Cyclic Peptide Analogues Research Articles

Structural basis of μ-opioid receptor targeting by a nanobody antagonist

The μ-opioid receptor (μOR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of μOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the μOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded μOR ligand and uncover the molecular basis for μOR antagonism by determining the cryo-EM structure of the NbE-μOR complex. NbE displays a unique ligand binding mode and achieves μOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a β-hairpin loop formed by NbE that deeply protrudes into the μOR, we design linear and cyclic peptide analogs that recapitulate NbE’s antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight μOR ligands that can serve as a basis for therapeutic developments.

Exploiting Hydrophobic Amino Acid Scanning to Develop Cyclic Peptide Inhibitors of the SARS-CoV-2 Main Protease with Antiviral Activity.

The development of novel antivirals is crucial not only for managing current COVID-19 infections but for addressing potential future zoonotic outbreaks. SARS-CoV-2 main protease (Mpro) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS-CoV-2 Mpro to enhance its cell permeability and in vitro antiviral activity. By identifying mutation-tolerant amino acid residues within the peptide sequence, we describe the development of a second-generation Mpro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS-CoV-2 Mpro.

Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase.

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.

All-Hydrocarbon Stapled Peptide Multifunctional Agonists at Opioid and Neuropeptide FF Receptors: Highly Potent, Long-Lasting Brain Permeant Analgesics with Diminished Side Effects.

Our previous study reported the multifunctional agonist for opioid and neuropeptide FF receptors DN-9, along with its cyclic peptide analogues c[D-Cys2, Cys5]-DN-9 and c[D-Lys2, Asp5]-DN-9. These analogues demonstrated potent antinociceptive effects with reduced opioid-related side effects. To develop more stable and effective analgesics, we designed, synthesized, and evaluated seven hydrocarbon-stapled cyclic peptides based on DN-9. In vitro calcium mobilization assays revealed that most of the stapled peptides, except 3, displayed multifunctional agonistic activities at opioid and neuropeptide FF receptors. Subcutaneous administration of all stapled peptides resulted in effective and long-lasting antinociceptive activities lasting up to 360 min. Among these stapled peptides, 1a and 1b emerged as the optimized compounds, producing potent central antinociception following subcutaneous, intracerebroventricular, and oral administrations. Additionally, subcutaneous administration of 1a and 1b caused nontolerance antinociception, with limited occurrence of constipation and addiction. Furthermore, 1a was selected as the final optimized compound due to its wider safety window compared to 1b.

The Cytotoxicity of Analogues Carnosine Dipeptide on Human Glioblastoma Multiforme

Background & Aims: The present study evaluated the effect of some synthesized linear and cyclic Carnosine analogues on the cells of human glioblastoma multiforme (GBM). The purpose of this research was to study the exposure of Carnosin analogues on astrocytoma using several experiments. Methods: The mass spectral measurements were performed on a 6410 Agilent LCMS triple quadrupole mass spectrometer (LCMS) with an electrospray ionization (ESI) interface. All tests were carried out six times. The concentration used for peptides (10 µg/mL) was selected based on MTT assay. The effect of peptides on the activity of SDH was assayed by MTT test. 100 μL mitochondrial suspension from GBM and normal groups were incubated with applied concentration of peptides (10μg/mL) at 37°C for 30 min. DCFH (final concentration, 10µM) was added to cells of both groups and incubated at 37°C for 15 min. The fluorescence intensity of DCFH which is an indicator of ROS concentration was then assayed by a Shimadzu RF-5000U fluorescence spectrophotometer. The cationic fluorescent dye, rhodamine 123 (concentration10 µM), from mitochondria into the cytosol have been used for the determination of MMP collapse. Mitochondria from astrocytoma and normal astocyte groups were suspended in corresponding assay buffer and incubated at 37°C with 10µg/mL peptides. The release was assayed by the Quantikine Cytochrome c. Carnosine peptide analogues on the activation of caspase-3 in the mitochondria isolated from GBM and normal groups using Sigma’s caspase-3 colorimetric assay kit. Results: Our study showed that a various range of toxicity on GBM cells was resulted by the linear and cyclic Carnosine analogues using MTT assay. Also, applying peptides on the mitochondria of GBM cells, a raise of mitochondrial reactive oxygen species (ROS) level, mitochondrial swelling, mitochondrial membrane potential (∆ψm) collapse, release of cytochrome c and caspase-3 activation of the affected mitochondria were detected. Conclusion: Based on the overall results, cyclic Carnosine analogues, especially compound 1c [Cyclo-(β-alanine-¬His-Pro-¬β-alanine-¬His)] showed more toxic activity than linear Carnosine analogues, which would be supporting to develop Carnosine cyclic peptide analogues as new anticancer and complementary therapeutic agents for the treatment of glioblastoma.

