J.Med. Chem. 1996,38,1974-1989
1974
Three-Dimensional Quantitative Structure-Activity Relationships of Somatostatin Analogues. 1. Comparative Molecular Field Analysis of Growth Hormone Release-Inhibiting Potencies Simon J. Hocart,* Vik Reddy,+William A. Murphy, and David H. Coy Peptide Research Laboratories, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112, and Tripos Associates Inc., St Louis, Missouri 63144 Received November 4, 1994@
Somatostatin is a hypothalamic hormone that inhibits the release of growth hormone (GH). It has also been shown to inhibit the release of a broad range of hormones including insulin, glucagon, and gastrin. Presently, five different receptor subtypes of somatostatin have been characterized and cloned. Our previous work on the structure-activity relationship of somatostatin and that of many others has generated a large database of analogues with different biological activities and receptor affinities. This present work is a n investigation of the growth hormone release-inhibiting potencies of somatostatin analogues by the three-dimensional quantitative structure-activity paradigm, comparative molecular field analysis (CoMFA). A total of 64 analogues were modeled in SYBYL using structural information from two NMR studies. The molecules were aligned by a root-mean-square fit of atoms and field-fit of the steric and electrostatic molecular fields and the resulting databases analyzed by partial least squares analysis with cross-validation to extract the optimum number of components. The analysis was then repeated without cross-validation to give the final QSAR models. Preliminary investigations with the CoMFA models led to the synthesis of a new somatostatin analogue. This compound together with five other newly synthesized compounds not included in the original training sets were used t o test the predictive ability of the CoMFA models. Two models with good predictive powers are presented. between a receptor and its ligand, or an enzyme and Introduction its substrate or inhibitor, are primarily noncovalent in Somatostatin, Ala1-Gly2-c[Cys3-Lys4-Asn5-Phe6-Phe7nature and shape-dependent.12J3 Therefore, a QSAR Trps-Lysg-Thr10-Phe11-Thr12-Ser13-Cys14], a hypothamay be derived by sampling the steric and electrostatic lamic factor that inhibits the release of growth hormone fields surrounding a set of ligands and correlating the (GH) was first isolated from ovine hypothalami in differences in those fields to biological activity. Partial 1972.1,2 It has since been shown to inhibit the release least squares (PLS) analysis is employed to extract a of a broad range of hormones including insulin, glucagon, and gastrin and has been located in many tissues QSAR from the large comparative molecular field including the gut, the pancreas, and the nervous system. analysis (CoMFA) data table produced. In PLS, the The broad spectrum of biological activity of somatostatin “best equation” is obtained by selecting the proper and its very short half-life in the blood stream reduce number of components (latent variables) using a crossthe therapeutic value of the native peptide. validation procedure. In this way the dimensionality Structure-activity studies have demonstrated that of the model is chosen according t o its ability to predict the sequence Phe7-Trp8-Lys9-Thr10 is essential for GH the data rather than to fit the data. Several successful release-inhibiting a ~ t i v i t y . ~These -~ studies have reCoMFA studies of small, conformationally constrained sulted in the synthesis of a large number of analogues systems have been reported including polyhalogenated of somatostatin which exhibit a variety of bioactivity dibenzo-p-dioxins,dibenzofurans, and biphenyls,14 cloprofiles. The various actions of these somatostatin dronic acid esters,15and progestin and androgen recepanalogues are mediated through different membranetor binding.16 Conformationally flexible systems which bound receptor subtypes. Recently, the existence of have been studied include inhibitors of protein-tyrosine several receptor subtypes has been conclusively demkinase,17 human rhinovirus 14,17thermoly~in,’~J~ reonstrated by molecular cloning. Presently, five receptor (I).2o nin,l* and human immunodeficiency virus protease subtypes (SSTRl-5) have been chara~terized.~-~ PreviThese studies used the X-ray crystallographic structure ous research in our laboratory and others has concenof the ligands bound to the enzymes to derive the trated on the identification of subtype-selective analogues of somatostatin.lOJ1 These structure-activity molecular conformation and alignment rules for the 3-D studies have resulted in a large database of noncongeQSAR models. Angiotensin-converting enzyme (ACE) neric somatostatin analogues in the literature. has been studied using an active analogue approach as The comparative molecular field analysis method of the basis for the molecular conformation and alignments three-dimensional structure-activity relationships (3-D of the ACE inhibitors.lg The results were comparable QSAR)is based on the assumption that the interactions to those obtained with the closely related thermolysin inhibitors using alignment rules derived from crystalt Current address: National Starch and Chemical Co., Central lographic data. In this paper we will present a prelimiAnalytical Group, Bridgewater, NJ 08807. nary CoMFA investigation of the GH release-inhibiting Abstract published in Advance ACS Abstracts, May 1, 1995. @
0022-2623/95/1838-1974$09.00/0 1995 American Chemical Society
Journal of Medicinal Chemistry,1995, Vol. 38, No. 11 1975
3-0 QSAR of Somatostatin Analogues
Table 1. Structure of Somatostatin Analogues and GH Release-InhibitionPotencies compd no 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64
code name BIM-23014 BIM-23030 BIM-23034 BIM-23042 BIM-23049 BIM-23050 BIM-23051 BIM-23052 BIM-23053 BIM-23055 BIM-23056 BIM-23057 BIM-23058 BIM-23059 BIM-23060 BIM-23063 BIM-23064 BIM-23065 BIM-23066 BIM-23067 BIM-23068 BIM-23069 BIM-23070 L-362-823 L-362-855 L-362-862 L-363-301 L-363-376 MK-678 NC4-28B NC8-12 5 SMS-201-99 DC23-60 EC5-21
modeled from 3 3
GHI' DNal-c[Cys-Tvr-DTr~-Lvs-Val-Cvsl-Thr-NH~ 0.83 c[Mpa-~-DTrp-Lys~V~l-Cysl-PhelNHz 23 structure
pGHIb
0.08 -1.36 DPhe-c[Cys-Tyr-DTrp-Lys-Val-Cysl-Nal-NHz 0.37 0.43 3 DNal-c[Cys-Tyr-DTrp-Lys-Val-Cysl-Nal-NHz 11 -1.04 DNal-Ala-Tyr-DTrp-Lys-Val-Ala-Thr-NHz 3 -2.64 435 NMeDAla-Tyr-DTrp-Lys-Val-Phe-NHz 3 -4.30 20000 DPhe-Ala-Phe-DTrp-Lys-Thr-Ala-Thr-NHz 2500 5 -3.40 3 DPhe-Phe-Phe-DTrp-Lys-Thr-Phe-Thr-NHz -1.89 77 -1.17 DPhe-Ala-Tyr-DTrp-Lys-Val-Ala-Nal-NHz 15 5 8 DPhe-Phe-Tyr-DTrp-Lys-Val-Phe-DPhe-NHz 11100 -4.05 DPhe-Phe-Tyr-DTrp-Lys-Val-Phe-DNal-NHz 12500 8 -4.10 -1.23 DPhe-Cpa-Tyr-DTrp-Lys-Val-Phe-Thr-NHz 17 8 DPhe-Phe-Tyr-DTrp-Lys-Val-Phe-Thr-NHz 111 8 -2.04 0.72 3 DNal-c[Cys-T'p-DTrp-Lys-Thr-Cysl-Thr-NHz 0.19 