Abstracts

Recent publications

1. A. Mansour, L.P. Taylor, J.L. Fine, R.C. Thompson, M.T. Hoversten, H.I. Mosberg, S.J. Watson, and H. Akil. Key residues defining the opioid receptor binding pocket: a site-directed mutagenesis study J. Neurochem., 68:344-353, 1997.
   Abstract.
   Structural elements of the rat m-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the mu, delta, and kappa receptors (Asn1⁵⁰ to Ala, His²⁹⁷ to Ala, and Tyr³²⁶ to Phe) and two designed to test for m/d selectivity (Ile¹⁹⁶ to Val and Val²⁰² to Ile). Mutation of His²⁹⁷ in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (³H-labeled D-Ala², N-Me-Phe⁴, Gly-ol5-enkephalin), [³H]bremazocine, or [³H]ethylketocyclazocine. Mutation of Asn150 in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, beta-endorphin1-31, JOM-13, deltorphin II, dynorphin1-13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr³²⁶ mutation in TM7 resulted in a decreased affinity for a wide spectrum of m, d and k agonists and antagonists. Altering Val²⁰² to Ile in TM4 produced no change on ligand affinity, but Ile¹⁹⁶ to Val resulted in a four- to fivefold decreased affinity for the m agonists morphine and DAMGO, with no change in the binding affinities of k and d ligands.

 

2. Lomize A.L. and Mosberg H.I. Thermodynamic model of secondary structure for a-helical peptides and proteins. Biopolymers. 42(2):239-269, 1997
   Abstract.
   A thermodynamic model describing formation of a-helices by peptides and proteins in the absence of specific tertiary interactions has been developed. The model combines free energy terms defining a-helix stability in aqueous solution and terms describing immersion of every helix or fragment of coil into a micelle or a nonpolar droplet created by the rest of protein to calculate averaged or lowest energy partitioning of the peptide chain into helical and coil fragments. The a-helix energy in water was calculated with parameters derived from peptide substitution and protein engineering data and using estimates of nonpolar contact areas between side chains. The energy of nonspecific hydrophobic interactions was estimated considering each a-helix or fragment of coil as freely floating in the spherical micelle or droplet, and using water/cyclohexane (for micelles) or adjustable (for proteins) side-chain transfer energies. The model was verified for 96 and 36 peptides studied by ¹H-nmr spectroscopy in aqueous solution and in the presence of micelles, respectively ([set 1] and [set 2]) and for 30 mostly a-helical globular proteins ([set 3]). For peptides, the experimental helix locations were identified from the published medium-range nuclear Overhauser effects detected by ¹H-nmr spectroscopy. For sets 1, 2, and 3, respectively, 93, 100, and 97% of helices were identified with average errors in calculation of helix boundaries of 1.3, 2.0, and 4.1 residues per helix and an average percentage of correctly calculated helix-coil states of 93, 89, and 81%, respectively. Analysis of adjustable parameters of the model (the entropy and enthalpy of the helix-coil transition, the transfer energy of the helix backbone, and parameters of the bound coil), determined by minimization of the average helix boundary deviation for each set of peptides or proteins, demonstrates that, unlike micelles, the interior of the effective protein droplet has solubility characteristics different from that for cyclohexane, does not bind fragments of coil, and lacks interfacial area.

 