Novel Isoindolinone-Based Analogs of the Natural Cyclic Peptide Fenestin A: Synthesis and Antitumor Activity.

Small- and medium-sized cyclopeptides have been found to have extensive bioactivities and have drawn much attention from medicinal chemists. In the work described in this paper, various cyclic peptide analogs of Fenestin A were synthesized by intramolecular photoinduced electron-transfer cyclization reactions to study the influence of slight structural changes on the bioactivity of small cyclopeptides. The incorporation of thiazole and rigid isoindolinone fragments was found to improve the bioactivity of the cyclopeptide. Detailed in vitro studies of the apoptosis mechanism, mitochondrial membrane potential, cell cycle, intracellular Ca2+ concentration, and lactate dehydrogenase activity following treatment with a cyclopeptide showed that the cyclopeptide could induce apoptosis of tumor cells and lead to cycle arrest in the G2/M phase. The research also suggested that the photoinduced reaction could be applied to construct cyclic peptides stereoselectively, and the introduction of rigid fragments could enhance the biological activity of cyclopeptide drugs.

Head-to-tail cyclization of side chain-protected linear peptides to recapitulate genetically-encoded cyclized peptides.

Genetically‐encoded cyclic peptide libraries allow rapid in vivo screens for inhibitors of any target protein of interest. In particular, the Split Intein Circular Ligation of Protein and Peptides (SICLOPPS) system exploits spontaneous protein splicing of inteins to produce intracellular cyclic peptides. A previous SICLOPPS screen against Aurora B kinase, which plays a critical role during chromosome segregation, identified several candidate inhibitors that we sought to recapitulate by chemical synthesis. We describe the syntheses of cyclic peptide hits and analogs via solution‐phase macrocyclization of side chain‐protected linear peptides obtained from standard solid‐phase peptide synthesis. Cyclic peptide targets, including cyclo‐[CTWAR], were designed to match both the variable portions and conserved cysteine residue of their genetically‐encoded counterparts. Synthetic products were characterized by tandem high‐resolution mass spectrometry to analyze a combination of exact mass, isotopic pattern, and collisional dissociation‐induced fragmentation pattern. The latter analyses facilitated the distinction between targets and oligomeric side products, and served to confirm peptidic sequences in a manner that can be readily extended to analyses of complex biological samples. This alternative chemical synthesis approach for cyclic peptides allows cost‐effective validation and facile chemical elaboration of hit candidates from SICLOPPS screens.

Preclinical assessment of Alzheimer's disease using novel designed 99mTc‐labeled RGD‐based pro‐apoptotic cyclic peptide as a promising SPECT agent

The increased prevalence of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and glioma results in increased risks to abolish the healthy life. This is because of inducing alternation causes into the intracellular compartment as well as noncell compartments. Therefore, herein, a simple and facile radiosynthesis strategy was employed for the development of a novel 99mTc (t1/2 = 6 h, Er = 140 keV) labeled diethylenetriamine pentaacetic acid (DTPA) conjugated cyclic heptapeptide (99mTc‐DTPA‐c[RGDKLAK]; cPT). The radiotracer was characterized using various analytical techniques to evaluate the identity, radiolabeling, biocompatibility, in vitro stability, and amenability for preclinical use. The biological specificity and efficacy of the radiolabeled cyclic peptide analog were confirmed by in vivo biodistribution and single‐photon emission computed tomography (SPECT) studies in Alzheimer disease (AD)‐induced animal models. The results demonstrated that 99mTc(CO)3‐DTPA‐cPT showed high radioactivity concentration at the site of αvβ3, α5β1‐integrins expressing brain regions while decreasing radioactivity levels in kidneys and increasing urinary bladder over the period of 3 h suggested the renal clearance route of the radiotracer. The diagnostic outcomes obtained from planar SPECT study are consistent with that of the biodistribution study with a target‐to‐nontarget ratio (T/NT = 28.47 ± 2.53) at 3‐h postinjection. The results are surprisingly supportive to our hypothesis that the small cyclic peptide‐based radiotracer is well‐oriented towards targeted drug delivery and envisaged that our proposed radiotracer 99mTc(CO)3‐DTPA‐cPT has potential diagnostic applications for Alzheimer's disease as a novel SPECT agent.

