Analysis of rhesus macaque leukocytes disclosed the presence of an 18-residue macrocyclic, tridisulfide antibiotic peptide in granules of neutrophils and monocytes. The peptide, termed rhesus theta defensin-1 (RTD-1), is microbicidal for bacteria and fungi at low micromolar concentrations. Antibacterial activity of the cyclic peptide was threefold greater than that of an open-chain analog, and the cyclic conformation was required for antimicrobial activity in the presence of 150 millimolar sodium chloride. Biosynthesis of RTD-1 involves the head-to-tail ligation of two alpha-defensin-related nonapeptides, requiring the formation of two new peptide bonds. Thus, host defense cells possess mechanisms for synthesis and granular packaging of macrocyclic antibiotic peptides that are components of the phagocyte antimicrobial armamentarium.

Theta-defensins (θ-defensins) are macrocyclic antimicrobial peptides expressed in leukocytes of Old World monkeys. The peptides are broad spectrum microbicides in vitro and numerous θ-defensin isoforms have been identified in granulocytes of rhesus macaques and Olive baboons. Several mammalian α- and β-defensins, genetically related to θ-defensins, have proinflammatory and immune-activating properties that bridge innate and acquired immunity. In the current study we analyzed the immunoregulatory properties of rhesus θ-defensins 1–5 (RTDs 1–5). RTD-1, the most abundant θ-defensin in macaques, reduced the levels of TNF, IL-1α, IL-1β, IL-6, and IL-8 secreted by blood leukocytes stimulated by several TLR agonists. RTDs 1–5 suppressed levels of soluble TNF released by bacteria- or LPS-stimulated blood leukocytes and THP-1 monocytes. Despite their highly conserved conformation and amino acid sequences, the anti-TNF activities of RTDs 1–5 varied by as much as 10-fold. Systemically administered RTD-1 was non-toxic for BALB/c mice, and escalating intravenous doses were well tolerated and non-immunogenic in adult chimpanzees. The peptide was highly stable in serum and plasma. Single dose administration of RTD-1 at 5 mg/kg significantly improved survival of BALB/c mice with E. coli peritonitis and cecal ligation-and-puncture induced polymicrobial sepsis. Peptide treatment reduced serum levels of several inflammatory cytokines/chemokines in bacteremic animals. Collectively, these results indicate that the anti-inflammatory properties of θ-defensins in vitro and in vivo are mediated by the suppression of numerous proinflammatory cytokines and blockade of TNF release may be a primary effect.

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Rhesus macaque -defensins (RTDs) are unique macrocyclic antimicrobial peptides. The three RTDs (RTD 1-3), isolated from macaque leukocytes, have broad-spectrum antimicrobial activities in vitro and share certain structural features with acyclic porcine protegrins, which are microbicidal peptides of the cathelicidin family. To understand the structural features that confer the respective cytocidal properties to -defensins and protegrins, we determined and compared the biological properties of RTD 1-3 and protegrin 1 (PG-1) in assays for antimicrobial activity, bacterial membrane permeabilization, and toxicity to human cells. RTD 1-3 and PG-1 had similar microbicidal potencies against Escherichia coli, Staphylococcus aureus, and Candida albicans in low-ionic-strength (10 mM) buffers at pH 7.4. The inclusion of physiologic sodium chloride partially inhibited the microbicidal activities of the RTDs, and the degree of inhibition depended on the buffer used in the assay. Similarly, the inclusion of 10% normal human serum partially antagonized the bactericidal activities of all four peptides. In contrast, the microbicidal activities of PG-1 and RTD 1-3 against E. coli were unaffected by physiologic concentrations of calcium chloride and magnesium chloride. Treatment of E. coli ML35 cells with RTD 1-3 or PG-1 rapidly rendered the bacteria permeable to -nitrophenyl-␤-D-galactopyranoside, and this was accompanied by the rapid entry of the RTDs. Finally, although PG-1 was toxic to human fibroblasts and caused a marked lysis of erythrocytes, the RTDs were not cytotoxic or hemolytic. Thus, compared to PG-1, RTD 1-3 possess substantially greater cytocidal selectivity against microbes. Surprisingly, the low cytotoxicity of the RTDs did not depend on the peptides' cyclic conformation.

We report the conformational analysis of a series of analogs of sandostatin (octreotide, D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys 7]-Thr8-ol) using 1H NMR spectroscopy and molecular modeling. Two active compounds in which the disulfide group is replaced by a monosulfide (lanthionine) bridge (D-Phe1-c[AlaL2-Phe3-D-Trp4-Lys5-Thr6-A laL7]-Thr8-ol and D-Phe1-c[AlaL2-Phe3-D-Trp4-Lys5-Thr6-Al aL7]-Thr8-NH2, where AlaL denotes each of the lanthionine amino acid ends linked by the monosulfide bridge) show different mSSTR2b/rSSTR5 receptor selectivities as compared to sandostatin. These new results have enabled us to reveal features of the somatostatin pharmacophore common to the model previously proposed in our laboratory on the basis of main chain and side chain chiral methylation studies. In addition, our studies provide new insight into the role of the disulfide bridge and of Thr8 in binding potency. We also show that the lanthionine group is a good mimetic of beta-VI turns and can be incorporated in sandostatin analogs maintaining the essential secondary structural features of sandostatin. These results facilitate the design of new sandostatin peptidomimetics.