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Three new peptidomimetics (1−3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-1, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 μM. Their activities against HIV-1 protease (Ki 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1−2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC50 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC50 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55^gag to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 Å (1) and 1.85 Å (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

Conotoxins, disulfide-rich peptides from the venom of cone snails, have created much excitement over recent years due to their potency and specificity for ion channels and their therapeutic potential. One recently identified conotoxin, MrIA, a 13-residue member of the -conotoxin family, inhibits the human norepinephrine transporter (NET) and has potential applications in the treatment of pain. In the current study, we show that the -hairpin structure of native MrIA is retained in a synthetic cyclic version, as is biological activity at the NET. Furthermore, the cyclic version has increased resistance to trypsin digestion relative to the native peptide, an intriguing result because the cleavage site for the trypsin is not close to the cyclization site. The use of peptides as drugs is generally hampered by susceptibility to proteolysis, and so, the increase in enzymatic stability against trypsin observed in the current study may be useful in improving the therapeutic potential of MrIA. Furthermore, the structure reported here for cyclic MrIA represents a new topology among a growing number of circular disulfide-rich peptides.

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the α-conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated α-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, α-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat α3β2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat α7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr¹⁶]AnIB showed a 2-fold and 10-fold decrease in activities at α3β2 (IC50 0.6 nM) and α7 (IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the α7 (IC50 367 nM) than at the α3β2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-α-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for α3β2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor α7 affinity.

The α-conotoxin MII is a two disulfide bridge containing, 16 amino acid long peptide toxin isolated from the marine snail Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors (nAChRs) of the subtype α3β2. To improve the bioavailability of this peptide, two lipidic analogues of MII have been synthesized, the first by coupling 2-amino-d,l-dodecanoic acid (Laa) to the N terminus (LaaMII) and the second by replacing Asn5 in the MII sequence with this lipoamino acid (5LaaMII). Both lipidic linear peptides were then oxidized under standard conditions. ¹H NMR shift analysis of these peptides and comparison with the native MII peptide showed that the tertiary structure of the N-conjugated analogue, LaaMII, was consistent with that of the native conotoxin, whereas the 5LaaMII analogue formed the correct disulfide bridges but failed to adopt the native helical tertiary structure. The N terminus conjugate was also found to inhibit nAChRs of the subtype α3β2 with equal potency to the parent peptide, whereas the 5LaaMII analogue showed no inhibitory activity. The active LaaMII analogue was found to exhibit significantly improved permeability across Caco-2 cell monolayers compared to the native MII, and both peptides showed negligible toxicity.

α-Conotoxin ImI derives from the venom of Conus imperialis and is the first and only small-peptide ligand that selectively binds to the neuronal α7 homopentameric subtype of the nicotinic acetylcholine receptor (nAChR). This receptor subtype is a possible drug target for several neurological disorders. The cysteines are connected in the pairs Cys2−Cys8 and Cys3−Cys12. To date it is the only α-conotoxin with a 4/3 residue spacing between the cysteines. The structure of ImI has been determined by ¹H NMR spectroscopy in aqueous solution. The NMR structure is of high quality, with a backbone pairwise rmsd of 0.34 Å for a family of 19 structures, and comprises primarily a series of nested β turns. Addition of organic solvent does not perturb the solution structure. The first eight residues of ImI are identical to the larger, but related, conotoxin EpI and adopt a similar structure, despite a truncated second loop. Residues important for binding of ImI to the α7 nAChR are all clustered on one face of the molecule. Once further binding data for EpI and ImI are available, the ImI structure will allow for design of novel α7 nAChR-specific agonists and antagonists with a wide range of potential pharmaceutical applications.

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [³H]MK-801 binding to human glutamate−N-methyl-d-aspartate (NMDA) receptors, and their structures have been examined using CD and ¹H NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [³H]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8,D17], and con-G[A7] have similar conformations in the presence of Ca²⁺. This consists of a helix for residues 2−16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7−10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [³H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca²⁺ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca²⁺-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.