A Cyclic BMP-2 Peptide Upregulates BMP-2 Protein-Induced Cell Signaling in Myogenic Cells.

In the current study, we designed four cyclic peptide analogues by incorporating two cysteine residues in a BMP-2 linear knuckle epitope in such a way that the active region of the peptide could be either inside or outside the cyclic ring. Bone morphogenetic protein receptor BMPRII was immobilized on the chip surface, and the interaction of the linear and cyclic peptide analogues was studied using surface plasmon resonance (SPR). From the affinity data, the peptides with an active region inside the cyclic ring had a higher binding affinity in comparison to the other peptides. To confirm that our affinity data are in line in vitro, we studied the expression levels of RUNX2 (runt-related transcription factor) and conducted an osteogenic marker alkaline phosphatase (ALP) assay and staining. Based on the affinity data and the in vitro experiments, peptide P-05 could be a suitable candidate for osteogenesis, with higher binding affinity and increased RUNX2 and ALP expression in comparison to the linear peptides.

Exploring Chemical Structures From Cortex Lycii, Based on Manual and Automatic Analysis of the HPLC-Q-TOF-MS Data

Cortex Lycii, the root barks of Lycium barbarum and L. chinense, known as “di gu pi” in traditional Chinese herbal drugs, is an important ingredient of formulations used for treating a variety of diseases. During the last 3 decades, more than 70 chemical entities have been separated and purified from either the aqueous or aqueous ethyl alcohol extracts of Cortex Lycii. In this study, high-performance liquid chromatography together with quadrupole-time-of-flight mass spectrometry (MS) was employed to explore new analog structures from aqueous ethyl alcohol extracts (50%, v/v), which led us to discover 4 new phenolic amides and a new cyclic peptide. The structure-based manual screening method, on the basis of the analysis of the fragmentation pathway of the previously known compounds, was used to make a preliminary analysis of the negative total ion chromatography and negative extract ion spectra. Three ions at m/ z 472.1, 314.1, and 445.2 were assigned to phenolic amides, and by further analysis of their MS/MS data, the structure of 1, corresponding to one of them ( m/ z 314.1), was illustrated as an analog of the known compound KN1. A parent ion at m/ z 856.1 was assigned to a cyclic peptide analog (2) in the manual analysis procedure. Furthermore, the MS/MS data were profiled on the Global Natural Product Social Molecular Networking (GNPS, https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp ) workflow to weave a visualization molecular network. Three more new analog ions ( m/ z 604.3 [3], 597.3 [4], and 611.3 [5]) were found in the aggregation of KN5 and KN7, and their structures were all determined by comparisons with known compounds. This manual and networking automatic screening method may provide a sensitive and efficient procedure to facilitate the mining of novel trace components.

Targeted melanoma radiotherapy using ultrasmall 177Lu-labeled α-melanocyte stimulating hormone-functionalized core-shell silica nanoparticles

Lutetium-177 (177Lu) radiolabeled ultrasmall (~6 nm dia.) fluorescent core-shell silica nanoparticles (Cornell prime dots or C′ dots) were developed for improving efficacy of targeted radiotherapy in melanoma models. PEGylated C′ dots were surface engineered to display 10–15 alpha melanocyte stimulating hormone (αMSH) cyclic peptide analogs for targeting the melanocortin-1 receptor (MC1-R) over-expressed on melanoma tumor cells. The 177Lu-DOTA-αMSH-PEG-C′ dot product was radiochemically stable, biologically active, and exhibited high affinity cellular binding properties and internalization. Selective tumor uptake and favorable biodistribution properties were also demonstrated, in addition to bulk renal clearance, in syngeneic B16F10 and human M21 xenografted models. Prolonged survival was observed in the treated cohorts relative to controls. Dosimetric analysis showed no excessively high absorbed dose among normal organs. Correlative histopathology of ex vivo treated tumor specimens revealed expected necrotic changes; no acute pathologic findings were noted in the liver or kidneys. Collectively, these results demonstrated that 177Lu-DOTA-αMSH-PEG-C′ dot targeted melanoma therapy overcame the unfavorable biological properties and dose-limiting toxicities associated with existing mono-molecular treatments. The unique and tunable surface chemistries of this targeted ultrasmall radiotherapeutic, coupled with its favorable pharmacokinetic properties, substantially improved treatment efficacy and demonstrated a clear survival benefit in melanoma models, which supports its further clinical translation.

