Synthesis of Novel Arginine Building Blocks with Increased Lipophilicity Compatible with Solid-Phase Peptide Synthesis.

Arginine, due to the guanidine moiety, increases peptides' hydrophilicity and enables interactions with charged molecules, but at the same time, its presence in a peptide chain might reduce its permeability through biological membranes. This might be resolved by temporary coverage of the peptide charge by lipophilic, enzyme-sensitive alkoxycarbonyl groups. Unfortunately, such a modification of a guanidine moiety has not been reported to date and turned out to be challenging. Here, we present a new, optimized strategy to obtain arginine building blocks with increased lipophilicity that were successfully utilized in the solid-phase peptide synthesis of novel arginine vasopressin prodrugs.

Myristoylation Confers Oral Bioavailability and Improves the Bioactivity of c(MyD 4-4), a Cyclic Peptide Inhibitor of MyD88.

Myeloid differentiation primary response 88 (MyD88) is an intracellular adaptor protein central to the signaling of multiple receptors involved in inflammation. Since innate immune inflammation promotes autoimmunity, MyD88 is an attractive target in autoimmune disease. We previously developed c(MyD 4-4), a novel cyclic peptide competitive inhibitor of MyD88 dimerization that is metabolically stable. Parenteral administration of c(MyD 4-4) reduces disease severity in a mouse model of the human autoimmune disease multiple sclerosis. We now show that N-terminal myristoylation of c(MyD 4-4) enhances the competitive inhibition of MyD88 dimerization in living cells, leading to improved inhibition of the Toll-like receptor and IL-1 receptor signaling. Importantly, myristoylation converts c(MyD 4-4) to an orally bioavailable inhibitor of MyD88. Oral administration of c(MyD 4-4) significantly lowered the inflammatory cytokines secreted by peripheral autoimmune T cells in mice immunized with myelin antigens and ameliorated disease severity in the mouse model of multiple sclerosis. Taken together, we show the conversion of a protein active region to a metabolically stable, selective cyclic peptide that is orally bioavailable.

The tripartite mechanism as the basis for a biochemical memory engram.

In this paper, we address the enigma of the memory engram, the physical trace of memory in terms of its composition, processes, and location. A neurochemical approach assumes that neural processes hinge on the same terms used to describe the biochemical functioning of other biological tissues and organs. We define a biochemical process, a tripartite mechanism involving the interactions of neurons with their neural extracellular matrix, trace metals, and neurotransmitters as the basis of a biochemical memory engram. The latter inextricably link physiological responses, including sensations with affective states, such as emotions.

Enhancing Oral Bioavailability of Cyclic RGD Hexa-peptides by the Lipophilic Prodrug Charge Masking Approach: Redirection of Peptide Intestinal Permeability from a Paracellular to Transcellular Pathway.

Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junctions (paracellular pathway) in the intestine. This permeability mechanism restricts the magnitude of their oral absorption and bioavailability. We hypothesized that concealing the hydrophilic residues of the peptide using the lipophilic prodrug charge masking approach (LPCM) can improve the bioavailability of hydrophilic peptides. To test this hypothesis, a cyclic N-methylated hexapeptide containing Arg-Gly-Asp (RGD) and its prodrug derivatives, masking the Arg and Asp charged side chains, were synthesized. The library was evaluated for intestinal permeability in vitro using the Caco-2 model. Further investigation of metabolic stability ex vivo models in rat plasma, brush border membrane vesicles (BBMVs), and isolated CYP3A4 microsomes and pharmacokinetic studies was performed on a selected peptide and its prodrug (peptide 12). The parent drug analogues were found to have a low permeability rate in vitro, corresponding to atenolol, a marker for paracellular permeability. Moreover, palmitoyl carnitine increased the Papp of peptide 12 by 4-fold, indicating paracellular permeability. The Papp of the prodrug derivatives was much higher than that of their parent peptides. For instance, the Papp of the prodrug 12P was 20-fold higher than the Papp of peptide 12 in the apical to basolateral (AB) direction. Whereas the permeability in the opposite direction (BA of the Caco-2 model) was significantly faster than the Papp AB, indicating the involvement of an efflux system. These results were corroborated when verapamil, a P-gp inhibitor, was added to the Caco-2 model and increased the Papp AB of prodrug 12P by 3-fold. The prodrug 12P was stable in the BBMVs environment, yet degraded quickly (less than 5 min) in the plasma into the parent peptide 12. Pharmacokinetic studies in rats showed an increase in the bioavailability of peptide 12 > 70-fold (from 0.58 ± 0.11% to 43.8 ± 14.9%) after applying the LPCM method to peptide 12 and converting it to the prodrug 12P. To conclude, the LPCM approach converted the absorption mechanism of the polar peptides from a paracellular to transcellular pathway that tremendously affects their oral bioavailability. The LPCM method provides a solution for the poor bioavailability of RGD cyclohexapeptides and paves the way for other active hydrophilic and charged peptides with poor oral bioavailability.

