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Nonpyrogenic Molecular Adjuvants Based on norAbuMuramyldipeptide and norAbu-Glucosaminyl Muramyldipeptide: Synthesis, Molecular Mechanisms of Action, and Biological Activities in Vitro and in Vivo Roman Effenberg,† Pavlína Turánek Knötigová,‡ Daniel Zyka,§ Hana Č elechovská,‡ Josef Mašek,‡ Eliška Bartheldyová,‡ František Hubatka,‡ Štěpán Koudelka,‡ Róbert Lukác,̌ ‡ Anna Kovalová,∥ David Šaman,∥ Michal Křupka,⊥ Lucia Barkocziova,⊥ Petr Kosztyu,⊥ Marek Šebela,# Ladislav Drož,§ Michal Hučko,§,∇ Mária Kanásová,§,○ Andrew D. Miller,‡,◆,¶ Milan Raška,*,‡,⊥ Miroslav Ledvina,*,† and Jaroslav Turánek*,‡ †

Department of Chemistry of Natural Compounds, University of Chemistry and Technology, Technická 5,166 28 Prague 6, Czech Republic ‡ Department of Pharmacology and Immunotherapy, Veterinary Research Institute vvi, Hudcova 70, 621 00 Brno, Czech Republic § APIGENEX s.r.o., Poděbradská 173/5, Prague 9, 190 00, Czech Republic ∥ Institute of Organic Chemistry and Biochemistry, AS CR vvi Flemingovo nám 2, 160 00 Prague, Czech Republic ⊥ Department of Immunology, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hněvotínská 3, 775 15 Olomouc, Czech Republic # Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University Olomouc, 775 15 Olomouc, Czech Republic ∇ Department of Organic Chemistry, University of Chemistry and Technology, Technická 5, 166 28 Prague 6, Czech Republic ○ Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 2030/8, 128 43 Prague 2, Czech Republic ◆ Institute of Pharmaceutical Science, King’s College London, London SE1 9NH, United Kingdom ¶ KP Therapeutics Ltd., Manchester M3 2ER, United Kingdom S Supporting Information *

ABSTRACT: Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDPLys(L18), norAbuGMDP-Lys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-L-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response).

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INTRODUCTION Vaccination has been proved to be one of the most effective medical interventions, allowing for the control or eradication of major infectious diseases. The progress in development of new recombinant vaccines has been assisted simultaneously by the © 2017 American Chemical Society

development of new biocompatible antigen carriers of antigens and potent adjuvants. Potent molecular adjuvants may be derived Received: April 21, 2017 Published: August 22, 2017 7745

DOI: 10.1021/acs.jmedchem.7b00593 J. Med. Chem. 2017, 60, 7745−7763

Journal of Medicinal Chemistry

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This research was initiated by the finding that N-acetylnormuramoyl-L-α-aminobutanoyl-D-isoglutamine (norAbuMDP) is less pyrogenic and possesses a higher immunoadjuvant activity in comparison with parent MDP.16,17 By making the same structural changes in GMDP, i.e., replacement of muramic acid to normuramic acid and of L-alanine with L-α-amininobutanoic acid, we thus obtained a nonpyrogenic and highly immunostimulatory norAbuGMDP (N-acetyl-β-D-glucosaminyl-(1→4)-N-acetylnormuramoyl-L-α-aminobutanoyl-D-isoglutamine).18 Following this, we demonstrated that the introduction of bulky lipophilic residues into norAbuMDP or norAbuGMDP enhanced their immunomodulatory activities while retaining the favorable pharmacological parameters of the parent structures.19−22 Accordingly, we became motivated to design and prepare new group of lipophilic fatty acyl analogues of norAbuMDP and norAbuGMDP, differing in the character and topology of their lipophilic residue in order to modify their immunopharmacological parameters.23,24 Here we now outline the synthetic pathways for the preparation of these novel norAbuMDP and norAbuGMDP muramylglycopeptide analogues and characterization of their basic biological activities in vitro and in vivo. In addition, we determine molecular mechanisms of action and biological effects pertinent to both stimulation of innate and adaptive immunity.