3. Pogozheva I.D., Lomize A.L. and Mosberg H.I. The transmembrane 7-a-bundle of rhodopsin: distance geometry calculations with hydrogen bonding constraints. Biophysical Journal. 72(5):1963-1985, 1997 .
   Abstract.
   A 3D model of the transmembrane 7-a-bundle of rhodopsin-like G-protein-coupled receptors (GPCRs) was calculated using an iterative distance geometry refinement with an evolving system of hydrogen bonds, formed by intramembrane polar side chains in various proteins of the family and collectively applied as distance constraints. The a-bundle structure thus obtained provides H bonding of nearly all buried polar side chains simultaneously in the 410 GPCRs considered. Forty evolutionarily conserved GPCR residues form a single continuous domain, with an aliphatic "core" surrounded by six clusters of polar and aromatic side chains. The 7-a-bundle of a specific GPCR can be calculated using its own set of H bonds as distance constraints and the common "average" model to restrain positions of the helices. The bovine rhodopsin model thus determined is closely packed, but has a few small polar cavities, presumably filled by water, and has a binding pocket that is complementary to 11-cis (6-s-cis, 12-s-trans, C = N anti)-retinal or to all-trans-retinal, depending on conformations of the Lys²⁹⁶ and Trp²⁶⁵ side chains. A suggested mechanism of rhodopsin photoactivation, triggered by the cis-trans isomerization of retinal, involves rotations of Glu¹³⁴, Tyr²²³, Trp265, Lys²⁹⁶, and Tyr³⁰⁶ side chains and rearrangement of their H bonds. The model is in agreement with published electron cryomicroscopy, mutagenesis, chemical modification, cross-linking, Fourier transform infrared spectroscopy, Raman spectroscopy, electron paramagnetic resonance spectroscopy, NMR, and optical spectroscopy data. The rhodopsin model and the published structure of bacteriorhodopsin have very similar retinal-binding pockets.

 

4. Mosberg H.I., Dua R.K., Pogozheva I.D. and Lomize A.L. Development of a model for the d-opioid receptor pharmacophore. 4. Residue 3 dehydrophenylalanine analogues of Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) confirm required gauche orientation of aromatic side chain. Biopolymers. 39(3):287-296, 1996.
   Abstract .
   We have previously proposed a model for the delta-opioid receptor binding conformation of the high affinity tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) based on experimental and theoretical conformational analysis of this peptide and a correlation of conformational preferences of further conformationally restricted analogues of this tetrapeptide with their receptor binding affinities. A key element of this model is the requirement that the Phe³ side chain exist in the c¹=-60 degrees conformation. Conformational calculations on the residue 3 dehydrophenylalanine analogues of JOM-13 suggest that while the dehydro(Z)phenylalanine analogue can be superimposed easily with the proposed binding conformer of JOM-13, the dehydro(E)phenylalanine analogue cannot. These results lead to the prediction that the dehydro(Z)phenylalanine analogue should display similar d-receptor binding affinity as JOM-13 while the dehydro(E)phenylalanine analogue is expected to bind less avidly. Synthesis and subsequent opioid receptor binding analysis of the dehydrophenylalanine analogues of JOM-13 confirms these predictions, lending support to the d-pharmacophore model.

 

5. Lomize A.L., Pogozheva I.D. and Mosberg H.I. Development of a model for the d-opioid receptor pharmacophore: 3. Comparison of the cyclic tetrapeptide, Tyr-c[D-Cys-Phe-D-Pen]OH with other conformationally constrained delta-receptor selective ligands. Biopolymers. 38(2):221-234, 1996.
   Abstract.
   We have previously proposed a model of the d-opioid receptor bound conformation for the cyclic tetrapeptide, Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) based on its conformational analysis and from conformation-affinity relationships observed for its analogues with modified first and third residues. To further verify the model, it is compared here with results of conformational and structure-activity studies for other known conformationally constrained delta-selective ligands: the cyclic pentapeptide agonist, Tyr-c[D-Pen-Gly-Phe-D-Phe]OH (DPDPE): the peptide antagonist, Tyr-Tic-Phe-PheOH (TIPP); the alkaloid agonist, 7-spiroindanyloxymorphone (SIOM); and the related alkaloid antagonist, oxymorphindole (OMI). A candidate d-bound conformer is identified for DPDPE that provides spatial overlap of the functionally important N-terminal NH3⁺ and C-terminal COO^- groups and the aromatic rings of the Tyr and Phe residues in both cyclic peptides. It is shown that all d-selective ligands considered have similar arrangements of their pharmacophoric elements, i.e., the tyramine moiety and a second aromatic ring (i.e., the rings of Phe³, Phe⁴, and Tic² residues in JOM-13, DPDPE, and TIPP, respectively; the indole ring system in OMI, and the indanyl ring system in SIOM). The second aromatic rings, while occupying similar regions of space throughout the analogues considered, have different orientations in agonists and antagonists, but identical orientations in peptide and alkaloid ligands with the same agonistic or antagonistic properties. These results agree with the previously proposed binding model for JOM-13, are consistent with the view that d-opioid agonists and antagonists share the same binding site, and support the hypothesis of a similar mode of binding for opioid peptides and alkaloids.