Orally Active Peptides: Is There a Magic Bullet?

For decades, the development of peptides as potential drugs was aimed solely at peptides with the highest affinity, receptor selectivity, or stability against enzymatic degradation. However, optimization of their oral availability is highly desirable to establish orally active peptides as potential drug candidates for everyday use. A twofold optimization process is necessary to produce orally active peptides: 1) optimization of the affinity and selectivity and 2) optimization of the oral availability. These two steps must be performed sequentially for the rational design of orally active peptides. Nevertheless, additional knowledge is required to understand which structural changes increase oral availability, followed by incorporation of these elements into a peptide without changing its other biological properties. Considerable efforts have been made to understand the influence of these modifications on oral availability. One approach is to improve the oral availability of a peptide that has been previously optimized for biological activity, as described in (1) above. The second approach is to first identify an intestinally permeable, metabolically stable peptide scaffold and then introduce the functional groups necessary for the desired biological function. Previous approaches to achieving peptide oral availability have been claimed to have general applicability but, thus far, most of these solutions have not been successful in other cases. This Review discusses diverse chemical modifications, model peptides optimized for bioavailability, and orally active peptides to summarize the state of the research on the oral activity of peptides. We explain why no simple and straightforward strategy (i.e. a "magic bullet") exists for the design of an orally active peptide with a druglike biological function.

Overcoming the Lack of Oral Availability of Cyclic Hexapeptides: Design of a Selective and Orally Available Ligand for the Integrin†αvß3.

A highly systematic approach for the development of both orally bioavailable and bioactive cyclic N-methylated hexapeptides as high affinity ligands for the integrin αvß3 is based on two concepts: a) screening of systematically designed libraries with spatial diversity and b) masking of the peptide charge with a lipophilic protecting group. The key steps of the method are 1) initial design of a combinatorial library of N-methylated analogues of the stem peptide cyclo(d-Ala-Ala5 ); 2) selection of cyclic peptides with the highest intestinal permeability; 3) design of sublibraries with the bioactive RGD sequence in all possible positions; 4) selection of the best ligands for RGD-recognizing integrin subtypes; 5) fine-tuning of the affinity and selectivity by additional Ala to Xaa substitutions; 6) protection of the charged functional groups according to the prodrug concept to regain intestinal and oral permeability; 7) proof of biological effects in mice after oral administration.

DMAP-assisted sulfonylation as an efficient step for the methylation of primary amine motifs on solid support.