from pathogen- and danger-derived molecular signatures, collectively referred to as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), respectively. PAMPs and DAMPs are recognized as main targets by receptors of antigen presenting cells. Therefore, PAMPs and DAMPs may be used as adjuvants or immunostimulants.1 One very specific and promising group of PAMP/DAMP-related adjuvants or immunostimulants are synthetic or semisynthetic muramylglycopeptides that derive from peptidoglycan (PGN) fragments of the bacterial cell wall. In 1974, Adam et al.2 described how muramyldipeptide (MDP; N-acetylmuramoyl-Lalanyl-D-isoglutamine) was the minimal structural unit deriving from PGN needed to mimic the immunostimulatory activities of Freund’s Complete Adjuvant (FCA). Unfortunately, the strong pyrogenicity of MDP has since hampered the possibility of using MDP in immunotherapy. On the other hand, the minimal PGN repeat unit known as N-acetyl glucosaminyl muramyldipeptide (GMDP; N-acetyl-β-D-glucosaminyl-(1→4)-N-acetylmuramoylL-alanyl-D-isoglutamine) was shown to be both more immunostimulatory than MDP and less pyrogenic, but the side effects were still significant.3,4 Mechanistic studies have recently suggested that MDP and GMDP are recognized by family members of the intracellular nucleotide-binding oligomerization domainlike receptors (NLRs) such as NOD2. NLRs play key roles in both adaptive and innate immunity by triggering a cascade of regulatory cytokines, chemokines, plus the production of antimicrobial peptides.5,6 Since first reports concerning the immunostimulatory activities of MDP and GMDP, many analogues have been designed and synthesized in order to optimize therapeutic profile and suppress undesirable side effects, especially strong pyrogenicity. Unfortunately, still only a very few such analogues have been introduced into clinical trials and thence into medical practice. In particular, one analogue of MDP modified by inclusion of dipalmitoylphosphatidylethanolamine (N-acetylmuramoyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy)]ethylamide) was approved as an immunotherapeutic for combined immunochemotherapy of osteosarcoma under the clinical designation Mifamurtide.7 Mifamurtide has also been assayed as a coadjuvant in combination with MF59-adjuvant in influenza and HIV-1 vaccines,8,9 but systemic side effects including fever, chill, and nausea have rendered such vaccine combinations inappropriate for clinical use. Alternatively, Romurtide, obtained by the prolongation of the MDP peptide chain with stearoyl-substituted lysine (N-acetylmuramoyl-L-alanyl-D-isoglutaminyl-ε-stearoyl-Llysine) is currently used in clinic as a pro-hemopoietic agent for treatment of leukopenia induced by chemotherapy and/or radiotherapy of cancers.10−12 The most common adverse reaction in cancer patients treated with Romurtide is fever accompanied by chill.13 Otherwise, GMDP is the active component of a preparation known as Likopid, registered in Russia and post-Soviet Republics as an immunotherapeutic with broad applicability, e.g., treatment of infectious diseases, and prevention of infections in patients post-trauma, postoperative, postchemotherapy, and postradiotherapy.14,15 Contrary to the case with Romurtide, the extension of the GMDP peptide chain with stearoyl-substituted lysine, to give N-acetyl-β-D-glucosaminyl-(1→4)-N-acetylmuramoyl-L-alanyl-D-isoglutaminyl-εstearoyl-L-lysine, led to significantly increased systemic toxicity.5 For over a decade, we have focused on developing muramylglycopeptide analogues with structural modifications in both saccharide and peptide moieties of MDP and GMDP parent structures.

