John Matsoukas

Round and Round we Go: Cyclic Peptides in Disease

There is a need for novel drugs for the treatment of infectious diseases, autoimmunity and cancer. Cyclic peptides constitute a class of compounds that have made crucial contributions to the treatment of certain diseases. Penicillin, Vancomycin, Cyclosporin, the Echinocandins and Bleomycin are well-known cyclic peptides. Cyclic peptides, compared to linear peptides, have been considered to have greater potential as therapeutic agents due to their increased chemical and enzymatic stability, receptor selectively, and improved pharmacodynamic properties. They have been used as synthetic immunogens, transmembrane ion channels, antigens for Herpes Simplex Virus, potential immunotherapeutic vaccines for diabetes and Experimental Autoimmune Encephalomyelitis - an animal model of Multiple Sclerosis, as inhibitors against alpha-amylase and as protein stabilizers. Herein, we review important cyclic peptides as therapeutic agents in disease.

Crucial Role of Granulocytic Myeloid-Derived Suppressor Cells in the Regulation of Central Nervous System Autoimmune Disease

There is a need in autoimmune diseases to uncover the mechanisms involved in the natural resolution of inflammation. In this article, we demonstrate that granulocytic myeloid-derived suppressor cells (G-MDSCs) abundantly accumulate within the peripheral lymphoid compartments and target organs of mice with experimental autoimmune encephalomyelitis prior to disease remission. In vivo transfer of G-MDSCs ameliorated experimental autoimmune encephalomyelitis, significantly decreased demyelination, and delayed disease onset through inhibition of encephalitogenic Th1 and Th17 immune responses. Exposure of G-MDSCs to the autoimmune milieu led to up-regulation of the programmed death 1 ligand that was required for the G-MDSC–mediated suppressive function both in vitro and in vivo. Importantly, myeloid-derived suppressor cells were enriched in the periphery of subjects with active multiple sclerosis and suppressed the activation and proliferation of autologous CD4+ T cells ex vivo. Collectively, this study revealed a pivotal role for myeloid-derived suppressor cells in the regulation of multiple sclerosis, which could be exploited for therapeutic purposes.

Hematocrit-lowering Effect Following Inactivation of Renin-Angiotensin System with Angiotensin Converting Enzyme Inhibitors and Angiotensin Receptor Blockers

Several clinical and experimental observations suggest that an intact and activated renin-angiotensin system (RAS) may be an important determinant of erythropoiesis in a variety of clinical conditions, including hypertension, chronic renal insufficiency or failure, chronic obstructive pulmonary disease, and congestive heart failure. Accordingly, RAS inactivation may confer susceptibility to the hematocrit-lowering effects of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers. Indeed, a dose-dependent decrease in hematocrit is observed within the first month of such therapy. In the majority of patients with hypertension decreases in hematocrit values after RAS inactivation are small and not clinically important. In extreme conditions, however, such as erythrocytosis after successful renal transplantation, secondary polycythemia of chronically hypoxemic COPD patients, erythrocytosis associated with renovascular hypertension, severe cardiac or renal failure, the hematocrit-lowering effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blocker may be profound and even lead to or worsen anemia. Hematocrit reaches its nadir value within three months, and then it remains stable during long-term observations. After discontinuation of RAS blockade, hematocrit values rise gradually over the next three to four months towards the pretreatment levels. The mechanism(s) related to this phenomenon is not yet fully understood, but angiotensin II seems to be responsible for inappropriately sustaining secretion of erythropoietin despite hematocrit elevation and capable to directly stimulate the erythroid progenitors in bone marrow to produce erythrocytes.

Recognition Pliability Is Coupled to Structural Heterogeneity: A Calmodulin Intrinsically Disordered Binding Region Complex

Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP(145-165)) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP(145-165)-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks.

Design of novel cyclic altered peptide ligands of myelin basic protein MBP83-99 that modulate immune responses in SJL/J mice.

