A stereospecifically 18O-labelled deoxydinucleoside phosphate block for incorporation into an oligonucleotide

Fully protected diastereoisomers of deoxyguanylyl (3' leads to 5') deoxyadenosine stereospecifically labelled on phosphorus with oxygen-18 have been synthesized by oxidation of phosphite triester intermediates in the presence of 18O-labelled water. The diastereoisomers have been chromatographically separated and their absolute configuration at phosphorus determined. (Rp)-[18O]deoxyguanylyl (3' leads to 5')deoxyadenosine has been prepared by complete deprotection of the parent diastereoisomer of the Sp configuration. Methylation of the former compound permits assignment of the absolute configurations of the methyl esters of N1-methyldeoxyguanylyl (3' leads to 5') N1-methyldeoxyadenosine.     

Synthesis and separation of diastereoisomers of 5'-O-(4,4'-dimethoxytrimethylphenyl)dithymidyl-(3,5')-4,4' -dimethoxytriphenylmethanephosphonate

"In solution" synthesis and separation of diastereoisomers of 5'-O-(4,4'-dimethoxytriphenylmethyl)dithymidyl (3',5') 4,4'-dimethoxytriphenylmethanephosphonate (DMTTDMTT) is described. One of diastereoisomers was successfully crystallized and characterized by means of HPLC.     

Photooxidation of d(TpG) by phthalocyanines and riboflavin. Isolation and characterization of dinucleoside monophosphates containing the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2''-deoxy-guanosine.

Phthalocyanine mediated photosensitization of 2'-deoxyguanosine (dG) in oxygen saturated aqueous solution has previously been shown to result in the addition of molecular oxygen to the guanine base generating the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine (dO) (the asterisk denotes unambiguous assignment of the 4R and 4S diastereoisomers). The data presented here show that the same guanine modified bases are generated in a 1:1 ratio when thymidylyl-(3',5')-2'-deoxyguanosine (d(TpG)) is similarly photo-oxidized. These modified dinucleoside monophosphates, labelled d(TpO)-A and -B, have been isolated by high performance liquid chromatography and characterized by proton NMR spectrometry, fast atom bombardment mass spectrometry, and enzymatic digestions. Photosensitization in D2O instead of H2O leads to an increase in the rate of d(TpO) formation that is consistent with a type II (singlet oxygen) reaction mechanism. Three interesting properties of these modified dinucleoside monophosphates are: i) the rate of their digestion with spleen phosphodiesterase is greatly reduced relative to d(TpG), ii) they are not digested by snake venom phosphodiesterase, and iii) they are stable to 1.0 M piperidine at 90 degrees C for 30 min. The latter observation indicates that 4,8-dihydro-4-hydroxy-8-oxoguanine is not a base lesion responsible for the strand breaks observed following hot piperidine treatment of DNA exposed to type II photosensitizers or chemically generated singlet oxygen.     

The stereochemical configuration of the natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone

Four possible diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone were chemically synthesized and compared with the natural metabolite by high-pressure liquid chromatography. The four synthetic diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone could be separated into three peaks by high-pressure liquid chromatography. The naturally occurring 1 alpha,25-dihydroxyvitamin D3-26,23-lactone isolated from dog serum and in vitro incubation of chick kidney homogenates comigrated with 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone. The four diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone were tested against naturally occurring 1 alpha,25-dihydroxyvitamin D3-26,23-lactone to determine their relative competition in the 1 alpha,25-dihydroxyvitamin D3-specific cytosol receptor binding assay for 1 alpha,25-dihydroxyvitamin D3. 23(S)25(S)-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone was the best competitor followed by 23(R)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone and 23(R)25(S)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone, and 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone was the poorest competitor. Natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone isolated from dog serum had almost the same binding affinity as that of 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone. These data unequivocally demonstrate that the stereochemistry of the natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone has the 23(S) and 25(R) configuration.     

Nonionic nucleic acid analogues. Synthesis and characterization of dideoxyribonucleoside methylphosphonates.