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RESULTS Chemistry. To prepare target norAbuMDP and norAbuGMDP muramylglycopeptide analogues comprising the normuramyl-L-α-aminobutanoyl (norAbu) structural moiety, we decided to employ an approach based on coupling of suitably protected normuramic or glucosaminyl normuramic building blocks and corresponding peptide building blocks previously prepared either in solution or by using solid-phase synthesis. All building blocks were designed with orthogonal protection, thus allowing for subsequent regioselective acylation of downstreamprotected muramyl glycopeptide analogue intermediates with selected fatty acids (e.g., stearic acid [L18] or 2-tetradecylhexadecanoic acid [B30]). The precursors of protected normuramic or glucosaminyl normuramic acid building blocks were N-acetyl-D-glucosamine (GlcNAc) derivatives with 3-OH allyl protection group. Such precursors are very appropriate for the construction of the saccharide portions of muramyl glycopeptides. Thereafter, the standard synthesis of normuramyl and muramyl glycopeptides has typically involved three main steps, starting from appropriate protected 3-O-allyl-D-GlcNAc intermediates. Generally, the allyl group was first isomerized to become a propenyl group, using Wilkinson’s catalyst [RhCl(PPh3)3], then subject to acid catalyzed cleavage, leaving the opportunity for attachment of a glycoloyl moiety by simple alkylation.18,20,21,25 All three steps could be conflated to one by conversion of the allyl group to a glycoloyl ether moiety by the action of RuO4 that can be generated in situ by catalytic amount of RuCl3 using NaIO4 as an oxidant under controlled conditions (Schemes 1 and 2).26 Bearing in mind that RuO4 is very powerful oxidizing agent reaction conditions were carefully adjusted for selective allyl group release in the presence of other protecting groups.27 Initially, allyl derivative 1 was subject to benzylation to give benzyl 2-acetamido-3-O-allyl-4,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside 2 by treatment with benzyl bromide and sodium hydride in DMF according to published procedure.18,23,24,28 Glucopyranoside 2 was then treated with NaIO4 and RuCl3 in CCl4−MeCN−H2O at 0 °C (Scheme 1).27 In early trial runs 7746



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characterized after their conversion to the diacetate 7 and methyl ester 8, respectively. In a corresponding manner, amine 9 and its acetyl derivative 10 were prepared as described previously.21 N-2,2,2-Trichloroethoxycarbonyl (Troc) derivative 11 was prepared by reaction of amine 9 with trichloroethoxycarbonyl chloride in pyridine. Thereafter, 10 and 11 were used to prepare intermediate glucosaminyl normuramic acid building blocks 12 and 13 (Scheme 2),18 then converted to their respective methyl esters 1427 and 15 by reaction with diazomethane in order to allow complete characterization of these compounds. The introduction of Troc protection into intermediate glucosaminyl normuramic acid building block 13 was intended to enable selective fatty acid acylization of the glucosaminyl moiety following selective deprotection, followed by global deprotection to yield novel norAbuGMDP muramylglycopeptide analogues (Scheme 3). The method of preparation of two norAbuGMDP muramylglycopeptide analogues was initiated by coupling of intermediate glucosaminyl normuramic acid building block 13 with a corresponding Abu-dipeptide building block by means of DCC in the presence of HOBt, giving a protected glucosaminyl normuramyl glycopeptide intermediate 16. Following N-Troc protecting group removal under mild conditions involving Zn in AcOH,29,30 the resulting free amino functional group of 17 could be acylated with DMAP and DIPEA in DMF using stearoyl (L18) chloride or 2-tetradecylhexadecanoyl (B30) chloride, giving N-acyl derivatives 18 and 19, respectively. Final catalytic hydrogenolysis to remove all benzyl-protecting groups yielded the desired norAbuGMDP muramylglycopeptide analogues 20 and 21. We observed that the Troc and benzyl protecting groups behaved in a perfectly orthogonal manner, i.e., they were removed independently of each other and without compromising other functional groups present in synthetic intermediates, leading to the target analogues 20 and 21. Target analogue 20 was reported prepared by a different synthetic route previously reported.20 The method of preparation of two novel norAbuMDP muramylglycopeptide analogues was initiated from protected normuramyl building block 6 by DCC/HOBt mediated coupling

Scheme 1. Synthesis of Protected Normuramyl Building Block by Oxidative Cleavage of 3-O-Allyl Protective Groupa

a

Reagents and conditions: (a) BnBr, NaH, DMF, rt, 24 h, 89%; (b) NaIO4, RuCl3, CCl4−MeCN−H2O, 0 °C, method A or method B; (c) Ac2O, Py, DMAP, DCM, rt, 24 h, 94%; (d) Pb(OAc)4, CHCl3, rt, 2 h, 63%; (e) toluene, reflux, 9 h, 52%; (f) CH2N2, Et2O, CH2Cl2MeOH, 0 °C, 86%.