0.05 DPhe-c[Cys-Tyr-DTrp-Lys-Thr-Cysl-Nal-NHz 3 1.30 DPhe-Cpa-Tyr-DTrp-Lys-Thr-Phe-Nal-NHz 8 100 -2.00 8 DPhe-Cpa-Tyr-DTrp-Lys-Val-Phe-DAla-NHz 4000 -3.60 17 3 DNal-Cpa-Tyr-DTrp-Lys-Thr-Phe-Thr-NHz -1.23 8 DPhe-Nif-Tyr-DTrp-Lys-Val-Phe-Thr-NHz 159 -2.20 5 DCpa-Ala-Tyr-DTrp-Lys-Val-Ala-DPhe-NHz 417 -2.62 8 DPhe-Cpa-Tyr-DTrp-Lys-Thr-Phe-Thr-NHz 3.4 -0.53 -1.65 5 DCpa-Ala-Tyr-DTrp-Lys-Val-Ala-Nal-NHz 45 5 DPhe-Ala-Tyr-DTrp-Lys-Thr-Ala-Nal-NHz 16 -1.20 3 0.09 c[Aha-c[Cys-Phe-DTrp-Lys-Thr-Cysll 0.81 24 c[Aha-Phe-Trp-DTrp-Lys-Thr-Phel -1.22 16.7 24 c[Aha-Phe-Cpa-DTrp-Lys-Thr-Phel -0.15 1.4 28 c[Pro-Phe-DTrp-Lys-Thr-Phel 0.23 0.59 3 c[Pro-Ala-DTrp-Lys-Thr-Phe] 5 -0.70 c[NMeAla-Tyr-DTrp-Lys-Val-Phe] 28 1.70 0.02 3 DPhe-c[Cys-Tyr-DTrp-Lys-Ser-Cysl-Nal-NHz 1.52 0.03 1.52 3 DPhe-c[Cys-Tyr-DTrp-Lys-Abu-Cysl-Nal-NHz 0.03 3 DPhe-c[Cys-Phe-DTrp-Lys-Thr-Cysl-Thr-OL 0.03 0.93 DNal-c[Cys-Tyr-DTrp-Lys-Val-Cysl-Thr-OH 3 -0.03 1.07 DPhe-c[Cys-Phe-DTrp-Lys-Thr-Cysl-Nal-NHz 3 -0.66 0.22 28 c[NMeAla-Phe-DTrp-Lys-Thr-PhelC 0.54 0.29 1.16 c[Pro-Tyr-DTrp-Lys-Thr-Phelc 0.07 28 0.54 c[Pro-Phe-DTrp-Lys-Val-PheE 28 0.29 28 0.15 c[Pro-Phe-DTrp-Lys-Ser-PheY 0.71 -0.10 c[Pro-Phe-DTrp-Lys-Abu-Phelc 1.25 28 24 c[ Aha-Phe-Phe-DTrp-Lys-Thr-Pheld 1.08 -0.03 24 c[Aha-Phe-Tyr-DTrp-Lys-Thr-Pheld -0.02 1.04 c[Aha-Phe-Fpa-DTrp-L~s-Thr-Phe]~ 24 20 -1.30 c[Aha-Phe-Nif-DTrp-Lys-Thr-PheP 24 -0.54 3.5 24 c[Aha-Phe-Amf-DTrp-Lys-Thr-Pheld -1.54 35 c[Aha-Phe-Thz-DTrp-Lys-Thr-Pheld 24 5 -0.70 c[ Aha-Phe-Leu-DTrp-L~s-Thr-Phe]~ 24 -0.02 1.05 c[Aha-PheAla-DTrp-L~s-Thr-Phe]~ 24 -0.80 6.25 24 -0.26 c[Aha-Phe-Phe-l-MeTrp-Amf-Thr-Pheld)e 1.8 24 -1.15 14 c[Aha-Phe-Phe-5-MeTrp-Lys-Thr-Pheldse 24 c[Aha-Phe-Phe-5-FTrp-Ly~-Thr-Phel~.~ 6.7 -0.83 c[Aha-Phe-Phe-6-FTrp-Lys-Thr-Pheldje 24 2.4 -0.38 24 c[Aha-Phe-Phe-5-BrTrp-Lys-Thr-Pheldae 50 -1.70 ~[Aha-Phe-Phe-5-0MeTrp-Lys-Thr-Phe]~~~ 50 -1.70 24 c[ Aha-Phe-Phe-DTrp-Orn-Thr-PheId 20 24 -1.30 24 c[Aha-Phe-Phe-DTrp-Ag-Thr-Pheld 14 -1.15 24 c[Aha-Phe-Phe-DTrp-L~s-MeAla-Phe]~ 50 -1.70 c[Aha-Phe-Phe-DTrp-Lys-Acp-Pheld 6.7 24 -0.83 16.7 -1.22 c[Pro-Phe-DTrp-Lys-Thr-AlaY 28 c[Pro-Phe-DTrp-Lys-Thr-ProY 125 -2.10 28 c[DPro-Phe-DTrp-Lys-Thr-DAlaY 28 166 -2.22 c[Pro-Phe-DTrp-Lys-Thr-DAla 28 500 -2.70 c[Phe-Phe-DTrp-Lys-Thr-PheY 3.71 -0.57 28 0.4 c[c[Cys-Phe-DTrp-Lys-Thr-Cys]F 3 0.40 c[c[Cvs-Tvr-DT~-Lvs-Val-Cvsllg 63 0.06 1.20 - - -a GH release-inhibitingpotency relative to somatostatin (=1.0). Expressed as the negative logarithm of the GH release-inhibiting potency. Reference 35. Reference 36.e Tryptophan derivative in this analogue was not resolved so peptide was modeled in the R (D) configuration.f Reference 37.8Reference 38. ~~
-
1
*
activity of somatostatin analogues based on structural data from solution phase NMR studies.21y22
Methods Abbreviations:Abbreviations of the common amino acids are in accordancewith the recommendationsofILJPAC-IuB.23 Additional abbreviationsinclude: Abu,2-aminobutanoicacid;
Acp, 1-amino-1-cyclopentanecarboxylic acid; Aha, 7-aminoheptanoic acid;Ahx, 6-aminohexanoicacid;Amf,p-aminophenylalanine;Cpa,p-chlorophenylalanine;Dip, 3,3-diphenylalanine; Fpa,p-fluorophenylalanine;Har, homoarginine; Nal, 3-(2-naphthyl)alanine;Mpa, mercaptoproprionic acid; Nif, p-nitrophenylalanine; Thz,3-(44hiazolyl)alanine;Tic,1,2,3,4tetrahydroisoquinoline-3-carboxylic acid. Following the suggestion of Clementi,24the abbreviation q2 is used in favor of
1976 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 11
