J . Am. CIteiit. Soc. 1995. 117, 5381F.5382
5381
A Transition State Analogue Inhibitor Conihinatorial Lihrar? D:i\ 111 . A C:imphcll.' J:ison C. Berm:ih. Tiiiioth! S. Rurhoth. ;md Dincsh V . Pitel
Cnmtvnaturial libraries are emerging a s an intesral part of the mcdicin.il chemist's repertoire in the search fkr therapeutic ;igents.' Traditionally the development of a new drug begins n tth the identification of a lead compound senerated from natural product collections or in-house chemical databases. Once a lead compound has been identified. medicinal chemists seriallv synthesize hundrcds to thousands of individual variants of the original structure, with each variant submitted for biological testing to optimize in vitro and hi vivo therapeutic efficacy. The ability to construct synthetic combinatorial libraries, which now include biopolymers.? nonnatural polymers.i and nonpolymeric organic compounds.' facilitates this process. Our strategy has becn to incorporate pharmacophores of proven therapeutic value into combinatorial libraries. Replacement of a peptide substrate's scissile carboxamide group with a phosphonic acid ester has yielded potent inhibitors of nietalloprotciiiases.' including a number of orally active ACE inhibitors developed by the Squibb proup." W e recently .~ described an impr&d prncedure for the synthesis of phosphonic acid esters' as well as the solid uhase svnthesis of Deotidvlohos. . ,. phonates.' We now report the constructinn of a peptidylphosphonaie combinatorial library and characterization of its interactinns with thennolysin. a well-studied zinc endopeptidase from B90%." The peptidylphosphonatc library was assayed for thermolysin inhibition while attached to the resin.'" After rank ordering of each mixture with a depletion assay," an iterative strategy for active sequence identification was employed." Thus. after each round of screening, the most active pool was selected for deconvolution. Each subpool was then synthesized and ahsayed with the most active mixtures then chosen for the next round of screening. The X' residue library consisted of six mixtures of peptidylphosphonate sequences (Cbz-XI'-"Y-Z-resin), with each mixture containing YO compounds. While all of the mixtures interacted with thermolysin t o some extent compared to thc control (acetylated resin), a definite rank ordering was observed I
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Voii. Acral. S C . ~ .: G ~ c m l c c W . . J.: Pxchcn. A.
I . .ticli. C I , ~ , , i~w, .x . .ii. xis. 171 !ai C;mlphell. D.A. .I. 0,i'. Clicrri. 1992. 57. 6331. Ihl C;mphell. D. .\.: 8tnn.1~.J . c .I. or,v.('IW,,~.I Y Y ~ .5 ~ 658. . I X l C;imphcll. D. h.: Hcrmak. J. C . J. hi.Clwm . h c . 1994. 110. hO39,
0002-i863/95/151T-53R1%090010
0 1995 American Chemical Society
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Leu, are I .OS,X.4, 360, and 57X. respectively. in agreement uith the P I ' library rank (rrdcr.?" The Z position library cnnsisted of I8 individual peptidylphosphonate sequences (Cbz-PheP-"Leu-Z-resin). This l i b brary contained a number of iimintr acids that exhibited a hish affinity for thermdysin (Figure 2c). In addition ti) identifyins the most potent peptidylphrisphonate sequence ftrr inhibition (if thermolysin described in the literature (P?' = Ala).'" thi\ combinatorial strategy uncovered additional active sequence\ containing the basic aminrr acids arginine and histidine and the carboxamide side chain amino acid glutamine at P i . Since the tethered library does not enable ii priori deteminiltion of carboxy terminus preference. Cbz-(R.S)-PheP-"LeuAla-X was synthesized as both the carboxylic acid 1 (X= OH) and the amide 2 (X = NH?). The amide 2 was 2.5 times more potent than the carboxylic acid 1 (49 versus 122 nM).?' Based I ' deconvolution (bl on this observation. all remaining peptidylphosphonates were synthesized as amides. Sequences containing the basic side C b z - X p a Z Q chains histidine 3 ( K , = 57 nM) and arginine 4 ( K , = 64 nM) at P i were equipotent to 2. while slightly lower activity was obtained with glutamine at P i 5 ( K , = 127 nM).?' These represent novel inhibiror.7 of rhermoly.sin, rind their discovery WOS unexpected sirice d l rhe hihihitors thnt hove hemi reporred in the literriture u " i n hydrophobic re.