Designing cyclic competence-stimulating peptide (CSP) analogs with pan-group quorum-sensing inhibition activity in Streptococcus pneumoniae

Streptococcus pneumoniae is an opportunistic human pathogen that utilizes the competence regulon, a quorum-sensing circuitry, to acquire antibiotic resistance genes and initiate its attack on the human host. Interception of the competence regulon can therefore be utilized to study S. pneumoniae cell-cell communication and behavioral changes, as well as attenuate S. pneumoniae infectivity. Herein we report the design and synthesis of cyclic dominant negative competence-stimulating peptide (dnCSP) analogs capable of intercepting the competence regulon in both S. pneumoniae specificity groups with activities at the low nanomolar range. Structural analysis of lead analogs provided important insights as to the molecular mechanism that drives CSP receptor binding and revealed that the pan-group cyclic CSPs exhibit a chimeric hydrophobic patch conformation that resembles the hydrophobic patches required for both ComD1 and ComD2 binding. Moreover, the lead cyclic dnCSP, CSP1-E1A-cyc(Dap6E10), was found to possess superior pharmacological properties, including improved resistance to enzymatic degradation, while remaining nontoxic. Lastly, CSP1-E1A-cyc(Dap6E10) was capable of attenuating mouse mortality during acute pneumonia caused by both group 1 and group 2 S. pneumoniae strains. This cyclic pan-group dnCSP is therefore a promising drug lead scaffold against S. pneumoniae infections that could be administered individually or utilized in combination therapy to augment the effects of current antimicrobial agents.

Design of Linear and Cyclic Mutant Analogues of Dirucotide Peptide (MBP82⁻98) against Multiple Sclerosis: Conformational and Binding Studies to MHC Class II.

Background: Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system. MS is a T cell-mediated disease characterized by the proliferation, infiltration, and attack of the myelin sheath by immune cells. Previous studies have shown that cyclization provides molecules with strict conformation that could modulate the immune system. Methods: In this study, we synthesized peptide analogues derived from the myelin basic protein (MBP)82–98 encephalitogenic sequence (dirucotide), the linear altered peptide ligand MBP82–98 (Ala91), and their cyclic counterparts. Results: The synthesized peptides were evaluated for their binding to human leukocyte antigen (HLA)-DR2 and HLA-DR4 alleles, with cyclic MBP82–98 being a strong binder with the HLA-DR2 allele and having lower affinity binding to the HLA-DR4 allele. In a further step, conformational analyses were performed using NMR spectroscopy in solution to describe the conformational space occupied by the functional amino acids of both linear and cyclic peptide analogues. This structural data, in combination with crystallographic data, were used to study the molecular basis of their interaction with HLA-DR2 and HLA-DR4 alleles. Conclusion: The cyclic and APL analogues of dirucotide are promising leads that should be further evaluated for their ability to alter T cell responses for therapeutic benefit against MS.

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87-99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis.

In this report, amide-linked cyclic peptide analogues of the 87–99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP87–99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP72–85-induced EAE in Lewis rats. The Lys91 and Pro96 of MBP87–99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91–99)[Ala96]MBP87–99, cyclo(87–99)[Ala91,96]MBP87–99 and cyclo(87–99)[Arg91, Ala96]MBP87–99, but not wild-type linear MBP87–99, strongly inhibited MBP72–85-induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87–99)[Arg91, Ala96]MBP87–99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.

The toxicity study of synthesized inverse carnosine peptide analogues on HepG2 and HT-29 cells.