with these reactions conditions, a range of reaction products were observed being diol 3, aldehyde 4, cyclic byproduct 5, and the desired protected normuramic acid 6 intermediate. The formation of undesired cyclic byproduct 5 was suppressed, and direct formation of carboxyl function was facilitated by carrying out the oxidation at moderate temperatures in the presence of an excess of NaIO4, thereby giving the protected normuramic acid 6 intermediate in high overall yield. The observed tendency of the intermediate aldehyde 4 to form the cyclic derivative 5 by the intramolecular reaction with the vicinal acetamido group was confirmed by heating of the abovementioned aldehyde 4 (prepared by reaction of diol 3 with Pb(OAc)4) in toluene, which resulted in the compound 5 as the only isolated product. The diol 3 and acid 6 were fully

Scheme 2. Synthesis of Protected Glucosaminyl Normuramyl Building Block by Allyl Oxidative Cleavagea

a

Reagents and conditions: (a) For 10: Ac2O, DCM, pyridine, rt, 73%. For 11: TrocCl, CH2Cl2, pyridine, DMAP, rt, 74%. (b) NaIO4, RuCl3, CCl4−MeCN−H2O, 0 °C, 57% for 12 or rt, 64% for 13. (c) CH2N2, Et2O, CH2Cl2−MeOH, 0 °C, 93% for 14 and 91% for 15. 7747



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Scheme 3. Synthesis of norAbuGMDP Muramylglycopeptide Analoguesa

a

Reagents and conditions: (a) DCC, HOBt, DCM, DMF, DIPEA, rt, 12 h, 60%. (b) Zn, AcOH, rt, 6 h, 79%. (c) For 18, CH3(CH2)16COCl; or for 19, [CH3(CH2)13]2CHCOCl, DMAP, DMF, DIPEA, 60 °C, 5 h/10 h, 69% (18) or 71% (19). (d) H2, Pd/C, rt, 15 h, 75% (20), and 69% (21).

Scheme 4. Synthesis of norAbuMDP Muramylglycopeptide Analogues Using Barlos Resina

a

Reagents and conditions. (a) CH2Cl2, DIPEA, rt, 1 h, MeOH, rt, 10 min, wash CH2Cl2, DMF, MeOH, Et2O, 1.56 mmol/dry gram. (b) 5% piperidine in DMF-DCM (1:1) 5 min, rt, 20% piperidine in DMF 20 min, rt, wash DMF, Fmoc-D-iGln-OH, HOBt, HBTU, DIPEA, DMF, rt, 90 min. (c) 5% piperidine in DMF−CH2Cl2 (1:1) 25 min, wash DMF, Fmoc-L-α-Abu-OH, HBTU, DIPEA, DMF, rt, 60 min. (d) 20% piperidine in DMF 20 min, rt. (e) HBTU, DIPEA, DMF, rt, 20 h (double coupling). (f) 2% N2H4.H2O, DMF, 10 min, rt. (g) For 25, CH3(CH2)16COOH, or for 26, [CH3(CH2)13]2CHCOOH; HBTU, DMF, DIPEA, rt, 6 h/6 h, 70% (25) or 87% (26). (h) For 27, AcOH−TFE−CH2Cl2 (1:1:8, v/v/v; 60 mL) for 1 h, rt, 35%; for 28, AcOH−TFE−CH2Cl2 (1:1:8; 60 mL) for 1 h, rt, 33%. (i) H2, Pd/C, rt, 24 h/24 h, 39% (29) and 35% (30).

of a protected N-Abu-tripeptide building block anchored on a solid phase chlorotrityl (Barlos) resin that could be cleaved at an appropriate time using mild AcOH−TFE−CH2Cl2 conditions (Scheme 4).6,31 The protected N-Abu-tripeptide building block attached to resin was prepared according to standard Fmoc solid-phase

synthesis protocols with standard orthogonal protecting groups,23,24,32 culminating in the free α-amino L-Abu-D-isoGlnL-Lys(Dde)-O-2-chlorotrityl conjugate 22. This conjugate was then coupled with protected normuramic acid 6 intermediate employing the “swelling volume” method,33 resulting in the formation of protected resin-bound normuramylglycopeptide 23. 7748