A series of dideoxyribonucleoside methylphosphonate analogues, dNpN and dNpNp, which contain a nonionic 3'--5' methylphosphonyl internucleoside linkage were prepared. The two diastereoisomers, designated isomers 1 and 2, of each dimer differ in configuration of the methylphosphonate group and were separated by column chromatography. The diastereoisomers of each dimer have different conformations in solution as shown by ultraviolet hypochromicity data and their circular dichroism spectra. For example, dApA isomer 1 is more highly stacked than isomer 2, although both isomers are less stacked than the dinucleoside monophosphate, dApA. The circular dichroism spectrum of isomer 1 is very similar to that of dApA, while the CD spectrum of isomer 2 shows a loss of molecular ellipticity, [theta], at 270 nm and a greatly diminished [theta] at 250 nm. These results suggest that the stacked bases of dApA isomer 1 tend to orient in an oblique manner, while those in isomer 2 tend to orient in a parallel manner. This interpretation is verified by the 1H NMR study of these dimers (L. S. Kan, D. M. Cheng, P. S. Miller, J. Yano, and P. O. P. Ts'o, unpublished experiments). Both diastereoisomers of dAaA form 2U:1A and 2T:1A complexes with poly(U) and poly(dT), respectively. The higher Tm (Tm of poly(U)--isomer 1, 15.4 degrees C; Tm of poly(U)--isomer 2, 19.8 degrees C; Tm of poly(dT)--isomer 1, 18.7 degrees C; Tm of poly(dT)--isomer 2, 18.4 degrees C) values of these complexes vs. those of the corresponding dApA--polynucleotide complexes (Tm of poly(U)--dApA, 7.0 degrees C; Tm of poly(dT)--DApA, 9.2 degrees C) result from decreased charge repulsion between the nonionic dimer backbone and the negatively charged polymer backbone. The difference in conformations between dApA isomer 1 and dApA isomer 2 is reflected in the Tm of the isomer 1-poly(U) complex which is 4.4 degrees C lower than that of the isomer 2-poly(U) complex. Since these nonionic oligonucleotide analogues are taken up by cells in culture, they show promise as molecular probes for the function and structure of nucleic acids inside living cells.     

The first enantioselective synthesis of the amavadin ligand and its complexation to vanadium

The ligand of the naturally occurring vanadium compound amavadin found in Amanita muscaria, (2S, 2'S)-N-hydroxyimino-2,2'-dipropionic acid (1), was synthesized stereoselectively in two steps with 43% overall yield. After complexation of this ligand to vanadyl acetate, amavadin was isolated in quantitative yield. Due to the chirality at vanadium amavadin consists of a mixture of delta and lambda diastereoisomers. Directly after its synthesis, the delta to lambda ratio of amavadin is 2.27 and it decreases to 0.80 after equilibrium has been reached. During this epimerization the optical rotation for V[(2S,2'S)-N-hydroxyimino-(2,2')-dipropionate]2 (=amavadin) changes from [alpha](D)25 = +36 degrees to +114.0 degrees (c = 0.5, H2O). For V[(2R,2'R)-N-hydroxyimino-(2,2')-dipropionate] the optical rotation changes from [alpha](D)25 = -36 degrees to -113.2 degrees (c = 0.5, H2O).     

Synthesis of nucleic acid methylphosphonates via the 1-hydroxybenzotriazole phosphotriester approach.

Dinucleoside methylphosphonates can easily be prepared starting from properly protected d-nucleosides and the bifunctional phosphorylating reagent methyl-O,O-bis(1-benzotriazolyl)phosphate. Separation of the diastereoisomers of 5'-DMTR-d-Ap(Me)T-3'-lev affords optically pure dinucleoside methylphosphonates which, after removal of the 3'-levulinoyl group, have been used for the synthesis of the two optically pure diastereoisomers of the hexamer d-CpGpAp(Me)TpCpG. Further, a one-pot procedure for the preparation of uridine-3',5'-cyclic methylphosphonate will be described. We also found that 3',5'-methylphosphonate linkages in RNA are not stable towards mild acid treatment.     

Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues.

Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.     

Proton magnetic resonance studies of 5,6-saturated thymidine derivatives produced by ionizing radiation. Conformational analysis of 6-hydroxylated diastereoisomers.

The conformational properties of ten 6-hydroxylated dihydrothymidine derivatives including the various diastereoisomers of 5,6-dihydroxy-5,6-dihydrothymidine, 6-hydroxy-5,6-dihydrothymidine and 5-bromo-6-hydroxy-5,6-dihydrothymidine have been studied by 250 MHz proton magnetic resonance in aqueous solutions. A close correlation has been established between the carbon-6 configuration and the osidic conformation. The increase in the amplitude of the puckering within the furanose ring compared to that of thymidine or 2'-deoxyuridine is more pronounced for the levorotatory (6S) nucleosides than for the dextrorotatory (6R) diastereoisomers. The importance of the 2' endo conformer population decreases in the following order: (-) greater than (+) greater than thymidine. The absence of destabilizing effects on the g+ rotameric population about the C(4')-C(5') bond denotes the lack of any interaction between the exocyclic hydroxymethyl group and the 6-hydroxyl function or the 2-keto group. The 5,6-saturated nucleosides adopt a preferential anti conformation. The comparison has been extended to syn nucleosides which show opposite trends in the sugar conformation and g+ distribution.     