The use of antagonist peptides derived from the myelin sheath constitutes a promising therapeutic approach for multiple sclerosis (MS). Cyclization of peptide analogues is of great interest, since the limited stability of linear peptides restricts their potential as therapeutic agents. Herein, we designed and synthesized a number of cyclic peptides by mutating TCR contact sites of the MBP 83-99 epitope. A number of cyclic analogues were tested for their ability to inhibit (antagonize) Th1 (IFN-gamma) responses, and cyclo(83-99)[A (91)]MBP 83-99 mutant peptide was found to be the most efficient inhibitor. We demonstrated that cyclo(83-99)[A (91)]MBP 83-99 peptide emulsified in CFA enhanced Th2 (IL-4) and antibody responses in vivo. Moreover, immunization of mice with antagonist cyclo(83-99)[A (91)]MBP 83-99 peptide conjugated to reduced mannan enhanced IL-4 responses compared to cyclo(83-99)MBP 83-99 peptide. Thus, cyclized peptides, which offer greater stability and enhanced responses, are novel leads for the immunotherapy of many diseases, such as MS. In particular, cyclo(83-99)[A (91)]MBP 83-99 is a promising mutant peptide analogue for the potential treatment of MS.

Citrullination of Linear and Cyclic Altered Peptide Ligands from Myelin Basic Protein (MBP87−99) Epitope Elicits a Th1 Polarized Response by T Cells Isolated from Multiple Sclerosis Patients: Implications in Triggering Disease

Derangement of cellular immunity is central in the pathophysiology of multiple sclerosis (MS) and is often manifested by abnormal cytokine production. We investigated cytokine secretion in peripheral blood mononuclear cells (PBMC) of 18 MS patients and 15 controls and correlated cytokine polarization with the nature of antigenic stimulus. We synthesized two novel citrullinated peptides, linear [Cit(91), Ala(96), Cit(97)]MBP(87-99) and cyclo(87-99)[Cit(91), Ala(96), Cit(97)]MBP(87-99) that resulted from citrullination of 91,97 Arg residues in antagonists, linear [Arg(91), Ala(96)]MBP(87-99) and cyclo(87-99)[Arg(91), Ala(96)]MBP(87-99) peptides. PBMC from MS patients and controls were cultured with citrullinated peptides, and both peptides caused a Th1 polarization in all MS patients studied. In contrast, culture with noncitrullinated MBP peptides resulted in heterogeneous cytokine secretion that differed between individual patients. Thus, citrullination of self-antigens may potentially trigger disease in susceptible individuals. This finding may open new avenues in drug design of new substances that inhibit citrullination and arrest epitope spreading and worsening of MS.

Treatment of experimental allergic encephalomyelitis (EAE) by a rationally designed cyclic analogue of myelin basic protein (MBP) epitope 72-85.

In this report the rational design, synthesis and pharmacological properties of an amide-linked cyclic antagonist analogue of the guinea pig myelin basic protein epitope MBP(72-85) are described. Design of the potent cyclic analogue was based on 2D NOESY nuclear magnetic resonance and molecular dynamics studies carried out in the linear antagonist Ala81MBP(72-85). The cyclic antagonist completely prevented the induction of experimental allergic/autoimmune encephalomyelitis when coinjected with linear and cyclic agonist analogues MBP(72-85) and cyclo(2-9)MBP(72-85).

The Design and Synthesis of a Potent Angiotensin II Cyclic Analogue Confirms the Ring Cluster Receptor Conformation of the Hormone Angiotensin II

The novel amide linked Angiotensin II potent cyclic analogue, c-[Sar1,Lys3,Glu5] ANG II 19 has been designed and synthesized in an attempt to test the aromatic ring clustering and the charge relay bioactive conformation we have recently suggested for ANG II. This constrained cyclic analogue was synthesized by connecting the Lys3 amino and Glu5 carboxyl side chain groups, and it was found to be potent in the rat uterus assay and in anesthetized rabbits. The central part of the molecule is fixed covalently in the conformation predicted according to the backbone bend conformational model proposed for Angiotensin II. The obtained results using a combination of 2D NMR, 1D NOE spectroscopy and molecular modeling revealed a similar Tyr4-Ile5-His6 bend, a His6-Pro7 trans configuration and a side chain aromatic ring cluster of the key aminoacids Tyr4, His6, Phe8 for c-[Sar1,Lys3,Glu5] ANG II as it has been found for ANG II (Matsoukas, J. H.; Hondrelis, J.; Keramida, M.; Mavromoustakos, T.; Markriyannis, A.; Yamdagni, R.; Wu, Q.; Moore, G. J. J. Biol. Chem. 1994, 269, 5303). Previous study of the conformational properties of the Angiotensin II type I antagonist [Hser(gamma-OMe)8] ANG II (Matsoukas, J. M.; Agelis, G.; Wahhab, A.; Hondrelis, J.; Panagiotopoulos. D.; Yamdagni, R.; Wu, Q.; Mavromoustakos, T.; Maia, H.; Ganter, R.; Moore, G. J. J. Med. Chem. 1995, 38, 4660) using 1-D NOE spectroscopy coupled with the present study of the same type of lead antagonist Sarilesin revealed that the Tyr4-Ile5-His6 bend, a conformational property found in Angiotensin II is not present in type I antagonists. The obtained results provide an important conformational difference between Angiotensin II agonists and type I antagonists. It appears that our synthetic attempt to further support our proposed model was successful and points out that the charge relay system and aromatic ring cluster are essential stereoelectronic features for Angiotensin II to exert its biological activity.