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Scheme 5. Synthesis of norAbuGMDP Muramylglycopeptide Analogues Using Barlos Resina

a

Reagents and conditions. (a) HBTU, DIPEA, DMF, rt, 3.5 h, +DMAP rt, 1 h. (b) 2% N2H4.H2O, DMF, 10 min, rt. (c) For 33, CH3(CH2)16COOH; or for 34, [CH3(CH2)13]2CHCOOH, HBTU, DMF, DIPEA, rt, 6 h/6 h, 33 or 34. (d) For 35: AcOH−TFE−CH2Cl2 (1:1:8, v/v/v; 60 mL) for 1 h, rt, 41%. For 36, AcOH−TFE−CH2Cl2 (1:1:8; 60 mL) for 1 h, rt, 32%. (e) H2, Pd/C, rt, 48 h/24 h, 78% (37) and 37% (38).

Characterization of Free (Micellar) and Liposomal Formulations of Lipophilic Analogues of norAbuMDP and norAbuGMDP. Solubilization of Lipophilic Analogues of norAbuMDP and norAbuGMDP and Characterization of Their Colloids. Our lipophilic analogues of norAbuMDP or norAbuGMDP are molecules of amphipathic character, and due to their physical−chemical properties they are surface-active compounds forming micelles in aqueous solutions. The analogues modified by stearoyl moieties were readily soluble in ethanol (96%, medical grade), and after dilution in water they formed micelles of the size of about 6−8 nm. B-30 modified analogues were also soluble in ethanol (96%, medical grade), and after dilution in water they formed micelles or vesicles of bimodal size distribution (35−50 and 80−120 nm). Notably, N-B30 norAbuGMDP 21 was only partially soluble in ethanol. Therefore, N-B30 norAbuGMDP 21 was solubilized in a minimal amount of dimethyl sulfoxide (DMSO) and subsequently diluted by phosphate-buffered saline (PBS). The procedure with DMSO was found generally applicable to all B30 modified analogues. Characterization of Liposomes by DLS. The size distribution, ζ potential, and structures of various liposomal preparations were analyzed by dynamic light scattering (DLS). Final size distributions and liposome polydispersities were not found to be a function of particular lipophilic analogues of norAbuMDP or norAbuGMDP (N-L18 norAbuGMDP 20, N-B30 norAbuGMDP 21, norAbuMDP-Lys(L18) 29, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, norAbuGMDP-Lys(B30) 38, B30 norAbuMDP 39, L18 norAbuMDP 40) (results not shown). Liposomes were prepared by lipid film hydration, freezing−thawing and extrusion through polycarbonate filters,

Thereafter, Dde deprotection with 2% hydrazine solution in DMF yielded resin-bound intermediate 24 that was acylated directly using stearic (L18) acid or 2-tetradecylhexadecanoic (B30) acid with HBTU and DIPEA in DMF, resulting in N-acyl derivatives 25 and 26, respectively. These were released from resin using AcOH−TFE−CH2Cl2 (1:1:8, v/v/v) to give protected and acylated normuramylglycopeptides 27 and 28, respectively. Finally, the desired norAbuMDP muramylglycopeptide analogues 29 and 30 were revealed following catalytic hydrogenolysis of remaining O-benzyl protecting groups. Following this, two novel norAbuGMDP muramylglycopeptide analogues were prepared in a comparable manner. First, protected glucosaminyl normuramyl building block 12 was coupled by DCC/HOBt mediated coupling to the same protected N-Abu-tripeptide building block attached to resin (as above), resulting in the formation of protected resin-bound glucosaminyl normuramylglycopeptide 31 (Scheme 5). Thereafter, Dde deprotection with 2% hydrazine solution in DMF yielded resin-bound intermediate 32 that was then acylated directly using stearic (L18) acid or 2-tetradecylhexadecanoic (B30) acid with HBTU and DIPEA in DMF, resulting in N-acyl derivatives 33 and 34, respectively. These were released from resin using AcOH−TFE−CH2Cl2 (1:1:8, v/v/v) to give protected and acylated glucosaminyl normuramylglycopeptides 35 and 36, respectively. Finally, the desired norAbuGMDP muramyl glycopeptide analogues 37 and 38 were revealed following catalytic hydrogenolysis of remaining O-benzyl protecting groups. Lipophilic Analogues and Derivatives Tested in This Study. Structural formulas, codes, and abbreviations are present in Figures 1 and 2 and Table 1. 7749



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Figure 1. Structural formulas of norAbuMDP or norAbuGMDP analogues and controls tested.