Several multistep strategies were developed to ensure single methylation of amines on solid support. These strategies rely on the introduction of the o-NBS protecting/activating group as a key step. We found that the state-of-the-art strategies fail for the methylation of several primary amine motifs, largely due to inefficient sulfonylation. Here we show that using the superior nucleophilic base DMAP instead of the commonly used base collidine as a sulfonylation additive is essential for the introduction of the o-NBS group to these amine motifs. DFT calculations provide an explanation by showing that the energy barrier of the DMAP intermediate is significantly lower than the one of the collidine. We demonstrate that using DMAP as a sole additive in the sulfonylation step results in an overall effective and regioselective N-methylation. The method presented herein proved highly efficient in solid-phase synthesis of a somatostatin analogue bearing three Nα-methylation sites that could not be synthesized using the previously described state-of-the-art methods.

cis-Peptide Bonds: A Key for Intestinal Permeability of Peptides? .

Recent structural studies on libraries of cyclic hexapeptides led to the identification of common backbone conformations that may be instrumental to the oral availability of peptides. Furthermore, the observation of differential Caco-2 permeabilities of enantiomeric pairs of some of these peptides strongly supports the concept of conformational specificity driven uptake and also suggests a pivotal role of carrier-mediated pathways for peptide transport, especially for scaffolds of polar nature. This work presents investigations on the Caco-2 and PAMPA permeability profiles of 13 selected N-methylated cyclic pentaalanine peptides derived from the basic cyclo(-D-Ala-Ala4 -) template. These molecules generally showed moderate to low transport in intestinal epithelia with a few of them exhibiting a Caco-2 permeability equal to or slightly higher than that of mannitol, a marker for paracellular permeability. We identified that the majority of the permeable cyclic penta- and hexapeptides possess an N-methylated cis-peptide bond, a structural feature that is also present in the orally available peptides cyclosporine A and the tri-N-methylated analogue of the Veber-Hirschmann peptide. Based on these observations it appears that the presence of N-methylated cis-peptide bonds at certain locations may promote the intestinal permeability of peptides through a suitable conformational preorganization.

Mechanistic studies of malonic acid-mediated in situ acylation.

We have previously introduced an easy to perform, cost-effective and highly efficient acetylation technique for solid phase synthesis (SPPS). Malonic acid is used as a precursor and the reaction proceeds via a reactive ketene that acetylates the target amine. Here we present a detailed mechanistic study of the malonic acid-mediated acylation. The influence of reaction conditions, peptide sequence and reagents was systematically studied. Our results show that the methodology can be successfully applied to different types of peptides and nonpeptidic molecules irrespective of their structure, sequence, or conformation. Using alkyl, phenyl, and benzyl malonic acid, we synthesized various acyl peptides with almost quantitative yields. The ketenes obtained from the different malonic acid derived precursors were characterized by in situ (1) H-NMR. The reaction proceeded in short reaction times and resulted in excellent yields when using uronium-based coupling agents, DIPEA as a base, DMF/DMSO/NMP as solvents, Rink amide/Wang/Merrifield resins, temperature of 20°C, pH 8-12 and 5 min preactivation at inert atmosphere. The reaction was unaffected by Lewis acids, transition metal ions, surfactants, or salt. DFT studies support the kinetically favorable concerted mechanism for CO2 and ketene formation that leads to the thermodynamically stable acylated products. We conclude that the malonic acid-mediated acylation is a general method applicable to various target molecules.

A small shared epitope-mimetic compound potently accelerates osteoclast-mediated bone damage in autoimmune arthritis.

We have recently proposed that the shared epitope (SE) may contribute to rheumatoid arthritis pathogenesis by acting as a ligand that activates proarthritogenic signal transduction events. To examine this hypothesis, in this study we characterized a novel small SE-mimetic compound, c(HS4-4), containing the SE primary sequence motif QKRAA, which was synthesized using a backbone cyclization method. The SE-mimetic c(HS4-4) compound interacted strongly with the SE receptor calreticulin, potently activated NO and reactive oxygen species production, and markedly facilitated osteoclast differentiation and function in vitro. The pro-osteoclastogenic potency of c(HS4-4) was 100,000- to 1,000,000-fold higher than the potency of a recently described linear SE peptidic ligand. When administered in vivo at nanogram doses, c(HS4-4) enhanced Th17 expansion, and in mice with collagen-induced arthritis it facilitated disease onset, increased disease incidence and severity, enhanced osteoclast abundance in synovial tissues and osteoclastogenic propensities of bone marrow-derived cells, and augmented bone destruction. In conclusion, c(HS4-4), a highly potent small SE-mimetic compound enhances bone damage and disease severity in inflammatory arthritis. These findings support the hypothesis that the SE acts as a signal transduction ligand that activates a CRT-mediated proarthritogenic pathway.