Objective:Cancer has risen as the main cause of diseases with the highest rate of mortality in the world. Drugs used in cancer, usually demonstrate side effects on normal tissues. On the other hand, anticancer small peptides, effective on target tissues, should be safe on healthy organs, as being naturally originated compounds. In addition, they may have good pharmacokinetic properties. carnosine, a natural dipeptide, has shown many biological functions, including anti-oxidant, anti-senescence, anti-inflammatory and anticancer activities. This study, with the aim of introducing new anticancer agents with better properties, is focused on the synthesis and cytotoxic evaluation of some peptide analogues of carnosine.Materials and Methods:The cytotoxic activity of the synthesized peptides, prepared by the solid-phase peptide synthesis method, was evaluated against two cell lines of HepG2 and HT-29 using MTT assay, lactate dehydrogenase (LDH) assay and flow cytometry analysis.Results:Linear and cyclic analogues of carnosine peptide showed cytotoxicity, demonstrated by several experiments, against HepG2 and HT-29 cell lines with mean IC50 values ranging from 9.81 to 16.23 µg/ml. Among the peptides, compounds 1c, 3c and 6b (linear analogue of 3c) showed a considerable toxic activity on the cancerous cell lines.Conclusion:The cyclic peptide analogues of carnosine with His-β-Ala-Pro-β-Ala-His (1c) and β-Ala-His-Pro-His-β-Ala (3c) sequences showed cytotoxic activity on cancerous cells of HepG2 and HT-29, better than carnosine, and thus can be good candidates to develop new anticancer agents. The mechanism of cytotoxicity may be through cell apoptosis.

Pheromone Research and Development in Israel 1975–2015

The review describes the history of pheromone research in Israel in 1975–2015. The research focused on sex pheromones of moths that were important agricultural pests. Identification, synthesis and field application of sex pheromones was performed. Synthetic procedures of several known sex pheromones were developed. Monitoring and control of key pest moths was evaluated. The interactions of pheromone components of closely related species were studied in field and laboratory experiments. The sex pheromones of three scale insects, two mealybug species and Matsucoccus josephi were studied. New syntheses were developed and the pheromones were implemented in pest management. Structure activity relationship of the pheromonal and kairomonal of the M. josephi pheromone was investigated. Different pherotypes of P. ficus were identified and evaluated. The aggregation pheromone of sap beetles in combination with food baits was evaluated. The aggregation pheromone of the almond bark beetle was identified and a stereospecific synthesis of its enantiomers was developed. Monitoring the pest in stone fruit orchards was implemented. The activity of the pheromone biosynthesis activating neuropeptide (PBAN) was studied in Helicoverpa armigera and Heliothis peltigera. The ligation technique was used to assess the effect of PBAN on the production of female and male pheromones. A structure-activity relationship study of PBAN indicated that shorter peptides display activity as the full length PBAN. A series of linear and cyclic peptide analogs was prepared, resulting in the discovery of a lead antagonist. The research and development activity facilitated the intensive integration of pheromones in the pest management regimes in Israeli agriculture.

Biologically relevant conformational features of linear and cyclic proteolipid protein (PLP) peptide analogues obtained by high-resolution nuclear magnetic resonance and molecular dynamics.

Proteolipid protein (PLP) is one of the main proteins of myelin sheath that are destroyed during the progress of multiple sclerosis (MS). The immunodominant PLP139-151 epitope is known to induce experimental autoimmune encephalomyelitis (EAE, animal model of MS), wherein residues 144 and 147 are recognized by T cell receptor (TCR) during the formation of trimolecular complex with peptide-antigen and major histocompability complex. The conformational behavior of linear and cyclic peptide analogues of PLP, namely PLP139-151 and cyclic (139-151) (L144, R147) PLP139-151, have been studied in solution by means of nuclear magnetic resonance (NMR) methods in combination with unrestrained molecular dynamics simulations. The results indicate that the side chains of mutated amino acids in the cyclic analogue have different spatial orientation compared with the corresponding side chains of the linear analogue, which can lead to reduced affinity to TCR. NMR experiments combined with theoretical calculations pave the way for the design and synthesis of potent restricted peptides of immunodominant PLP139-151 epitope as well as non peptide mimetics that rises as an ultimate goal.