(according to the Ph. Eur. 2005; protocol EP5.0 2.6.8 (EP 2005a)), then free (micellar) and liposomal formulations of norAbuMDP, norAbuGMDP, and all their lipophilic analogues were here declared nonpyrogenic. In addition, no other adverse or toxic effects were observed (e.g., weight lost, skin irritation and necrosis at site of application, behavioral changes), over a period of 2 months in rabbits post administration of either norAbuMDP, norAbuGMDP, or their lipophilic analogues. Stimulation of Innate Immunity. Stimulation of NOD2 Receptor. Biological assay studies revealed the ability of all synthesized analogues to stimulate NOD2 receptor in the in vitro model based on RAW-Blue Reporter Cells. The extent of stimulation was comparable to lipophilic analogue L18 MDP 41, to MDP 42, N-glycolyl-MDP 43, and to norAbu-MTP 44. Lipophilic norAbuMDP or norGMDP analogues B30 norAbuMDP 39, N-L18 norAbuGMDP 20, L18 norAbuMDP 40, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, and norAbuGMDP-Lys(B30) 38 each exerted high levels of receptor stimulation comparable with L18 MDP 41 if not better.

and size distributions were measured in the range 95−120 nm with polydispersity indexes in the range 0.09−0.12. Biological Studies. Pyrogenicity Tests on Rabbits. The pyrogenicities of free (micellar) or liposomal formulations of lipophilic norAbuMDP or norAbuGMDP analogues and controls were tested. Standard pyrogenicity tests (iv application or modified for sc application) were performed with rabbits. These tests demonstrated that both free and liposomal formulations of MDP 42 were pyrogenic. The sum + ΔTmax (3 rabbits per group) was shown to be 2.7 and 2.9 °C, respectively. In comparison, the sum + ΔTmax for control liposomes was only 0.2 °C. In comparison, both free and liposomal formulations of norAbuMDP, norAbuGMDP, and their lipophilic analogues (N-L18 norAbuGMDP 20, N-B30 norAbuGMDP 21, norAbuMDP-Lys(L18) 29, norAbuMDP-Lys(B30) 30, norAbuGMDPLys(L18) 37, norAbuGMDP-Lys(B30) 38, B30 norAbuMDP 39, and L18 norAbuMDP 40), exhibited values of +ΔTmax only within the range 0.4−0.6 °C. Accordingly, because pyrogenicity is defined by the situation if the sum + ΔTmax > 1.15 °C 7750



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Figure 2. Expanded structures of analogues 21 and 30 showing explicit linkages and stereocenters.

norAbuMDP-Lys(L18) 29, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, norAbuGMDP-Lys(B30) 38, B30 norAbuMDP 39, and L18 norAbuMDP 40) did not stimulate NOD1 to any significant degree (Figure 4). Stimulation of NLRP 3 Receptor. MDP 42, norAbu-MTP 44, and norAbuMDP or norAbuGMDP analogues (N-L18 norAbuGMDP 20, N-B30 norAbuGMDP 21, norAbuMDP-Lys(L18) 29, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, norAbuGMDP-Lys(B30) 38, B30 norAbuMDP 39, L18 norAbuMDP 40) were each found to stimulate NLRP3 receptor using an in vitro detection system based on a THLP1 cell line expressing NLRP3 and HEK-Blue Cells used for detection of IL-1β secreted by THLP1. Stimulatory activities were comparable to ATP, which is a standard positive control for NLRP3 stimulation (Figure 5). All compounds tested stimulated NLRP3 well with the exception of N-B30 norAbuGMDP 21, which was found to be only a weak NLRP3 agonist. Likewise, N-B30 norAbuGMDP 21 was among the weakest agonists of NOD2 as well (Figure 3). Proliferation-Stimulating Activity of Sera from Mice Treated by sc Administration of Various MDP Analogues. MDP analogues formulated into liposomes were shown to induce cellular proliferation in sera of mice after 6 h (sc application). Only N-B30 norAbuGMDP 21 did not exert significant effects within a short period (6 h) post administration. The cellular proliferation effects of L18 norAbuMDP 40 and norAbuGMDPLys(L18) 37 were comparable to MDP 42 (Figure 6). Adjuvant Activity of Liposomal norAbuMDP-Lys(B30). Immunization of Experimental Mice with rOspA Proteoliposomes Loaded with norAbuMDP-Lys(B30) 30. Following previously reported experiments, recombinant protein rOspA from Borrelia burgdorferi was evaluated as an antigen to induce Th1- versus Th2-type OspA-specific antibodies in experimental mice post immunization with vaccination nanoparticles comprised of norAbuMDP-Lys(B30) 30 formulated as an adjuvant in