A highly efficient in situ N-acetylation approach for solid phase synthesis.

We describe a new general N-acetylation method for solid phase synthesis. Malonic acid is used as a precursor and the reaction proceeds by in situ formation of a reactive ketene intermediate at room temperature. We have successfully applied this methodology to peptides and non-peptidic molecules containing a variety of functional groups. The reaction gave high yields compared to known acetylation methods, irrespective of the structure, conformation and sequence of the acetylated molecule. Computational studies revealed that the concerted mechanism via the ketene intermediate is kinetically favorable and leads to a thermodynamically stable acetylated product. In conclusion, our method can be easily applied to acetylation in a wide variety of chemical reactions performed on the solid phase.

A tandem in situ peptide cyclization through trifluoroacetic acid cleavage.

We present a new approach for peptide cyclization during solid phase synthesis under highly acidic conditions. Our approach involves simultaneous in situ deprotection, cyclization and trifluoroacetic acid (TFA) cleavage of the peptide, which is achieved by forming an amide bond between a lysine side chain and a succinic acid linker at the peptide N-terminus. The reaction proceeds via a highly active succinimide intermediate, which was isolated and characterized. The structure of a model cyclic peptide was solved by NMR spectroscopy. Theoretical calculations support the proposed mechanism of cyclization. Our new methodology is applicable for the formation of macrocycles in solid-phase synthesis of peptides and organic molecules.

Development of Clarstatin, a Novel Drug Lead for the Therapy of Autoimmune Uveitis.

We describe the design, synthesis, and activity of a potent thiourea-bridged backbone cyclic peptidomimetic known as Clarstatin, comprising a 5-amino acid sequence (Q/D)1-(R/K)2-X3-X4-A5-(Gln/Asp)1-(Arg/Lys)2-AA3-AA4-Ala5-based on a motif called "shared epitope (SE)", specifically present in specific alleles of the HLA-DRB1 gene. This SE binds to a particular site within the proline reach domain (P-domain) of the cell surface-calreticulin (CS-CRT). CS-CRT is a multifunctional endoplasmic reticulum (ER) calcium-binding protein that is located on the cell surface of T cells and triggers innate immune signaling, leading to the development of inflammatory autoimmune diseases. The development of Clarstatin was based on the parent peptide W-G-D1-K2-S3-G4-A5- derived from the active region of the SE. Following the design based on the cycloscan method, the synthesis of Clarstatin was performed by the Fmoc solid phase peptide synthesis (SPPS) method, purified by HPLC to 96% homogeneity, and its structure was confirmed by LC-MS. Clarstatin reduced calcium levels in Jurkat lymphocyte cultures, ameliorated uveitis in vivo in the experimental autoimmune uveitis (EAU) mice model, and was safe upon acute toxicity evaluation. These findings identify Clarstatin as a promising lead compound for future drug development as a novel class of therapeutic agents in the therapy of uveitis.

Structure-Activity Relationship of Synthetic Linear KTS-Peptides Containing Meta-Aminobenzoic Acid as Antagonists of α1ß1 Integrin with Anti-Angiogenic and Melanoma Anti-Tumor Activities.