Treatment of experimental allergic encephalomyelitis (EAE) induced by guinea pig myelin basic protein epitope 72–85 with a Human MBP87–99 analogue and effects of cyclic peptides

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory and demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). In the present report, a linear analogue and a series of cyclic semi-mimetic peptides were designed and synthesized based on the human myelin basic protein (MBP(87-99)) epitope (Val87-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro90) and on Copolymer I (a mixture of random polymers of Ala, Gln, Lys and Tyr used to treat MS). These analogues were designed looking for suppressors of EAE induced by guinea pig MBP(72-85) epitope (Gln-Lys-Ser-Gln-Arg-Ser-Gln-Asp-Glu-Asn-Pro-Val) in Lewis rats. The linear analogue [Arg91,Ala96]MBP(87-99), in which Arg substitutes Lys91 and Ala substitutes Pro96, was found to be a strong inhibitor which when administered to Lewis rats together with the encephalitogenic agonist MBP(72-85) completely prevented the induction of EAE. In contrast, three N- and C-termini amide-linked cyclic semi-mimetic peptides, [cyclo-Phe-Arg-Asn-Ile-Val-Thr-Ala-Acp (1), cyclo-Phe-Ala-Arg-Gln-Acp (2), cyclo-Tyr-Ala-Lys-Gln-Acp (3)] as well as a Lys side chain and C-terminous cyclic semi mimetic peptide cyclo(Lys, Acp)-Phe-Lys-Asn-Ile-Val-Thr-Ala-Acp (4) which contain segments of MBP(87-99) or are constituted from immunophoric residues of copolymer 1, were ineffective in inducing or inhibiting EAE in Lewis rats. However co-injection of cyclic analogues with MBP(72-85) delayed the onset of EAE indicating a modulatory effect on the EAE activity of MBP(72-85). These findings suggest that molecule length, size of cyclic moiety and backbone conformation are important elements for immunogenic activity. Moreover blockade of MBP(72-85) induced EAE by the unrelated peptide [Arg91,Ala56]MBP(87-99) could indicate that the mechanism of inhibition is not due to binding competition but rather due to the delivery of a negative signal by the antagonist which overcomes the agonist response possibly through the activation of antigen specific regulatory T cells.

Design and Pharmacology of Peptide Mimetics

Publisher Summary This chapter analyzes the revolutionary changes that are at the forefront of the new pharmaceutical era, and also attempts to extrapolate the likely outcomes in the not-too-distant future. Although new conceptual and technological advances in the area of biomolecule mimetics includes all conceivable types of biomolecules, the most prolific effects both in the short and long terms is expected in the area of peptide mimetics because of the widespread distribution of peptides at the active sites of proteins including receptors, enzymes, antibodies, and so on. In fact the main thrust of the new technologies revolves around the ability to invoke a strategy of epitope identification, modeling, and mimicry, which is able to focus on a single biomolecule of interest, thus changing the scenario from random screening to targeted screening. A fairly ordered process is beginning to emerge for obtaining a peptide mimetic from a protein or biological peptide, which is outlined in the chapter. The interpretation of pharmacological data for peptide mimetics, such as the peptides themselves, is complicated by a number of factors including multiple binding sites, receptor subtypes, and issues relating to receptor cooperativity. Small-molecule mimetics may invoke another layer of complexity because of interactions with restricted binding sites not available to peptides, as well as effects resulting from increased membrane solubility.