Table 1. Codes and Abbreviations of Lipophilic Analogues of norAbuMDP or norAbuGMDP Evaluated Biologically ref no.

abbreviations

20 21 29 30 37 38 39 40

N-L18 norAbuGMDP N-B30 norAbuGMDP norAbuMDP-Lys(L18) norAbuMDP-Lys(B30) norAbuGMDP-Lys(L18) norAbuGMDP-Lys(B30) B30 norAbuMDP L18 norAbuMDP

N-B30 norAbuGMDP 21 and norAbuMDP-Lys(L18) 29 were weaker receptor agonists in the model used for testing. By contrast, adamantyl dipeptide (AdDP) 45, belonging to the family of desmuramyl dipeptides, did not stimulate NOD2 in RAW cells as expected. In conclusion, this experiment demonstrated that structural changes leading to norAbuMDP or norAbuGMDP muramylglycopeptide analogues did not affect their activation potential for NOD2 receptor activation. On the other hand, the character of lipophilic moieties and their position in norAbuMDP or norAuGMDP muramylglycopeptide analogues did appear to impact at least in part on receptor stimulation, as demonstrated with N-B30 norAbuGMDP 21 and norAbuMDPLys(L18) 29 (Figure 3) Stimulation of NOD1 Receptor. MDP 42 and analogues are not typical NOD1 ligands, therefore norAbuMDP and norAbuGMDP muramylglycopeptide analogues were tested only to confirm their NOD2 selectivty. Diaminopimelic acid derivatives (C12-iE-DAP 46, Tri-DAP 47, M-Tri-DAP 48) did activate NOD1 as expected, given that they are NOD1-specific ligands. Stimulation of NOD1 by C12-iE-DAP 46 was less effective than by Tri-DAP 47 and M-Tri-DAP 48. As anticipated, MDP 42, norAbu-MTP 44, and norAbuMDP or norAbuGMDP analogues (N-L18 norAbuGMDP 20, N-B30 norAbuGMDP 21, 7751



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Figure 3. Stimulation of NOD2 receptors by various MDP analogues. (A) Stimulation of NOD2 receptor. Testing system RAW-Blue Reporter Cells NOD2 (Invivogen) was used for assaying stimulatory activity of lipophic analogues of norAbuMDP and norAbuGMDP (N-L18 norAbuGMDP 20, N-B30 norAbuGMDP 21, norAbuMDP-Lys(L18) 29, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, norAbuGMDP-Lys(B30) 38, B30 norAbuMDP 39, L18 norAbuMDP 40). L18 MDP 41, MDP 42, N-glycolyl MDP 43, and norAbu-MTP 44 were used as positive standards. Desmuramyl derivative adamantyl dipeptide (AdDP) 45 was used as negative control. Cells were exposed for 48 h to equimolar concentration of tested compounds (10 μM), and induction of the reporter gene was measured by color enzymatic reaction at 650 nm. ANOVA method was used to compare MDP 42 and RAW-Blue Cells to other compounds. Statistics were calculated using GraphPad Prism version 5.0. Dunett’s Multiple Comparison Test negative control column (RAW-Blue Cells) versus the other columns: * p < 0.05, ** p < 0.01; positive control column (MDP) versus the other columns, ■ p < 0.05, ■■ p < 0.01. (B) Inhibition of NOD2 receptor interaction with MDP analogues by GSK 717. Testing system RAW-Blue Reporter Cells NOD2 (Invivogen) was used for assaying stimulatory activity of lipophic analogues of norAbuMDP/norAbuGMDP (N-L18 norAbuGMDP 20, N-B30 norAbuGMDP 21, norAbuMDP-Lys(L18) 29, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, norAbuGMDP-Lys(B30) 38, B30 norAbuMDP 39, and L18 norAbuMDP 40). L18 MDP, MDP 42. The cells were treated by the inhibitor (0.8 μM) 2 h prior addition of the tested analogues. The cells were exposed for 24 h to equimolar concentration of tested compounds (10 μM), and induction of reporter gene was measured by color enzymatic reaction at 650 nm. As positive control served MDP and L18 MDP without the inhibitor GSK717. Induction of reporter gene was measured by color enzymatic reaction at 650 nm. ANOVA method was used to compare MDP 42 and RAW-Blue Cells to other compounds. Statistics were calculated using GraphPad Prism version 5.0. Dunett’s Multiple Comparison Test positive control column (MDP and L18MDP without GSK 717) versus the other columns, * p < 0.05, ** p < 0.01; negative control column (MDP with GSK 717) versus the other columns, ■ p < 0.05, ■■ p < 0.01.

