`
`Lupln Ex. 1058 (Page 1 of b)
`Lupin Ex. 1058 (Page 1 of 9)
`
`

`

`Editorial Correspondence
`M an uscrip/s trod editorial correspondence
`~hould be ~m~t 1o:
`
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`
`~,im and Scope
`The I NFERNATIO:NAL JOURNAL O1, PEPTIDE & PROTEIN RESEARCH will
`be of lhe I~i~hesi possible scientific and tee}mica[ standard and wfl] cover not only
`proteins as such bul also pcpfide~ and amino acids. The Journal will be open 1o
`originaI papers lhnl contribule ~o the fi.u{her development o{ peptide and protein
`research.
`
`Rapid Publicatio~
`-[’hi.~; i,~ indec’d a l:nge field :,rod there is need for a journal R]at caa pt~b!~sh the In{est
`
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`
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`Victor J. Hruby, LiSA
`
`Ediloria! iBoard
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`H. Y~jima, Japa~
`
`Lupin Ex. 1058 (Page 2 of 9)
`
`

`

`~trate~ Ac.-’l:hr4 !e-Nle-Nle-Gbri-Arg-N 1-1.~, {ncc)rporai.ior, ol72-amirlobc:r~zoic acid il~ place of the acct3<l group
`
`dru~:~;, lf~ ordei’ to allow the oxploralio~] of’ potel)ti.al
`l~flfil:~itor~ of HIV pro~ase and to opdmis~ lead
`
`the itqhibitor which allowed R~r automauou and hmh
`
`{HYiO intervals, l’h{~ {-t-PL(: atlsay ,:luicklv became the
`rate4irnlti~/g step in lhe dovelc~l:>m(,m of HIV
`inhibitoJs wilh desirabk~ therapet~.dc proper~ie~. A
`
`kit~etic ch~u’aclerJzafion of l.he h-~[eraotJo,} o.f the
`hibttors with HIV protease was needed. The ~;tra.tegy
`of developing either a chromo,~et~ic or a
`
`va,,e sites
`Our initial auenlpts ff~ctl~ed on develo!:,mg a
`
`modalion of Phe and Tvr a~ position PI in HIV pro-
`tease s~tbsirates, we prepared a series of potential
`
`544
`
`Lupin Ex. 1058 (Page 3 of 9)
`
`

`

`[)I PI?A for 31] mJ n, {. c)m.i)l~lod p¢lJlM,2s
`with H i:i::misolc. 9:t. (-:rude popIicit?$ wcio
`
`¢Olt]i!i!l II’3iIIP. {t {.),{)5".’o T{:A H,O and 1}.05%
`
`<A,;:ls 81t<:mpt~xi Tl’l~2 p<-p idc~. Acilhr-/t¢-;’~lo-Phetp-
`
`dihltod w)th walo{. ::uld/yopl~i]izod .’>m~l pOi’lJOl~S ot
`
`The Phcfm--NO:...) used
`
`CH CI-,
`I)MV
`
`;1] ill
`
`Lupin Ex. 1058 (Page 4 of 9)
`
`

