1013, p. 006
`
`

`

`NEUTRAL
`
`ENDOPEPTIDASE
`
`24.11
`
`93
`
`V. Design
`24.11
`
`of Selective
`and
`Aminopeptidase
`
`Neutral
`
`Endopeptidase
`N Inhibitors
`
`NEP
`860
`
`=
`
`devoted
`have
`reviews
`recent
`Several
`inhibitors
`and
`aminopeptidase
`thesis
`of NEP
`and Wyvratt,
`and
`Fourni#{233}-Zaluski,
`1986;
`Thorsett
`in the
`active
`the
`Rich,
`1990)
`and
`to
`differences
`Given
`the
`large
`NEP
`and ACE
`(Roques,
`1985).
`ties
`in the
`active
`sites
`of Zn metallopeptidases
`of
`by Matthews,
`1988),
`the
`rational
`design
`selective
`NEP
`inhibitors
`and
`mixed
`inhibitors
`on
`and
`APN
`was
`based
`the
`synthesis
`of molecules
`that
`contain
`a strong
`metal-coordinating
`group
`and
`favorable
`to
`able
`satisfy
`of
`the
`possible
`energetically
`all
`surrounding
`interactions
`at
`least
`one
`of
`the
`subsites
`with
`the
`the
`catalytic
`site. Moreover,
`for
`the
`exopeptidases,
`presence
`of
`a positively
`charged
`group
`on
`the
`P1 residue
`or
`a negatively
`charged
`group
`the
`P2’
`residue
`can
`used
`to
`increase
`the
`selectivity
`inhibitors
`for
`amino-
`or peptidyl-dipeptidases,
`respectively
`(Roques
`peptidases
`Rogues,
`and
`Fourni#{233}-Zaluski,
`1986;
`1988b).
`Because
`enkephalins
`ACE
`is also
`able
`to cleave
`the
`at
`their
`Gl?-
`Phe4
`bond,
`showing
`that
`its
`active
`site
`some
`similar-
`ities
`with
`that
`of NEP,
`a comparison
`the
`inhibitory
`potencies
`of
`some
`inhibitors
`will
`for
`both
`zymes
`illustrate
`how this
`problem
`been
`overcome
`to
`used
`or
`how
`this
`information
`now
`design
`mixed
`NEP/ACE
`inhibitors.
`the most
`representative
`corn-
`Only
`in each
`series
`of
`inhibitors,
`characterized
`by
`the
`pounds
`of
`the
`coordinating
`group,
`are
`reported.
`nature
`
`been
`
`on
`of
`
`has
`of
`given
`has
`
`be
`
`to
`
`is
`
`Zn
`
`syn-
`to the
`(Roques
`1987;
`sites
`of
`similari-
`(reviewed
`potent
`and
`of NEP
`that
`are
`
`be
`
`en-
`
`A. Development
`Endopeptidase
`
`of Selective
`24.11
`
`Inhibitors
`
`of Neutral
`
`of
`or
`
`et
`
`enkephalin
`of
`studies
`inhibitors.
`Thiol
`1.
`Extensive
`specificity
`the
`shown
`that
`have
`and
`dipeptides
`ana!ogues
`subsite,
`which
`by
`the
`S(cid:1)’
`ensured
`essentially
`is
`of NEP
`large
`hydropho-
`aromatic
`or
`with
`preferentially
`interacts
`poor
`specificity,
`subsite
`has
`a
`bic moieties.
`The
`52’
`TLN,
`a proline
`but
`to ACE,
`like
`although
`in contrast
`(Fourni#{233}-Zaluski
`to poor
`inhibitors
`this
`position
`leads
`It was
`also
`observed
`a!.,
`1979,
`1981a;
`Llorens
`et a!.,
`1980).
`amide
`bond
`that
`N-rnethylation
`the
`Phe4-Met5
`enkephalin
`analogues
`in various
`dipeptides
`decreased
`their
`affinities
`for NEP
`but
`not
`for ACE
`(Fourni#{233}-Zalu-
`ski
`al.,
`1979,
`1981a).
`This
`observation
`is in agreement
`with
`the
`good
`affinity
`for ACE
`of
`inhibitors
`bearing
`N-methylated
`amino
`acid
`or
`