Hocart et al.
Table 2. Conformation of BIM-23034 from Each CoMFA Model model A DPhe5 Cy@ 5r7 DTrp8 Lys9 VallO Cys" Nail2
d
w
X1
-64 -61 76 -139 58 -60 -73
-39 161 174 -72 41 16 -179 67
50 -34 64 -179 -54 -170 93 -52
X2
6
76 81 113 --173 105
w
X1
61 28 -69 68 -63 -82 -80 -141
the SYBYL standard "cross-validated rz"to distinguish it from the analogous conventional r2. Peptide Synthesis. Peptides were synthesized by a solidphase synthesis methodology as described p r e v i ~ u s l y .Briefly, ~~ the peptide amides were synthesized on a 4-methylbenzhydrylamine substituted, 1% cross-linked polystyrene resin (Advanced ChemTech, Louisville, KY) using butyloxycarbonyl Na protection. Peptide acids were elongated on a conventional Merrifield resin (Advanced ChemTech). The crude peptides were cleaved and deprotected with anhydrous hydrogen fluoride a t 0 "C. The cysteine containing analogues were cyclized in 90% acetic acid with a slight excess of 12. DC-S-10-96 was synthesized using BocLys(Fmoc). The protected peptide was cleaved from the Memifield resin by HF, cyclized with @BOP, and deprotected with piperidine. All the analogues were purified t o homogeneity by reversed-phase liquid chromatography and gave satisfactory molecular weights by matrixassisted laser desorption mass spectrometry (LaserMat, Finnegan MAT, San Jose, CA) and amino acid analyses. The analytical data will be published elsewhere.26 GH Release Inhibiting Potency. Assays to determine the in vitro GH release inhibiting potency (IC501 were performed as described p r e v i o u ~ l y . Anterior ~~ pituitaries from adult male rats (200-250 g) were dispersed aseptically by a trypsin/DNase method. The dispersed cells were diluted with sterile Dulbecco's modified Eagles's medium (GIBCO, Grand Island, NY)supplemented with 2.5% fetal calf serum (GIBCO), 3% horse serum (GIBCO), 10% fresh rat serum from the pituitary donors, 1% minimum essential medium nonessential amino acids (GIBCO), 10 ng/ml gentamycin (Sigma Chemical Co., St Louis, MO), and 10 000 units/mL nystatin (GIBCO). The cells were counted with a haemocytometer and randomly plated a t a density of 2 x lo5 cells/well (Costar Cluster 24, Rochester Scientific, Rochester, NY). The plated cells were maintained in the Dulbecco's medium described above in a humidified atmosphere of 95% air and 5% C02 at 37 "C for 96 h. In preparation for hormone challenge, the cells were washed three times with medium 199 (GIBCO). Each dose of analogue (diluted in siliconized test tubes) was tested in quadruplicate wells, in a total volume of 1 mL of medium 199 containing 1%bovine serum albumin. Cells were pulsed in the presence of 1 nM GRF(1-29)NH2, in the presence or absence of various concentrations of somatostatin analogues. After 3 h a t 37 "C in a 95% air/5% C02 atmosphere, the medium was removed and stored a t -20 "C until the time of the GH radioimmunoassay. GH in plasma and media was measured by a standard double-antibody radioimmunoassay using components generously supplied through NHPP, NIDDK, NICHHD, and USDA. Potencies relative to somatostatin (=LO) were calculated by four-point assay28from the mean of a least three experiments or by comparison of ICso's from doseresponse curves. Additional data and analogues were obtained from the literature as indicated by the references in Table 1. Molecular Modeling. All molecular modeling and CoMFA studies were performed on a Silicon Graphics Personal Iris 4D/35TG+ computer. Molecular databases containing 64 somatostatin analogues (see Table 1)were modeled in SYBYL 6.0329using the standard TRIPOS force field.30 The first 34 compounds were taken from an earlier publication from this group,'O and the remainder were obtained from the literature as indicated in Table 1. The first CoMFA model (model A) was based on the conformational inferences of Van Binst and T 0 u n v 6 . ~Compound ~ 3
