Peteris Romanovskis

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.

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.

Expanding molecular diversity with pseudopeptides and macrotorials: Synthesis, characterization, and biological activities of macrocyclic combinatorial libraries

Combinatorial mixtures represent a potentially rich source of drug leads if problems involving deconvolution and structure optimization can be overcome. Cyclic peptides and their analogs are partially constrained systems that reduce the complexities inherent with linear macromolecules. Through careful optimization of synthetic procedures, we have been able to prepare, test, and interpret biological results from both small and large (>1000 component) cyclic peptide libraries.

Cyclic Octapeptide Inhibitors of the Acetylcholinesterase Peripheral Site: Implications for Inhibition of Nerve Toxins

The primary physiological role of acetylcholinesterase (AChE) is to hydrolyze the neurotransmitter acetylcholine at cholinergic synapses. AChE is among the most efficient enzymes, with a turnover number of >104 s-1 [1]. Ligand binding studies and X-ray crystallography have focused on the structural basis for the enzyme’s high catalytic efficiency and revealed that the AChE active site consists of a narrow gorge some 20 A deep with two separate ligand binding sites: an acylation site at the bottom of the gorge where substrate hydrolysis occurs, and a peripheral site at the gorge mouth.

By-Product Formation During SPPS of Linear, Cyclic, and Novel Bicyclic Peptides as AChE Inhibitors

We have been interested in the synthesis of loop II of the AChE inhibitor of the fasciculin (a “three-finger” snake venom polypeptide neurotoxin from green mambas (genus Dendroaspis)) [1]. Our parent sequence involves amino acids 27–34 or Arg-Ala-His-Pro-Pro-Lys-Met-Gln- as the base peptide from which cyclic and bicyclic analogues have been prepared. These may serve as chemical probes to study peripheral site acetylcholinesterase (AChE) inhibition.