 

6. Lomize A.L. and Mosberg H.I. Thermodynamic model of a -helix in aqueous solution and micelle-bound state. in Peptides: Chemistry, Structure, and Biology, Proceedings of the 14^th American Peptide Symposium, P. Kaumaya and R.S. Hodges, Eds., Mayflower Scientific, England, pp. 474-476, 1996.
   Abstract.
   The theoretical model of a -helix formation presented here is the first part of a more general approach to calculation of the lowest free energy partition of peptides and proteins into elements of regular secondary structure and coil under different experimental conditions. Estimations of secondary structure stability in the method are based on unfolding free energies measured in peptide substitution and protein engineering experiments and on transfer free energies of model compounds. In the simplest case of linear peptides, considered here, when there is no intra- and intermolecular aggregation of a -helices, the lowest energy partition of peptide into helices and coil can be calculated using the dynamic programming algorithm. However, for flexible peptides, the situation is complicated by averaging of many helix-coil partitions. Recent ¹H NMR studies of more than a hundred peptides in aqueous solution and complexes with micelles are used here to verify the model.

 

7. Pogozheva I.D., Lomize A.L., and Mosberg H. I. Modeling of the d-opioid receptor transmembrane a-bundle, in Peptides: Chemistry, Structure, and Biology, Proceedings of the 14^th American Peptide Symposium, P. Kaumaya and R.S. Hodges, Eds., Mayflower Scientific, England, pp.350-351.1996.
   Abstract.
   The d-opioid receptor belongs to the large family of G-protein coupled receptors (GPCRs), transmembrane proteins which transduce external signals to the activation of G-proteins. All members of the rhodopsin-like GPCR family share common spatial structure of the transmembrane 7-a-bundle which represents the most evolutionarily conserved part of these receptors. A new approach for GPCR structure modeling originates from the observation that the content of hydrophilic residues in the GPCR family is unusually high for membrane proteins. It is known that polar groups buried from water in the protein interior have a strong tendency to form hydrogen bonds with each other. The pairs of buried hydrogen-bonded polar residues that appear and disappear in a correlated manner in amino acid sequences can be identified from analysis of multisequence alignments. Hydrogen bonds thus obtained can be used as distance constraints for the packing of the transmembrane a-helices using a distance geometry algorithm.

 

8. Wang C. and Mosberg H.I. Synthesis of a novel series of topographically constrained amino acids: benzo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids. Tetrahedron Lett., 36:3623-3626, 1995.
   Abstract.
   The incorporation of conformational constraints into biologically active peptides can provide them with very useful chemical and biological properties, e.g. well defined backbone conformation, specific topographical properties, high potency and selectivity at biological receptors, and increased stability against enzymatic degradation. In the design of bioactive peptides using the concept of topographical constraints, the conformationally restricted phenylalanine analog 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) has been utilized in many cases. With its bicyclic structure formed via cyclization of the phenolic aromatic ring to the amine nitrogen, Tic provides two potential advantages for replacement of phenylalanine in bioactive peptide ligands: conformational constraints are introduced in peptide backbone by the pipecolic acid bridge and the orientation of the aromatic side chain is restricted. The greatly reduced flexibility of the Tic side chain, in particular, can provide important insights into the bioactive conformation of the peptide ligand and can allow features of the complementary region of the receptor binding site to be inferred. In order to further probe details of the region of the receptor responsible for interacting with the Tic side chain, the benzoTic series was designed by fusing an additional benzo ring to the restricted aromatic system of Tic. Benzo[f]Tic, benzo[g]Tic and benzo[h]Tic corresponding to the three possible extension orientations, were prepared.