The stereochemical configuration of the natural 23,25,26-trihydroxyvitamin D3

Four possible diastereoisomers of 23,25,26-trihydroxyvitamin D3 were synthesized and compared with the natural metabolite. The 4 synthetic diastereoisomers could be separated into 4 peaks by high-performance liquid chromatography. The natural 23,25,26-trihydroxyvitamin D3 comigrated with 23(S),25(R),26-trihydroxyvitamin D3. This result unequivocally demonstrates that the stereochemistry of the natural 23,25,26-trihydroxyvitamin D3 has the 23(S) and 25(R) configuration.     

E. coli Ada regulatory protein repairs the SP diastereoisomer of alkylated DNA

Using HPLC and 31P NMR spectroscopy on a chemically synthesized asymmetric mixture of the diastereoisomers of thymidyl(3'----5')thymidyl-O-methyl phosphate absolute configuration has been correlated with chromatographic mobility. The methyl phosphotriester system in alkylated DNA which is repaired by the Ada regulatory protein of E. coli has consequently been established to possess the Sp configuration.     

The difference of biological activity among four diastereoisomers of 1 alpha,25-dihydroxycholecalciferol-26,23-lactone

All four possible diastereoisomers of 1 alpha,25-dihydroxycholecalciferol-26,23-lactone (1 alpha,25-(OH)2D3-26,23-lactone) were chemically synthesized and were compared to 1 alpha,25-dihydroxycholecalciferol (1 alpha,25(OH)2D3) in terms of their stimulation, in vivo, of intestinal calcium transport and mobilization of calcium from bone in vitamin D-deficient rats (the two classic vitamin D-mediated responses), and their relative binding to the chick intestinal cytosol receptor for 1 alpha,25-(OH)2D3. The receptor binding affinity results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1 alpha,25(OH)2D3. The RCI obtained for 23(S)25(S)-1 alpha,25(OH)2D3-26,23-lactone was 7.90, for 23(R)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 2.27, 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 0.17, for 23(R)25(S)-1 alpha,25(OH)2D3-26,23-lactone 0.22 and for the in vivo produced 1 alpha,25(OH)2D3-26,23-lactone the RCI was only 0.17. Also the four diastereoisomers of 1 alpha,25(OH)2D3-26,23-lactone all stimulated intestinal calcium transport, reaching a maximum 8 h after administration. Compared with the stimulation of intestinal calcium transport by 1 alpha,25(OH)2D3, 23(S)25(S)-1 alpha,25(OH)2D3-26,23-lactone was 1/4 as effective, 23(R)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 1/20 as effective, 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 1/74 as effective and 23(R)25(S)-1 alpha,25(OH)2D3-26,23-lactone was 1/53 as effective. Similarly, 23(S)25(S)-1 alpha,25(OH)2D3-26,23-lactone and 23(R)25(R)-1 alpha,25(OH)2D3-26,23-lactone were estimated to be 3 and 20 times less active than 1 alpha,25-(OH)2D3 in elevation of serum calcium. However, 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone and 23(R)25(S)-1 alpha,25(OH)2D3-26,23-lactone decreased the serum calcium levels 24 h after administration. 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone reduced serum calcium concentrations to a greater extent than 23(R)25(S)-1 alpha,25(OH)2D3-26,23-lactone. These results indicate that the biological activities of the diastereoisomers of 1 alpha,25(OH)2D3-26,23-lactone were quite different among four stereochemical configurations.     

Subsite substrate specificity of midgut insect chymotrypsins

Insect chymotrypsins are distinctively sensitive to plant protein inhibitors, suggesting that they differ in subsite architecture and hence in substrate specificities. Purified digestive chymotrypsins from insects of three different orders were assayed with internally quenched fluorescent oligopeptides with three different amino acids at P1 (Tyr, Phe, and Leu) and 13 amino acid replacements in positions P1', P2, and P3. The binding energy (DeltaG(s), calculated from K(m) values) and the activation energy (DeltaG(T)++, determined from k(cat)/K(m) values) were calculated. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results showed that except for S1, the other subsites (S2, S3, and S1') vary among chymotrypsins. This result contrasts with insect trypsin data that revealed a trend along evolution, putatively associated with resistance to plant inhibitors. In spite of those differences, the data suggested that in lepidopteran chymotrypsins S2 and S1' bind the substrate ground state, whereas only S1' binds the transition state, supporting aspects of the present accepted mechanism of catalysis.     