`

`0,A6 x 25 cm!. developed with 0.05% TFA lk>r 5
`fl.~llowed by a ~radienl of 0-40% acetoniiri.le in
`40rain. For inhibitor studies, (O.~(_, of the
`~qution was p~eincubated at 25" h)r/(} rain wi~h 20 uL
`oK0 { m~.l inhi bil.or ~d issoi veal in DMSO and diluted to
`
`same concentration shows dramatically d~e tenfold
`increase in excitation and sixfl-)ld increase m emissio~
`lt]~.~(}[l en2vnmtic hydrolysis. Initml kinetics were
`itored a~ 25~) with magnetic utirdng :n opth~ml con-.
`difion.s {excitation. = 337nm and emi>~mn = 41
`
`stone. Ac-Tlnr--lk~.Met.-Met-GIn--Arg-.NH:, or Ab2.-
`Thr-lle-Nle--Phe-,/~-NO~)-GIt>Arg-.NH:
`6h was added h} order ro delermirte inhibition
`c!.ea.vage. Reactions were stopped alld elea.va~ze
`were monitoced by HPLC as above. In oroer to
`contirm the cleavage pa.ttero, of Abz-I’hr-Ile~Nle-
`Plne{p-NO~)-Gh>Acg-NH:, cleav~ge was allowed
`fimsh as judged by HPLC and the two product peaks
`were isolated by HPLC. Incubation with ehher syn~
`the:it HIV protease or enzyme expressed and purified
`{’tom g. ¢oli ~kindly supplied by Dr. George Glovcr
`Monsanto~ ga~’e the same HPLC pattern IFig.
`Retention time of the substratm was 42.1 rain. while
`Abz-.Thr-Ile--Nle--OH was 3?.4rain and !f-.Phe~p--
`N% ~-GIn-A.rg-NH., w;:ts 1.7,Tmin Identity o{" the
`product peplides was confirmed by FABMS and
`amino z~cid analysis,
`
`The exciladon and emission spectra were measured
`an SLM 8000 C apectro!luorome{er, Both the sub-
`strafe. Abz--N!-1**6. and the ?%~.crminal prod~tct, Abz-
`Thrqlc*Nle-OH. show absorption maxima at 337 nm
`and broad elnission maxuna between 390 and 440
`The comparison between substrate and producl at the
`
`The rfl)sorption-ts ~e~’tr’~..~. of’ the sub~trate.
`and the N-termimfl product. Abz-Thr--lle-.Nle-OH
`were recorded on a Be:ckma~, I)L;--~ speclrophoto..
`nu-,t-,r.~e ".l"he subshate ghowg maxuna at 284 nm and
`258 am. whil.e the cleavage produc~ has
`318 nm aad 252nm.
`
`F’hmrescence measurements oa 96-well EMSA plates
`were made wil.h dm Titertek Fluoroskan II. version
`3.1. An excttadon lilter o£ 355nm ~bandwid.th
`35 - 4 nm~ and an enussion filter of 430 nm {band-
`wid{~ 25 4- 3 n n~ were used. Ten microliters of
`stock solutio~ (0.05rag/miD o1" HIV pro~.ease were
`iacubated with five di~}:rent concentrations of
`NF’*-6 i~ a tinal w~lume of 100/.~I_. of" assay bufl)r a.t
`37u with the i~}.crease in itta~rescence m.on.itored in each
`we!] every 2rain fbr 20 rain. A stock 3olution of
`Abz~NF*~6 in DMSO was dilmed to 0.1 m~.~ witl~
`aa:~ay ~ult?r and used fi>r the assay. The highest con--
`ce~tra{io~ of I)MSO tesled was 5%. which shows
`eft)ors of cleavage. A standard curve (Fig, 2} relating
`
`Calibration Curve
`
`546
`
`})’I(3U RE 2
`
`cea~ration:, The fluorogenic Substra~e (,¢bz-N]eF*-:6} at eJgh~ di~;,
`
`prolease in t{ssay lmfti~r (final volun-~e ::. 100 !~L} on a micrtditer
`
`Lupin Ex. 1058 (Page 5 of 9)
`
`

