Arno F. Spatola

Helix-stabilized cyclic peptides as selective inhibitors of steroid receptor–coactivator interactions

The interaction between nuclear receptors and coactivators provides an arena for testing whether protein–protein interactions may be inhibited by small molecule drug candidates. We provide evidence that a short cyclic peptide, containing a copy of the LXXLL nuclear receptor box pentapeptide, binds tightly and selectively to estrogen receptor α. Furthermore, as shown by x-ray analysis, the disulfide-bridged nonapeptide, nonhelical in aqueous solutions, is able to adopt a quasihelical conformer while binding to the groove created by ligand attachment to estrogen receptor α. An i, i+3 linked analog, H-Lys-cyclo(d-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln–NH2 (peptidomimetic estrogen receptor modulator 1), binds with a Ki of 25 nM, significantly better than an i, i+4 bridged cyclic amide, as predicted by molecular modeling design criteria. The induction of helical character, effective binding, and receptor selectivity exhibited by this peptide analog provide strong support for this strategy. The stabilization of minimalist surface motifs may prove useful for the control of other macromolecular assemblies, especially when an amphiphilic helix is crucial for the strong binding interaction between two proteins.

Potent Inhibitors of LXXLL‐Based Protein–Protein Interactions

Protein–protein interactions between estrogen receptors, ERα and ERβ, and their coactivators (CoAs) are an attractive target for drug intervention. This interaction is mediated by a small pentapeptide motif (LXXLL), termed the NR box. Based on this motif, a variety of cyclic and linear peptides were synthesized in order to gain a better understanding of the association of CoA proteins with the ER isoforms. Utilizing a time‐resolved florescence‐based coactivator interaction assay, we determined the abilities of these peptides to inhibit this interaction. Using molecular modeling and CD spectroscopy, we have examined the structural basis of their bioactivities with both hormone receptor isoforms. Either homocysteine or penicillamine was utilized as a substitute for cysteine in the disulfide‐bridged peptides, while tertiary leucine and neopentyl glycine were used as the surrogates for the NR box leucines. The most potent disufide‐bridged peptide (Ki= 70 pM, with ERα) incorporates neopentyl glycine in the NR box, while the most active peptide in this series with ERβ (Ki=350 pM) incorporates tertiary leucine. Surprisingly, several linear peptides containing a single cysteine residue showed activities with low nanomolar Ki values. Collectively, our results suggest a synthetic approach for designing potent and selective peptidomimetics for ERα and ERβ interactions with CoA proteins effecting estrogen action.

Structure-activity relationships of enkephalins containing serially replaced thiomethylene amide bond surrogates

An isomeric series of four leucine-enkephalin analogs containing the thiomethylene ether unit as an amide bond replacement in all positions have been prepared by solid phase methods. The resulting pseudopeptides divulged widely differing retentive behaviors on reversed phase high performance liquid chromatography (HPLC). An analog containing the Phe psi[CH2S]Leu dipeptide replacement at the 4-5 position exhibited binding close to the parent, leucine enkephalin; its guinea pig ileum (GPI) activity was the highest of the analogs tested. Another compound, Tyr psi[CH2S]Gly1-2]-Leu-enkephalin, also displaced 3H-etorphine well in the binding assay, but caused increased contractions in the GPI assay at low concentrations. The Phe psi[CH2S]Leu results are not compatible with the necessity of a beta-turn structure for agonist activity in the GPI assay.

Applications of ammonium formate catalytic transfer hydrogenolysis--IV1: a facile method for dehalogenation of aromatic chlorocarbons

Abstract Ammonium formate catalytic transfer hydrogenolysis, in the presence of palladium on carbon, has shown utility for the rapid dehalogenation of mono-or polychlorinated aromatic compounds in neutral media under ambient conditions of temperature and pressure.

An approach to cyclic peptide libraries: Reducing epimerization in medium sized rings during solid phase synthesis

Abstract Using an amino acid linked by its side chain to a solid support, head-to-tail cyclic peptide libraries of varying ring size have been prepared via resin-bound cyclization. These mixtures complement the use of linear peptide libraries for drug lead discovery and extend molecular diversity to conformationally constrained systems. During our synthesis of cyclic peptide mixtures, C-terminal epimerization was identified as a problem during chain elongation. This was significantly overcome by coupling with preactivated amino acid pentafluorophenyl esters.

Evidence of a peptide backbone contribution toward selective receptor recognition for leucine enkephalin thioamide analogs.

The in vitro opioid activities of a series of leucine enkephalin analogs containing a thioamide linkage in place of the peptide bond at various positions of the backbone were determined in mu- and delta-receptor-selective bio- and binding-assays. Thioamide substitution in the 1-2 position resulted in an inactive compound, whereas the same modification in the 2-3 and 4-5 position produced potency enhancement. Most interestingly, the 2-3 modified analog showed a 3 to 5 times higher preference for delta- over mu-receptors than natural leucine enkephalin. These results suggest that subtle backbone modifications can have a profound effect on receptor affinity and selectivity of biologically active peptides.

Inhibitors tethered near the acetylcholinesterase active site serve as molecular rulers of the peripheral and acylation sites.

The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants KI2 for propidium and tacrine, inhibitors specific for the P- and A-sites, respectively. Values of KI2 for propidium increased 30- to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of KI2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations.

Metal binding peptide inhibitors of vertebrate collagenase

Abstract A series of metal binding peptide analogues of the C-terminal sequence of collagen adjacent to its known site of cleavage by vertebrate collagenases has been synthesized by solid phase methods and tested as inhibitors both of collagenolysis and of hydrolysis of the model substrate DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg by tadpole backskin collagenase. The peptide analogue inhibitors have the general structure R-Ala-Gly-Gln-D-Arg-NH2. Using an HPLC assay system, I50 values of 70 μM, 50 μM and 10 μM were obtained for the three most potent inhibitors for which R = HSCH(CH2C6H5)CO-, HSCH2CH(NH2CO- and HSCH(CH2CH(CH3)2)CO-, respectively, when the above octapeptide was used as substrate. In contrast, an I50 value of 3 mM was found for cysteine under the same conditions. The mercaptophenylalanyl derivative was also highly effective in inhibiting collagen degradation. Using calf skin collagen as substrate, inhibition was obtained at concentrations of peptide analogue below 10 μM.

A rapid non-acidolytic method for deprotection and removal of peptides from solid phase resins - applications of ammonium formate catalytic transfer hydrogenation

Abstract Ammonium formate aided catalytic transfer hydrogenation has been employed in the cleavage, and concommitant deprotection, of the pentapeptide leucine enkephalin from the Merrifield peptide resin under ambient conditions of temperature and pressure in a neutral medium.

Synthesis and biological activities of pseudopeptide analogues of LH-RH: agonists and antagonists.

Using solid phase methods, seven agonist and antagonist analogues of LH-RH have been prepared containing enzyme-resistant CH2S linkages as selected amide bond replacements. Agonists modified at the 5–6, 6–7 and 9–10 position had 2, < 0.1, and 10% of the invitro activity of LH-RH, respectively. Among potential antagonists, 6–7 position analogues showed only minimal inhibitory activity but N- and C-terminal modified analogues retained substantial LH-RH-LH and FSH inhibitory activity. In addition, a 1–2 position methylene thioether analogue of the parent [Ac-Pro1, D-Phe2, D-Trp3,6]LH-RH antagonist was completely inhibitory at 30 ng invitro and represents the first such structure-modification that may be at least as active as its corresponding amide linked congener. However, neither 1–2 nor 9–10 methylene thioether position antagonists showed invivo antiovulatory activity at the 250 μg level.