model C
model B
50
81 -139 -33 77 173 154
41 -71 -52 176 -63 -59 -58 -67
X2
6
74 97 -87 -,179 --104
Y 56
32 -73 80 -60 -67 -77 -146
61 71 -144 -31 74 163 158
X1
39 -61 -44 177 -68 -65 -67 -66
X2
71 120 -94 176 86
(BIM-23034;DPhe5-c[Cys6-Tyr7-D~rp8-Lysg-Va110-Cys111-Na11 NH2) was built from the predefined amino acids and its conformation set to /3 sheet. The type 11' bend was introduced around DTrp8-Lysgand the cysteine bridge formed. Partial atomic charges were calculated for the molecule with a charged amino terminus using the Pullman m e t h ~ d . The ~ ~ ,geom~~ etries of the extra cyclic residues, DPhe5 and Nail2, were adjusted visually to facilitate hydrogen bond formation between DPhesCO and Na112NH.21 The structure was then optimized by energy minimization using the Broyden, Fletcher, Goldfarb, and Shannon (BFGS) algorithm to a final root mean square (rms) gradient of 0.1 kcal mol A-l. A distancedependent dielectric function33 was employed together with the default settings for all the other minimization options. This resulted in a conformation for compound 3 in which the N and C termini were hydrogen bonded (see Table 2 and Figure la). The remaining compounds were modeled by mutating the residues of compound 3 where possible or by replacing the residues for the cases in which a perfect backbone fit was not possible. The actual analogue used t o form each compound is indicated in Table 10. The unchanged residues of the new analogue were defined as an aggregate to constrain their conformation during the initial minimization of the modified residues. The constraints were then removed and the minimization repeated. The second CoMFA model was based on the folded conformation of He and Huang et al.22,34Since the minimization of model A to a final gradient of 0.1 kcal mol A-1 had been time consuming, this CoMFA database was minimized in two stages. Initially the analogues were minimized to an intermediate gradient and then the database reminimized to a final gradient using a simple SYBYL Programming Language (SPL) script. The cyclic hexapeptide c[Pro6-Phe7-(2R,3S)-/3-MeTrp8L y ~ ~ - T h r ~ O - Pwas h e ~modeled ~l from the published 4, q, and ~1 values,22 cyclized and minimized with Pullman partial atomic charges to an rms gradient of 1 kcal mol A-1. This structure was used as the basis for the conformation of the template compound 3. The cyclic hexapeptide was mutated to H-Cys6-Phe7-DTrp8-Lyss-~10-Cys11-OH. The cysteine bridge was formed and minimized with the remainder of the sequence (Phe7:Thr10)defined as an aggregate. The peptide was elongated to the structure of compound 3 and the pendant amino acids and cystine bridge minimized to an rms gradient of 1 kcal mol A-1 with the Phe7:Thr10aggregate present t o remove any bad steric contacts between the extracyclic amino acids while maintaining the conformation of the ring. Finally, the aggregate constraint was removed and the analogue reminimized to a final rms gradient of