`

`t-IlV protease fluorometric assay
`
`changes h~ fluorescent intensity to changes m con-
`centration of product was used to convert fluoFescence
`changes into molar velocitieS. In order to predete>-
`mine the concentration range for K~ determination of
`inhibitors, we do a preliminary screening assay by
`HPLC. Twenty microliters of 0.~ mM inhibitor in.
`assay but~br (pH 6.4) at~d IOHL of HIV protease
`(stock solutio~x of 0.05 mg/mL) were preh~cubated for
`5-10rain at 25°. Twenty microlite~s of 0.1 mM Abz-
`NF*.-6 were added and the reaction continued lk~r l h,
`Sixty microliters of !0% TFA were added to stop the
`reaction and the amount of cleavage determined by
`H PLC. in the absence of inhibition, almost complete
`cleavage occm:s. By the amount of substrate remain-
`ing, an estimated K~ allows apwopriate choice of
`concentn~tion ranges R~r the inhibitor during the
`fluorescence assay.
`
`RI-TSULTS AND DISCUSSION
`
`Six peptidcs were prepared as potemial chromogenic
`substrate:~ with the l\~llowing sequences::
`
`H-Ser-Phe-Ast>Phe(p-N O_, ),.Pro,-GIn-,Va [-T h r--OH
`H-A rg~LysoIle-Leu-Phe( p-NOx)..Leu-Asp-GlyoOH
`H-Thr-Leu-Asn~-Ph¢( !>N% )-.Pro.,ID-Se r-Pro-OH
`
`Ac-Thr-lle-Phe(l>NO~)-Nle-.GI[:- A rg--NH
`Ac-.Thr-lle..Nle-Phe( p-NO....)-.G h>A rg-N H~
`
`Of lhese six peptides, onJy the last with the Phe(p-
`NO2) residue in the PI’ position showed cleavage
`under our conditions of HPLC assay and with incuba-
`tion times of I h. Tomaszek ~’i aL (6) have reported a
`
`chromogenic octapeptide substrate, Ac-Arg-Lys-Ile-
`Phe{/)-NO:.,.)-Leu-Asp-Gly-NH,.,, which is similar to
`those we tried, but with blocked amino and carboxyl
`terminals. Nashed el aL {5) have reported two chro-
`mogenic peptide substrates, Ao-Lys-.Ala-Ser-Gln--
`Phe(t>NO:)-Pro-Val-Val--NHx and H-Thr,Phe.-Gln--
`Ala--Phe(t>NO:)-Pro--Leu-Arg--Ala-OH, wMch can
`form the basis of a spectrophotometric assay. In these
`three cases, d~e hydrolysis leaves the chromogenic
`residue at the C-termim.ts, resulting in greater spectral
`changes, Because of the small spectral changes seen
`
`wid~ the chromo~enic._ residue at the ~X’-ternunus,~ ’ we
`decided to try the fiuorogenic substrate strategy, By
`preparing a Gonger substrate, [ys-~Ala,-Arg~Va/-.Leu-.
`Phe(/~-NO~)-Olu-Ala-Met, with the chromogenic
`residue at: the Pl’ position, Richards ~’I aL (7) have
`developed a spectrophommetric assay,
`Modification of the hexapepfide buba[rate: Ac--Thr-
`Ile-Nle--Phe( !~-.NO~)..GIn..Arg--NI-{~. with the acceptot
`residue in the C-terminal product only required the
`addition of a donor fluorescent group to the N-termi-
`ha1 producl. Replacing {l~e ace@ group with 2-amino-
`benzoic acid (Abz) resulting in the lluorogenic
`peptide, Abz-Thr-lle-Nle~Phe { p-NO:~ .)-GIn-Arg-
`NH:~ gave the same combination o17 donor and accep~
`tot used by Carmel & Yai:on (10) in their substrate for
`angiotensin converting enzyme. Since those auil~ors
`had shown l.hal quenching etliciency depends on the
`proximity of the donor a~td acceptor chrom.opho~:es,
`we synthesized an an:alog with the Abz group at posi-
`tion P3, Abzqle-.Nle-.Phe(p--NO~>Gh>Arg-NHe, but
`the cleavage etliciency was dramatically reduced. The
`hexapeptide substrate above reR’rred to as Abz-NF*-6
`
`38,00
`
`Excitation=337
`
`ABZ-TIN-OH
`
`<350
`
`400.
`Ernission Wavelength
`
`450,
`
`500>
`
`FIGURE 3
`Fluorescence cmi.,,sion spcclra of
`O. lm~i aubstratc (Abz-NF*-O)
`and 0.1 m~.[ N-terminal cle~ivage
`product (Abz- Th r..lle-N ~e-OH)
`as:say bugler, showing quenched
`
`proximiU of donor (Abz)and
`ceptor (Phe-(p-.NO~))
`phore:,. Fluorescence maguiitldc
`plolted versus eritissioll wave-.
`~e,~gth at 337 nm excitation wave-
`length.
`
`547
`
`Lupin Ex. 1058 (Page 6 of 9)
`
`