To develop peptide drugs targeting integrin receptors, synthetic peptide ligands endowed with well-defined selective binding motifs are necessary. The snake venom KTS-containing disintegrins, which selectively block collagen α1ß1 integrin, were used as lead compounds for the synthesis and structure-activity relationship of a series of linear peptides containing the KTS-pharmacophore and alternating natural amino acids and 3-aminobenzoic acid (MABA). To ensure a better stiffness and metabolic stability, one, two and three MABA residues, were introduced around the KTS pharmacophore motif. Molecular dynamics simulations determined that the solution conformation of MABA peptide 4 is more compact, underwent larger conformational changes until convergence, and spent most of the time in a single cluster. The peptides' binding affinity has been characterized by an enzyme linked immunosorbent assay in which the most potent peptide 4 inhibited with IC50 of 324 ± 8 µM and 550 ± 45 µM the binding of GST-α1-A domain to collagen IV fragment CB3, and the cell adhesion to collagen IV using α1-overexpressor cells, respectively. Docking studies and MM-GBSA calculations confirmed that peptide 4 binds a smaller region of the integrin near the collagen-binding site and penetrated deeper into the binding site near Trp1. Peptide 4 inhibited tube formation by endothelial cell migration in the Matrigel angiogenesis in vitro assay. Peptide 4 was acutely tolerated by mice, showed stability in human serum, decreased tumor volume and angiogenesis, and significantly increased the survival of mice injected with B16 melanoma cells. These findings propose that MABA-peptide 4 can further serve as an α1ß1-integrin antagonist lead compound for further drug optimization in angiogenesis and cancer therapy.

Treatment with αvß3-integrin-specific 29P attenuates pressure-overload induced cardiac remodelling after transverse aortic constriction in mice.

Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin targeting has been used in the treatment of ocular disease and cancer, but little is known about its utility in the treatment of heart failure. Here we sought to determine whether the second generation orally available, αvß3-specific RGD-mimetic, 29P , was cardioprotective. Male mice were subjected to transverse aortic constriction (TAC) and treated with 50 µg/kg 29P or volume-matched saline as Vehicle control. At 3 weeks post-TAC, echocardiography showed that 29P treatment significantly restored cardiac function and structure indicating the protective effect of 29P treatment in this model of heart failure. Importantly, 29P treatment improved cardiac function giving improved fractional shortening, ejection fraction, heart weight and lung weight to tibia length fractions, together with partial restoration of Ace and Mme levels, as markers of the TAC insult. At a tissue level, 29P reduced cardiomyocyte hypertrophy and interstitial fibrosis, both of which are major clinical features of heart failure. RNA sequencing identified that, mechanistically, this occurred with concomitant alterations to genes involved molecular pathways associated with these processes such as metabolism, hypertrophy and basement membrane formation. Overall, targeting αvß3 with 29P provides a novel strategy to attenuate pressure-overload induced cardiac hypertrophy and fibrosis, providing a possible new approach to heart failure treatment.

Backbone cyclic helix mimetic of chemokine (C-C motif) receptor 2: a rational approach for inhibiting dimerization of G protein-coupled receptors.

The transmembrane helical bundle of G protein-coupled receptors (GPCRs) dimerize through helix-helix interactions in response to inflammatory stimulation. A strategy was developed to target the helical dimerization site of GPCRs by peptidomimetics with drug like properties. The concept was demonstrated by selecting a potent backbone cyclic helix mimetic from a library that derived from the dimerization region of chemokine (C-C motif) receptor 2 (CCR2) that is a key player in Multiple Sclerosis. We showed that CCR2 based backbone cyclic peptide having a stable helix structure inhibits specific CCR2-mediated chemotactic migration.

Quantitative analysis of influenza M2 channel blockers.