Proteolytically Stable Cyclic Decapeptide for Breast Cancer Cell Targeting.

Starting with a previously reported linear breast cancer targeting decapeptide WxEAAYQkFL, here we report the synthesis of a novel cyclic peptide analogue cyclic WXEAAYQkFL. The N- to C-terminus amide cyclized peptide with one d-amino acid (k) displayed higher uptake by breast cancer cells, with minimal uptake by the noncancerous cells compared to the linear peptide with two d-amino acids (x and k), and was stable toward proteolytic degradation. When immobilized on gold microcantilever surface, the cyclic peptide was able to capture breast cancer cells specifically and sense samples with ≥25 cancer cells/mL. Animal studies using mice carrying orthotopic breast MDA-MB-231 tumors showed that the cyclic peptide preferentially accumulates in tumor (2 h after injection) and is rapidly cleared from all other organs except kidneys and liver. The study highlights the discovery of a novel proteolytically stable cyclic peptide that can be used for targeted drug delivery or for enumerating circulating breast tumor cells.

Cyclic citrullinated MBP87–99 peptide stimulates T cell responses: Implications in triggering disease

Amino acid mutations to agonist peptide epitopes of myelin proteins have been used to modulate immune responses and experimental autoimmune encephalomyelitis (EAE, animal model of multiple sclerosis). Such amino acid alteration are termed, altered peptide ligands (APL). We have shown that the agonist myelin basic protein (MBP) 87–99 epitope (MBP87–99) with crucial T cell receptor (TCR) substitutions at positions 91 and 96 (K91,P96 (TCR contact residues) to R91,A96; [R91,A96]MBP87–99) results in altered T cell responses and inhibits EAE symptoms. In this study, the role of citrullination of arginines in [R91,A96]MBP87–99 peptide analog was determined using in vivo experiments in combination with computational studies. The immunogenicity of linear [Cit91,A96,Cit97]MBP87–99 and its cyclic analog – cyclo(87–99)[Cit91,A96,Cit97]MBP87–99 when conjugated to the carrier mannan (polysaccharide) were studied in SJL/J mice. It was found that mannosylated cyclo(87–99)[Cit91,A96,Cit97]MBP87–99 peptide induced strong T cell proliferative responses and IFN-gamma cytokine secretion compared with the linear one. Moreover, the interaction of linear and cyclic peptide analogs with the major histocompatibility complex (MHC II, H2-IAs) and TCR was analyzed using molecular dynamics simulations at the receptor level, in order to gain a better understanding of the molecular recognition mechanisms that underly the different immunological profiles of citrullinated peptides compared to its agonist native counterpart MBP87–99 epitope. The results demonstrate that the citrullination of arginine in combination with the backbone conformation of mutated linear and cyclic analogs are significant elements for the immune response triggering the induction of pro-inflammatory cytokines.

Synthesis and Kinetic Analysis of Two Conformationally Restricted Peptide Substrates of Escherichia coli Penicillin-Binding Protein 5.

Escherichia coli PBP5 (penicillin-binding protein 5) is a dd-carboxypeptidase involved in bacterial cell wall maturation. Beyond the C-terminal d-alanyl-d-alanine moiety, PBP5, like the essential high-molecular mass PBPs, has little specificity for other elements of peptidoglycan structure, at least as elicited in vitro by small peptidoglycan fragments. On the basis of the crystal structure of a stem pentapeptide derivative noncovalently bound to E. coli PBP6 (Protein Data Bank entry 3ITB ), closely similar in structure to PBP5, we have modeled a pentapeptide structure at the active site of PBP5. Because the two termini of the pentapeptide are directed into solution in the PBP6 crystal structure, we then modeled a 19-membered cyclic peptide analogue by cross-linking the terminal amines by succinylation. An analogous smaller, 17-membered cyclic peptide, in which the l-lysine of the original was replaced by l-diaminobutyric acid, could also be modeled into the active site. We anticipated that, just as the reactivity of stem peptide fragments of peptidoglycan with PBPs in vivo may be entropically enhanced by immobilization in the polymer, so too would that of our cyclic peptides with respect to their acyclic analogues in vitro. This paper describes the synthesis of the peptides described above that were required to examine this hypothesis and presents an analysis of their structures and reaction kinetics with PBP5.