 

9. Mosberg H.I., Omnaas J.R., Lomize A.L., Heyl D.L., Nordan I., Mousigian C., Davis P. and Porreca F. Development of a model for the d-opioid receptor pharmacophore. 2. Conformationally restricted Phe³ replacements in the cyclic delta receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13). Journal of Medicinal Chemistry. 37(25): 4384-4391, 1994.
   Abstract.
   The in vitro pharmacological properties and conformational features of analogs of the delta opioid receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) in which the Phe³ residue was replaced by each of the four stereoisomers of b-methylphenylalanine (b-MePhe) were investigated. Both analogs in which the a carbon of the Phe³ replacement has L-stereochemistry display high affinity for d receptors with the (2S,3S)-MePhe³ analog exhibiting approximately 8-fold higher affinity than the (2S,3R)-MePhe³ diastereomer. Surprisingly, one analog with D-stereochemistry in residue 3, the (2R,3R)-MePhe³ analog, also displays high affinity for the delta receptor and is extraordinarily selective for this receptor. All analogs were agonists in the mouse vas deferens (MVD) and guinea pig ileum (GPI) smooth muscle bioassays, displaying MVD and GPI potencies consistent with their d and m opioid receptor affinities, respectively. The use of b-MePhe as a replacement for Phe³ was based upon the desire to reduce the conformational flexibility of the Phe³ side chain by imposing a steric rotational constraint in the form of the b-methyl substituent and to thus deduce the residue 3 side chain orientation in the d receptor-bound conformation from the correlation between delta receptor binding affinities and conformational preferences. Molecular mechanics computations revealed, however, that the conformational constraints imposed by the b-methyl group in the (2S,3S)-MePhe³ and (2S,3R)-MePhe³ analogs were too modest to allow unequivocal determination of delta receptor-bound residue 3 side chain conformation. However, analysis of the high-affinity (2R,3R)-MePhe³ analog revealed a strong preference for a single side chain conformer (c¹ approximately 60 degrees). Low-energy conformers of this analog could only be effectively superimposed with low-energy conformers of the parent peptide in which the Phe³ side chain conformation was limited to c¹ approximately -60 degrees. This observation eliminates the last remaining uncertainty regarding conformational features of the pharmacophore elements in the d receptor-bound state, allowing the proposal of a complete model.

 

10. Mosberg H.I., Lomize A.L., Wang C., Kroona H., Heyl D.L., Sobczyk-Kojiro K., Ma W., Mousigian C. and Porreca F. Development of a model for the d-opioid receptor pharmacophore. 1. Conformationally restricted Tyr¹  replacements in the cyclic delta receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13).  Journal of Medicinal Chemistry. 37(25):4371-4383, 1994.
    Abstract.
    A series of analogues of the conformationally restricted delta opioid receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]OH (JOM 13) was prepared in which the conformationally labile Tyr residue was replaced with several less flexible tyrosine analogues. Among these tyrosine analogues were the bicyclic structures 1,2,3,4-tetrahydro-7-hydroxyisoquinoline-3-carboxylic acid (HO-Tic), 2-amino-6-hydroxytetralin-2-carboxylic acid (Hat), and 2-amino-5-hydroxyindan-2-carboxylic acid (Hai) in which rotations about the C a-C b and C b-C g bonds are restricted due to cyclization of the side chain to the backbone. Also examined were analogues in which tyrosine was replaced with either trans-3-(4'-hydroxyphenyl)proline (t-Hpp) or cis-3-(4'-hydroxyphenyl)proline (c-Hpp), residues in which rotations about C alpha-C beta, but not C beta-C gamma, are restricted. Both the t-Hpp¹ and c-Hpp¹ analogues displayed d receptor binding affinity similar to the parent Tyr¹-containing peptide, while the D-Hat¹, L-Hat¹, and L-Hai¹ analogues exhibited somewhat lower affinity. The results observed for the t-Hpp¹ and c-Hpp¹ analogues are particularly significant since these two residues have little accessible conformational space in common. Since the binding conformation of residue 1 must be included in this limited conformational intersection, its elucidation is facilitated. Bioassay results from guinea pig ileum and mouse vas deferens preparations are in general agreement with the binding results; however some potency discrepancies are observed. These discrepancies may reflect different selectivities among delta receptor subtypes for the analogues or may represent differing efficacies among these conformationally restricted peptides. The conformational properties of the parent tetrapeptide and the residue 1-modified analogues were studied by molecular mechanics computations. All these peptides share a common rigid tripeptide cycle with a single energetically preferred backbone conformation and three different conformers of the D-Cys, D-Pen disulfide bridge, two of which are observed in the solid state and in aqueous solution, as previously determined from X-ray crystallography and ¹H NMR spectroscopy data (Lomize, A; et al. J. Am. Chem. Soc. 1994, 116, 429-436). All the peptides have similar sets of low-energy conformations of their common flexible elements, the Phe³ side chain and the peptide group between the first residue and the rigid tripeptide cycle. However, possible conformations of the first residue differ and depend on the covalent constraints incorporated into the side chain.