Extended binding inhibitors of chymotrypsin that interact with leaving group subsites S1'-S3'

We have synthesized inhibitors of chymotrypsin, based on fluoromethyl ketones, that bind at S and S' subsites. "Small" inhibitors of serine proteases, which have previously been synthesized, only interact with S subsites. The parent compound is Ac-Leu-ambo-Phe-CF2H (1) (Ki = 25 X 10(-6) M). This inhibitor was modified by successively replacing H of the -CF2H group by -CH2CH2CONHCH3, (4), -CH2CH2CONH-Leu-NHMe (5), -CH2CH2CONH-Leu-Val-OEt (6), and -CH2CH2CONH-Leu-Arg-OMe (7). Corresponding Ki values are 7.8 (4), 0.23 (5), 0.21 (6), and 0.014 (7) microM. Extending 5 to 6 by addition of Val-OEt at P3' does not decrease Ki. In contrast, extension of 5 to 7 by incorporating Arg-OMe at P3' decreases Ki approximately 15-fold, suggesting interaction between Arg and the S3' subsite but no corresponding interaction at that subsite with Val. These results are in accordance with results obtained with the homologous family of avian ovomucoid third domain proteins. Proteins with Arg at the P3' position show highly favorable interactions with the protease at the S3' subsite [Park, S. J. (1985) Ph.D. Thesis, Purdue University; M. Laskowski, Jr., personal communication]. These results establish that incorporation of residues which interact with S' subsites significantly increases the efficacy of inhibitors and that valuable information concerning the most effective amino acid composition of small inhibitors can be obtained from the amino acid sequence of protein inhibitors.     

Combining mutations in HIV-1 protease to understand mechanisms of resistance

HIV-1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV-1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2-1.2 A to determine the associated molecular changes. Sequence-dependent changes in protease-inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2', P3/P3'/P4/P4', and P1/P1', respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6(pol)-PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag-Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2-P2' and S2-S2' sites. The complex effects of combining mutations are discussed.     

2',5'-Oligoadenylates chiral at phosphorus: enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioate trimer and tetramer analogues synthesized from (SP)-ATP alpha S

The enzymatic synthesis and characterization of (RP)-2',5'-AMPS trimer and tetramer (SP)-5'-O-(1-thiotriphosphates) from chirally substituted (SP)-[alpha-35S]ATP alpha S by 2',5'-oligoadenylate synthetase from interferon-treated L cell extracts are described. The (RP)-ATP alpha S isomer is not a substrate for the synthetase. The identification of the trimer and tetramer analogues (molar ratio 70:30) was accomplished by high-performance liquid chromatography and subsequent separation by charge using DEAE-cellulose thin-layer chromatography. The digestion of the analogue by snake venom phosphodiesterase I (SVPD) to [alpha-35S]ATP alpha S and [35S]AMPS but not by T2 RNase demonstrated the presence of the 2',5' linkage. The assignment of RP configuration of the 2',5'-phosphorothiodiester linkage was based on the highly specific stereoselectivity of SVPD for RP diastereomers [Burgers, P. M. J., & Eckstein, F. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4978-4800; Bryant, F. R., & Benkovic, S. J. (1979) Biochemistry 18, 2825-2828; Nelson, P. S., Bach, C. T., & Verheyden, J. P. H. (1984) J. Org. Chem. 49, 2314-2317]. This suggests that the synthesis of the phosphorothioate analogues proceeded via inversion of configuration at the chiral phosphorus of (SP)-ATP alpha S. The putative (RP)-2',5'-AMPS tetramer (SP)-5'-O-(1-thiotriphosphate) displaced the 2',5'-p3A4[32P]pCp analogue from 2',5'-oligoadenylate-dependent endonuclease 5 times more efficiently than did equimolar concentrations of authentic 2',5'-adenylate tetramer triphosphate. Furthermore, in studies using the calcium phosphate coprecipitation technique, the 2',5'-phosphorothioate trimer and tetramer analogues inhibited protein synthesis better than did 2',5'-adenylate trimer and tetramer triphosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.     

A molecular modeling analysis of novel non-hydroxamate inhibitors of TACE

Recently, an X-ray co-crystal structure of our hydroxamate inhibitor IK682 and TACE [Niu, X.; Umland, S.; Ingram, R.; Beyer, B. M.; Liu, Y.-H.; Sun, J.; Lundell, D.; Orth, P. Arch. Biochem. Biophys. 2006, 451, 43-50] was published that explicitly shows the orientation of the hydroxamate and the TACE-selective 4-[(2-methyl-4-quinolinyl)methoxy]phenyl P1' group in the S1' and S3' sites. The preceding paper described a novel series of potent and TACE-selective hydantoins and we previously described pyrimidinetrione (barbiturate) inhibitors of TACE, both of which contain the same P1' group as IK682. Using this TACE-selective P1' group as an anchor, stereochemical and conformational constraints in the inhibitors, and restrictions to the active site Zn coordination geometry, we developed a highly plausible and predictive pharmacophore model that rationalizes the observed TACE activity of all three inhibitors.