~idi!e.~ o f the P.' porition.2i This study emphasizes some important advantages of the cumbinatorial library approach to inhibitor discovery. As a consequence of the limited number of compounds a medicinal chemist can synthesize in a reasonable amount of time, the compounds chosen for synthesis are constrained by pre-existing knowledge about the system under study. As a result, the work P2' deconvolution (Cl takes place in a local minimum that probably constrains most competitors working in the field also. However, by judicious choice of the mnnomer basis set. combinatorial strategies investigate a far greater region of space and are more likely t n discover configurations that are closer to the absolute local minima or exist in divergent Inca1 minima. An additional advantage is that besides identifying a lead crimp~iundisl. significant structure-activity data are generated which can then be used for k vivo activity optimization. In summary, a peptidylphrisphonate combinatorial library has been constructed and used to idcntiSy a number of potent themolysin inhibitors. The mint active peptidylphosphonates are Cbr-(R,S)-Phel'-"Leu-Arg(NH?) ( K , = 64 nM), Cbz-(R.S)6. ns PheP-"Leu-His(NH?) ( K , = 57 nM). and Cbz-(R.S)-PheP~"LeuAla(NH2) ( K , = 49 nM). The histidine and arginine residues Figurc 2. l)~q,lc:t,m .tn:t! r c w l t \ . I',>ICIII lpqm+ l p l ~ ~ ~ y ~ l inhihi~cm,~t~ at P i represent a significant deviation from themolysin ton 01 t h ~ r i n dw ~r e~ iduntiiicd ~ ~ ~ win: .in i t ~ l i i t i i u \trategy 10 inhibitors previously described, which typically contain hydrod e i ~ m i the i ~ ~ c q i i i l i i c ~ofs the i m m t i i c t i w lihrary cmixtuccs. The y-axis phobic groups at that position. The strategy US constructing \hen\ the h!drolysi< v.w ImAimin). Error h s s indicate standard combinatorial libraries that incorporate a pharmacuphore within J c i iimoiii a biopolymer to increase inhibitor potency, as well as to reduce (Figure ?a). The rank order indicated the following preference the size of the inhibitors, has now been validated and should at thc P I position: Phe" > Leu" > llel', Ala" > Val" > FlyP, be applicable to other pharmacophores and enzymes. ujhich is i n agreement" with a series of peptidylphosphonate Supplementary Material Availahle: linperimenial prooedurc\ Ibr inhibitors that have been described in the literature (Cbz-XI'construction nf thc peptidylphosphon;Ite lihrary. the d r c m v d u t i o n "Leu-AMOH). where X = Phe. Leu, Ala. Gly have K, = 45 pnm,col. and the inhihitor K , dclcmmin;ilion pmcedurc 14 page'!. Thn nhl. hXO nM. 1.X pM. and I ? ,uM. respectively).',5 miterial i s contained in many I i h m r m on micmlichc. immcdi;itely The "Y prrsition library consisted of five mixtures, each follows this article in thc microfilm vcrcion 01 the j w m a l . can he containing I X peptidylphosphonate sequences (Cbz-PheP-"Yordered lmm the ACS. and can he downloaded Irom thc Internct: w e any current m;icthead p a y for c,rdcrmg inlirnmatim and Intcmet iiccc\\ Z-resin). The depletion assay indicated that the a-hydroxy acid instructions. analogue of leucine was the preferred PI' residue (Figure 2b). Although a series of peptidylphosphonate inhibitors with vxying JAY44133U PI' residues has not been described in the literature. obviating I201 Morihara. K.: T w m k i . H. liw .I. liindion. 1970. I S . 374. direct comparisons with the "Y position library rank order, I21 I K , YBIUCI fbr hoth eiimtioiiicr\ of Chr-Fhel'-"Lru~AI;~lOHihme hem reponcd I45 nM and 30 !'M for the K and 5 m m t ~ o m c r ~ . Bxtlett has shown that ii linear cnrrelation exists between the re\prcuvely!." HP1.C' a n a l p \ indic;aed rhat the I U O dia\rereomer