`

`{:rvsta] structure+ (22) of’ the {:+-~mplex of s~ u.thdic HIV
`
`NH..
`
`MerJlen bd}avior wuh lh+lear kirteuc+~ over the 20 rain
`
`[40; {l~v v~rsus l[S]} of the inJihfl vdoc~des~ c’a!-
`
`culated {’tom du-: linear phase o1" 1he reactions, gave
`K.,,, :: 37 ::: 81z~.~ (Fig. 5~. V, ...... was catculmed to be
`090 :~: 7{}nmol,min ’m2Lrolcasc . TI~ese
`are similar lo th,:}se ret)oM(~d fbr longer peptid(~ sub-
`
`K ,, = 0.29 --£ ).{ 3 * was calculated {)ore ’v ...... .
`
`observe f}recip~tauon duri~i~ reR)lding of HIV pro-
`
`m.u.}]enl:i<: standard, The chromc)geni{: 5ubstral.es of
`
`450mx.i. Fhc ratio K..._.IK... ]br Abz-N.!:’*-6 with
`thdic HIV protoas¢ is 7.8 -1_: / ,,t~l~.l s . This v’41ue
`is somewhat lower i.]~.an fl-~r longer sllbstl’atc~
`r<:conlbJi]a]-lt 1-tlV iJrol.case expressed in £ coli 123j,
`whiJm fine r;:tl.lO [Ot’ 1:1~¢ l:~c)I~;:l]’tOl-)t~de chromogcnic
`
`SJucc this ~.o~’~ was compIeccd. Mata,.’oshi #t
`{I ? }. and Geogheg ]. ; e~ ~t. (18) have published sh]fiJar
`
`l-n+?l.)arcd 4-14-dil-nd h ylamhioph.enyla7o the nzolc acid-
`Sor..G1]J ~A sn :Tv r- Pio~ Iie-.Val-.(7l i].-’5-[(2-
`}. T inoethvl)an~h~o]naf~l-ithcllono-l-sulfonic aci~ widl a
`l.(~: =:: 103 7: 8,U~,l. a{]d reported a 40-,l%1d increase
`fluorescer]ce yield oi;. deava~je. This is solyl0wt!at
`trlsirig as quenchiru7 is rdated ro distance_ which
`
`/
`
`/
`
`,%00,
`
`Seconds
`
`i000>
`
`FIGURE 4
`
`t~orfion off assay under coaditi{ms used. Jmliat ]dneJics were mon-
`
`!-:1(7 U I-I E $
`
`at ol)dmal excitation (337n~1} mxd emisst(m (4It} ran) waveleng[h.
`Arrow.s indk:aie tht: addilioi~ of HIV protease (i 0 Itg aud 30
`HIV proteasc sloCk :solulion, ]~spectively) Io two dJJ~2:!ent
`cenhafio~s of Abz-NEs-6 fluorogenic substrate {201ts] Jower
`40y~] upper line)in final volume of 2 mL assay buffer, Afier the
`addition nf die HIV protease, lhe stibstr3l.e is immedialeh, cleaved,
`aBd the l]uoresce~c¢ emission Iinearly iucresses {27r up
`Fluor~scence emission plotled versus time.
`
`548
`
`Linew~’avcv+Burk plot of 5tlbstr~ttc kmelics, The I]tl(}rogol-lic: sub-
`
`with (}.Syg ofH1V pro~oase i~a assay builder (final voluine =
`on a microfiler plaie; Fluorescen0e ~ncreases were mo~fitored every
`
`sI;~nd{~rd curve (Fig. 2). A pMt of !/~, versus l/IS] is shown. The
`
`Lupin Ex. 1058 (Page 7 of 9)
`
`

