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.

A series of cyclic somatostatin analogs containing a lanthionine bridge have been subjected to studies of structure-activity relationships. A direct synthesis of the thioether bridged analog (1) of sandostatin (SMS 201,995) and several lanthionine hexa-, hepta-, and octapeptides was carried out by using the method of cyclization on an oxime resin (PCOR) followed by condensation reactions in solution. The structures of the target peptides were analyzed by liquid secondary ion mass spectrometry (LSIMS) and subjected to high-energy collision-induced dissociation (CID) studies after opening of the peptide ring by proteolytic cleavage. The biological activities of these compounds have been evaluated by assaying their inhibitory potencies for the release of growth hormone (GH) from primary cultures of rat anterior pituitary cells, as well as by their binding affinities to cloned somatostatin receptors (SSTR1-5). The structural modification of sandostatin by introducing a lanthionine bridge resulted in a significantly increased receptor binding selectivity. The lanthionine octapeptide with C-terminal Thr-ol (1) showed similar high affinity for rat SSTR5 compared to somatostatin[1-14] and sandostatin. However, it exhibits about 50 times weaker binding affinity for mSSTR2b than sandostatin. Similarly, the lanthionine octapeptide with the C-terminal Thr-NH2 residue (2) has higher affinity for rSSTR5 than for mSSTR2B. Both peptides (compounds 1 and 2) have much lower potencies for inhibition of growth hormone secretion than sandostatin. This is consistent with their affinities to SSTR2, the receptor which is believed to be linked to the inhibition of growth hormone release by somatostatin and its analogs. The metabolic stability of lanthionine-sandostatin and sandostatin have been studied in rat brain homogenates. Although both compounds have a high stability toward enzymatic degradation, the lanthionine analog has a 2.4 times longer half-life than sandostatin. The main metabolites of both compounds have been isolated and identified by using an in vivo technique (cerebral microdialysis) and mass spectrometry.

-Defensins are macrocyclic antimicrobial peptides that were previously isolated from leukocytes of a single species, the rhesus macaque. We now report the characterization of baboon -defensins (BTDs) expressed in bone marrow and peripheral blood leukocytes. Four cDNAs encoding -defensin precursors were characterized, allowing for the prediction of 10 theoretical -defensins (BTD-1 to BTD-10) produced by binary, headto-tail splicing of nonapeptides excised from paired precursors. Five of the predicted -defensins were purified from baboon leukocytes, and synthetic versions of each were prepared. Anti--defensin antibody localized the peptides in circulating neutrophils and monocytes and in immature and mature myeloid elements in bone marrow. Each of the BTDs possessed antimicrobial activity against bacterial and fungal test organisms in vitro. Peptide activities varied markedly despite a high degree of sequence conservation among the -defensins tested. Thus, baboons express numerous -defensins which appear to differentially contribute to host defense against diverse pathogens.Antimicrobial peptides (AMPs) are effectors of the innate immune system. AMPs are expressed in cells (epithelia, neutrophils, and macrophages) that come into contact with potentially invasive microorganisms (17). In mammals, the two major classes of AMPs are defensins and cathelicidins. Cathelicidins are characterized by a conserved cathelin prodomain which lies N terminal to highly variable mature peptides that are released by activating proteases (27). Defensins are small, cationic peptides that are composed of three structural subclasses, ␣-, ␤-, and -defensins, differentiated by the spacing and pairing of their six disulfide-bonded cysteines (7, 9, 18). -Defensins are further distinguished by their macrocyclic backbone and as such represent the only known cyclic protein motif expressed in animals (16).The biosynthesis of -defensins requires head-to-tail splicing of two 9-amino-acid sequences derived from -defensin precursors (16). -Defensins were first identified in neutrophils and monocytes of the rhesus monkey (21). Subsequently, Nguyen et al. (15) conducted a phylogenetic survey that revealed the existence of -defensin genes in other Old World monkeys and two apes (the siamang and orangutan), but none in New World monkeys or prosimians. Humans, chimpanzees, bonobos, and gorillas express -defensin pseudogenes in which the precursor mRNA contains a mutation producing a stop codon in the signal sequence, thus preventing translation of the -defensin precursor (15).Rhesus -defensin-1 (RTD-1) is produced from the heterodimeric splicing of two -defensin precursors, proRTD1a and proRTD1b. Homodimeric excision/ligation reactions involving proRTD1a and proRTD1b were revealed by the isolation of RTD-2 and RTD-3 (12, 23). RTD-1, -2, and -3 have potent microbicidal activities against bacteria and fungi (23) and have antiviral activities against human immunodeficiency virus type 1 (3, 24) and herpes simplex virus (26). A synthetic -defensin designed b...

Mammalian defensins are cationic, antimicrobial peptides that play a central role in innate immunity. The peptides are composed of three structural subfamilies: α-, β-, and θ-defensins. θ-defensins are macrocyclic octadecapeptides expressed only in Old World monkeys and orangutans and are produced by the pair-wise, head-to-tail splicing of nonapeptides derived from their respective precursors. The existence of three active θ-defensin genes predicts that six different RTDs (1-6) are produced in this species. In this study, we isolated and quantified RTDs 1-6 from the neutrophils of 10 rhesus monkeys. RTD-1 was the most abundant θ-defensin, constituting ~50% of the RTD content; total RTD content varied by as much as threefold between animals. All peptides tested were microbicidal at ∼1 μM concentrations. The contribution of θ-defensins to macaque neutrophil antimicrobial activity was assessed by analyzing the microbicidal properties of neutrophil granule extracts after neutralizing θ-defensin content with a specific antibody. θ-defensin neutralization markedly reduced microbicidal activities of the corresponding extracts. Macaque neutrophil granule extracts had significantly greater microbicidal activity than those of human neutrophils, which lack θ-defensins. Supplementation of human granule extracts with RTD-1 markedly increased the microbicidal activity of these preparations, further demonstrating a prominent microbicidal role for θ-defensins.