 

11. Lomize A.L., Flippen-Anderson J.L, George C., and Mosberg H.I. Conformational analysis of the d receptor-selective, cyclic opioid peptide, Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13). Comparison of X-ray crystallographic structures, molecular mechanics simulations and ¹H NMR data. J. Amer. Chem. Soc., 116:429-436, 1994.
    Abstract.
    The conformational features of the d -selective, cyclic opioid peptide, Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) were investigated using a combination of experimental (X-ray crystallography, 1H NMR spectroscopy) and theoretical (molecular mechanics computations) techniques. Energy calculations with the CHARMm force field show the existence of a single energetically preferred backbone conformation for the cyclic tripeptide portion of the molecule. Several distinct low-energy conformations were calculated, however, for the disulfide bridge linking the D-Cys and D-Pen residues, for the single Tyr residue outside the cycle, and for the Tyr and Phe sidechains. The two calculated lowest-energy conformers of the D-Cys, D-Pen disulfide bridge (A and B) differ in values of D-Cys c ¹ and c ³(S-S) torsion angles (the -60° , 90° and 180° , -90° combinations). This is consistent with the observation of two 1H NMR signal sets in aqueous solution (in the ratio 68:32) with distinctly different vicinal coupling constants for protons H-CaCb-H that correspond to a D-Cys c ¹ angle ~-60° for the first set and ~ 180° or +60° for the second. X-ray crystallographic analysis of crystals grown from aqueous solution also revealed two independent conformers which are in excellent agreement with the computational and NMR data for the rigid, cyclic part of the molecule including the disulfide bridge. However, ¹H NMR data and computational results indicate that the flexible elements of JOM-13 (the exocyclic Tyr residue and the Tyr and Phe sidechains) have no fixed structure in water solution. In the crystalline environment they adopt conformations that are stabilized mainly by intermolecular interactions and which do not correspond exactly to any local energy minimum identified in the molecular mechanics calculations.

 

12. Mosberg H.I., Omnaas J.R., Sobczyk-Kojiro K., Ho J.C., Ma W., Bush P., Mousigian C., and Lomize A.L. Pharmacophore elements of the TIPP (Tyr-Tic-Phe-Phe) class of d-opioid receptor antagonists, Letters in Peptide Science, 1:69-72, 1994.
    Abstract.
    A series of tri- and tetrapeptides sharing the amino terminal dipeptide unit Tyr-Tic found in the high affinity delta opioid receptor antagonist Tyr-Tic-Phe-Phe (TIPP) was prepared and evaluated in receptor binding assays to explore the role(s) of the Phe residues in position 3 and 4. It was found that aromaticity of residues 3 and 4 is not required for high affinity, a lipophilic side chain in either location being sufficient, as evidenced by the high delta receptor binding affinities observed for the tetrapeptide, Tyr-Tic-Ala-Leu and the tripeptide, Tyr-Tic-Leu. These results support the suggestion of Temussi et al. (Biochem. Biophys. Res. Commun., 198 (1994) 933) that the aromatic sidechain of the Tic residue corresponds to the aromatic sidechain found in residues 3 or 4 in other delta selective peptide series.