The influenza M2 H(+) channel enables the concomitant acidification of the viral lumen upon endosomic internalization. This process is critical to the viral infectivity cycle, demonstrated by the fact that M2 is one of only two targets for anti-flu agents. However, aminoadamantyls that block the M2 channel are of limited therapeutic use due to the emergence of resistance mutations in the protein. Herein, using an assay that involves expression of the protein in Escherichia coli with resultant growth retardation, we present quantitative measurements of channel blocker interactions. Comparison of detailed K(s) measurements of different drugs for several influenza channels, shows that the swine flu M2 exhibits the highest resistance to aminoadamantyls of any channel known to date. From the perspective of the blocker, we show that rimantadine is consistently a better blocker of M2 than amantadine. Taken together, such detailed and quantitative analyses provide insight into the mechanism of this important and pharmaceutically relevant channel blocker system.

Intestinal permeability of cyclic peptides: common key backbone motifs identified.

Insufficient oral bioavailability is considered as a key limitation for the widespread development of peptides as therapeutics. While the oral bioavailability of small organic compounds is often estimated from simple rules, similar rules do not apply to peptides, and even the high oral bioavailability that is described for a small number of peptides is not well understood. Here we present two highly Caco-2 permeable template structures based on a library of 54 cyclo(-D-Ala-Ala(5)-) peptides with different N-methylation patterns. The first (all-trans) template structure possesses two ß-turns of type II along Ala(6)-D-Ala(1) and Ala(3)-Ala(4) and is only found for one peptide with two N-methyl groups at D-Ala(1) and Ala(6) [(NMe(1,6)]. The second (single-cis) template possesses a characteristic cis peptide bond preceding Ala(5), which results in type VI ß-turn geometry along Ala(4)-Ala(5). Although the second template structure is found in seven peptides carrying N-methyl groups on Ala(5), high Caco-2 permeability is only found for a subgroup of two of them [NMe(1,5) and NMe(1,2,4,5)], suggesting that N-methylation of D-Ala(1) is a prerequisite for high permeability of the second template structure. The structural similarity of the second template structure with the orally bioavailable somatostatin analog cyclo(-Pro-Phe-NMe-D-Trp-NMe-Lys-Thr-NMe-Phe-), and the striking resemblance with both ß-turns of the orally bioavailable peptide cyclosporine A, suggests that the introduction of bioactive sequences on the highly Caco-2 permeable templates may result in potent orally bioavailable drug candidates.

Studying protein-peptide interactions using benzophenone units: a case study of protein kinase B/Akt and its inhibitor PTR6154.

Protein-protein interactions (PPIs) govern nearly all processes in living cells. Peptides play an important role in studying PPIs. Peptides carrying photoaffinity labels that covalently bind the interacting protein can be used to obtain more accurate information regarding PPIs. Benzophenone (BP) is a useful photoaffinity label that is widely used to study PPIs. We developed a one-pot two-step synthesis for the preparation of novel BP units. To map the binding site more thoroughly, linkers of various lengths were attached to the BP moiety. These units can be incorporated into peptide sequences using well-established solid phase peptide synthesis (SPPS) protocols. As a proof of concept, we studied the interaction between protein kinase B (PKB/Akt) and its synthetic peptide inhibitor, PTR6154. The methodology is general and can be implemented to study PPIs in a variety of biological systems.

Developing potent backbone cyclic peptides bearing the shared epitope sequence as rheumatoid arthritis drug-leads.

Rheumatoid arthritis (RA) is a common human leukocyte antigen-associated disease. Most RA patients have a five-residue sequence motif called the shared epitope (SE) in the DRß-chain of the HLA-DRB1 protein. The SE was found to activate nitric oxide (NO) production, suggesting a possible mechanism for RA development. The native conformation of the SE is presumed to be an α-helix, thus using cyclic peptides to stabilize this conformation may produce a potent SE mimetic which will have drug-like properties. We present the development of a backbone cyclic SE mimetic that activates NO production in the low nM range. Circular dichroism analysis revealed a conformational change from for the parent linear peptides to the cyclic analogs. The most active cyclic analog is completely stable towards trypsin/chymotrypsin degradation while the linear 15-mer analogs completely degraded within 30 min. The outcome of this study is a potent cyclic peptide with drug-like properties that can be used as a template for drug development.