`

`ditious, however, o:}c wouJd pro 0ably ~:tm
`iu duplicate and include :.m inhibitor
`
`per bottr. UfiJ{ziilg lhk~ assay procedure.
`nt.uu her of’ inJlJbitorso1" H I’"~ prolca so h~-~v6 been q uctn-
`
`NOW
`
`Pro~Tc,,im ~44353K t and the Coopeiullve
`Pl’o{tFall] o[tltt ::~;ttlonal ]tlstJl tiics of He;~lth (A}27302) h, gta/cfuli)
`
`;:wkl~owh’.decd Analviica/ data wclx:
`
`R VI:EIt KN(’I:S
`
`~ !)::rke. l’.{ .... bh.ltl ILl:.. fh:<lv S.F G:::’~kv. V.M: Cw-
`c::mne, F,M.. Let:. C.-:F,. l.ummn P,K_ F:e~di:~:e: R.M.,
`
`Moore. M.{.. {{r),an. W.M.. }"ak]tour)’. 7~,/k ~.’~a~.!;~l’d. V.~k:..
`{u~laa . W.[.. {)av~c>n. B.].). Meek /,D..
`
`[)ebc-~c}~. C.: ]9£9~ ~’ioch<’;m 17;~:::7~ R,’.v (’,’>~nmg,?7. 159,
`.120 425
`I foxapeptidcs derived from known <:l<:avage
`
`:I
`
`Sl]bS1.L;~{es Toth. M .k.. Chiu. F,. C]awsol~. L.. Sv:lk. I... E<ul.
`
`5. Nashcd. N.T., l~mis, JM.. Saver../.M.. WorK~<ak I-:M..
`
`/)i<,,dsv~ Rv< (’omm~u~ f.63.
`
`su..,..ffcs[s that ~t l’oldcd con{o;nlatJou mus~
`nat¢ for the substrate, (_}coghegarJ v[ u], {iS) plspa, rod
`%dJ.mech~ larmnona phlhaicne.-/,-> u lfo ~vl-Sc~- (; Ir/--
`,,ksn:J.’yr-Pro-Ile-Val:l.’rp. bul were umfl)lc ~o dc~er-
`mb~e ibe K,,,. v<hich wa~ estimated at ,/~reace~ t.han
`100 m~. The increase in the Trp emission peak upon
`hydrolysis wa~ estimated to be ~.5-,fl~ld -[he ttuores~
`cent substral:c reported here has ~ 1<,, :::: ~7
`arid ~tn hu:rea~c in fluorescence o1" sixlk}ld. While thif~
`
`cd by Matayo~lfi ~’f a!./17), its ~;ma!ler ~izc and ease of
`
`Toma:~zek. T.A .Is.. Maaaard. V.W Bry’:m. H.G.. Mo~,rc..
`
`t.ftilizi~g ttne fluoron:enic stlbstrate, A.bz..NF~..0.
`screening procedure tbr potential HIV prmease in--
`t)i )ilors waq esi.;tblJslJed which allowed the determitm..
`non of inhibitor af]]nity, Eight wells allowed cite deter-
`min;~tion of" the e~)c:t o1: I])HI tlJfl]N’orJf
`Of JrJhibJtor ol] l.wo stlbstlate cotlcellt.ratlOliS which
`was nih~hmi!ly sufficient to cal:ulate a K value t} b~- 6)
`based on a Dixon ploc ~l,v vs, inhibitor
`
`20 rain. Thus. it is f~a~ibIe to determine the aliinity of
`12 inlqMtor~ m 20rain Thi:~is to beccmtrasted with
`the H PLC analysis, wMch requires about 30 separate
`
`for lhc detcrtrdnatio~i of one K,. !_Jltder touline cot)..
`
`R]c]3ards, A.I).. } hv]lf. L.]-].. }:at[i}e]ics,.
`P.E.. A]tatcz, A.. {)unu. B.M. H:i:d.
`
`(.’h~sm. 265,,
`Hylaud. I_,J,. Toma:,~.cq<, -f’.A., .b’,. Dayi~m B,D.
`
`M.I,., Magaa:’d. V W }-]ut]]:]au. W.]" l{eys. I.R,. Sh,.,
`A.Y,I,.. Motcalf. B.W. c~: Meek. ].D
`
`New Yo]’k Acad, Sci { 20
`[’alI][Klrilli. P.P D]’tvcr. R,N,, Itai]sos~ 3.. l_c’tsilT~z<::’, J..
`[Zldnr7. J. Gore-Wiltse. A. l-):dt,,. R.. Hunko. R
`l).. Kail/arztt:k. ~,1.{{. <~: V’ots-.~VarlxTlt.
`
`~U. Carmel. A & Yaron. A. ~. ,,..~ Eur,@~’a~
`2(,5 -273
`Yaron A.. (.;un-~-iel. A & Katdiaist,:A-K,llzir. E.
`/Ti,><’iwm. 95. 22~--235
`
`11
`
`5-19
`
`Lupin Ex. 1058 (Page 8 of 9)
`
`

`

`M.V. Toth and G.R. MarsMtll
`
`12, VcnvilL C,F., Rasnick, D., Crumiey. K.V:, NisMno,
`Powe~-s. J.C. (i985) tYoct~o~i.s’tr..~., 24, 3149--3157
`13 Stack, M,£. & Gray, R.D. (198q) g. !~hd. C’hem, 2{~{, 4277-
`4281
`Malf~’oy, B. & gumier, .I. II987) Biochem. l~i,~l~&,s. Rea’.
`(h)¢*~*m~. 143, 58-66
`
`32, )5-40
`16. Weidn:er~ J.R., Rober*s: R.F. & Dunn. I~.M. {1990)i~
`t ]t], Am. 1)~7~ij~ .~!’~P. (Rivier, J, & Mars[roll. G.R., eds,), pp.
`83l---832, ESCOM. Leiden
`17. Matayoshi, E.D., Wang, G.T.~ Kr~ffL G,A, & Eri~:kson. J.
`
`18, Geoghegan, K.~,, Spencer~ R.W., Darnley. D.E,. Cont~illo=
`L.G,: Jr. & Andrews, G,C, (1980/ FElYS Le~. 262, 119-t22
`19. Kaiser, g., CoJescott, R.L., ~ossmger, C,D, & Cook, P.I.
`(! 970) A~al: 8io(]-~eo,. 34, 595-598
`20. Castro, B.. l)ormoy, .!.--R,, Evin, G. & Selw~, C.
`
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`S.B.I-I, (1990) ,1 Meal. Cl~,,m. 33, 1285-1288
`
`Address:
`
`Prof. Garhgmt R.
`Dept. of Pharmacology
`Washington University School of Medicine
`Box 8103
`~60 Somh Euclid Avenue
`S1. Loui,~. MO 631
`USA
`
`550
`
`Lupin Ex. 1058 (Page 9 of 9)
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