 

13. Lomize A.L. and Mosberg H.I. A conformational modelling of the d-receptor bound conformation that is similar for the cyclic opioid tetrapeptides and d selective alkaloids, in Peptides: Chemistry, Structure and Biology, Proceedings of the 13^th American Peptide Symposium, R.S. Hodges and J.A. Smith, Eds., ESCOM. Leiden, pp 905-907.1994.
    Abstract
    Low-energy conformations have been calculated for the delta opioid receptor selective deltorphin analog Tyr-c[D-Cys-Phe-D-Pen]OH and for related analogs with conformationally constrained Tyr¹ replacements. The cyclic part of the parent tetrapeptide has a rigid mainchain structure, but the side chains of Tyr and Phe are flexible and there are three different conformations of the Cys-Pen disulfide bridge, in agreement with ¹H NMR data. A remarkable feature of the lowest-energy conformations for the high affinity delta selective agonists (residue 1 = Tyr, t-3-(4'-hydroxyphenyl)proline, or 2',6'-dimethyl-Tyr, all with Ki ~ 1 nM) is an overlap in space of the Phe³ aromatic ring with the indole ring of the delta-selective alkaloid oxymorphindole (OMI) after superposition of the Na , Ca and Cb atoms of Tyr¹ and the corresponding N17, C9 and C10 atoms of the alkaloid tyramine moiety. The overlap is observed only for the g- rotamer (c ¹=-60) of the Phe³ side chain. A common low-energy conformation of the Tyr¹ residue can be identified as well. It has an identical relative position of the NH3⁺ group and Tyr ring for all these peptides and the OMI tyramine moiety. The only difference between the candidate receptor-bound conformation for the peptides and the alkaloid is the twist of Tyr¹ aromatic ring around Cb -Cg bond (c ² torsion angle). At the same time, analogs with reduced delta receptor binding affinity (Hai1 and Hat1 replacements with Ki ~ 10-20 nM) have exactly "OMI-like" c ² angles, but have the Phe³ ring shifted slightly in space relative to the OMI indole ring. The results obtained suggest that the binding modes of these delta selective peptides and alkaloids are very similar.

 

14. Mosberg H.I., Kroona H.B., Omnaas J.R., Sobczyk-Kojiro K., Bush P., and Mousigian C. Cyclic deltorphin analogues with high d opioid receptor affinity and selectivity. in Peptides: Chemistry, Structure and Biology, Proceedings of the 13^th American Peptide Symposium, R.S. Hodges and J.A. Smith, Eds., ESCOM. Leiden, pp 514-516.1994.
    Abstract.
    The heptapeptides deltorphin I and II, Tyr-D-Ala-Phe-Xxx-Val-Val-GlyNH₂, where Xxx = Asp or Glu for deltorphin I and II, respectively, are naturally occurring opioids isolated from frog skin which have been shown to exhibit high selectivity for the d type of opioid receptor. This high selectivity is due largely to the anionic side chain of residue 4 which diminishes m but not d receptor affinity, while high d binding affinity is dependent upon an intact C-terminal tripeptide. It has been proposed that this tail plays a conformational role by inducing a b-turn involving residues 3-6. We have previously suggested that a similar conformational element exists in a series of cyclic, d selective tetrapeptide opioids, exemplified by Tyr-c[D-Cys-Phe-D-Pen] (JOM-13), which we described prior to the discovery of the deltorphins. In JOM-13, which, like the deltorphins has a Tyr-D-Yyy-Phe N-terminal sequence, the disulfide cyclization has a similar conformational effect as the tripeptide tail in the deltorphins and results in the high d binding affinity displayed by this tetrapeptide. In an effort to better define the role of C-terminal conformation on d selectivity and affinity, we have examined a series of cyclic heptapeptide and related truncated, pentapeptide deltorphin analogs and report these results here.