classical approach of peptide mass fingerprinting, the dominant protein bands were identified by matching the OspA amino acid residue sequence in the NCBInr database (gi|11496927) with the

rOspA proteoliposomes. Alum-rOspA and MDP 42-rOspA proteoliposomes were used for controls. The identity of the rOspA was initially confirmed by MALDI-TOF MS. Using a 7752



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Figure 4. Stimulation of NOD1 receptors by various MDP analogues. Testing system RAW-Blue reporter cells NOD1 (Invivogen) was used for assaying selectivity of MDP 42, norAbu-MTP 44, and all lipophilic norAbuMDP and norAbuGMDP analogues (N-L18 norAbuGMDP 20, N-B30 norAbuGMDP 21, norAbuMDP-Lys(L18) 29, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, norAbuGMDP-Lys(B30) 38, B30 norAbuMDP 39, and L18 norAbuMDP 40) toward NOD1. MDP 42 and norAbu-MTP 44 were used as positive standards for NOD2 stimulation as both are not recognized by NOD1. Diaminopimelic acid derivatives (C12-iE-DAP 46, Tri-DAP 47, M-Tri-DAP 48) were used as positive controls for stimulation of NOD1 receptor. Cells were exposed for 48 h to equimolar concentration of tested compounds (10 μM) and induction of reporter gene was measured by color enzymatic reaction at 650 nm. ANOVA method was used to compare MDP 42 and RAW-Blue Cells to other compounds. Statistics were calculated using GraphPad Prism version 5.0 Dunett’s Multiple Comparison Test, negative control column (RAW-Blue Cells) versus the other columns, * p < 0.05, ** p < 0.01, *** p < 0.001; positive control column (MDP) versus the other columns, ■ p < 0.05, ■■ p < 0.01, ■■■ p < 0.001.

Figure 5. Stimulation of NLRP3 receptor by various MDP analogues. Testing system based on THP1-Null Cells and HEK-Blue IL-1β Cells (Invivogen) was used for assaying activation of NLRP3 receptor. L18 MDP 41, MDP 42, norAbu-MTP 44, and all lipophilic norAbuMDP and norAbuGMDP analogues (N-L18 norAbuGMDP 20, N-B30 norAbuGMDP 21, norAbuMDP-Lys(L18) 29, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, norAbuGMDP-Lys(B30) 38, B30 norAbuMDP 39, L18 norAbuMDP 40) were tested for NLRP3 activation. ATP was used as positive standard. ANOVA method was used to compare control untreated HEK-BlueLI-1β Cells to cells stimulated with tested compounds. Statistics were calculated using GraphPad Prism version 5.0. Dunett’s Multiple Comparison Test negative control column (HEK-BlueLI-1β Cells) versus the other columns: * p < 0.05, ** p < 0.01, *** p < 0.001.

of rOspA. Immunizations with soluble rOspA without adjuvant elicited very low levels of OspA-specific antibodies in total Ig, IgG1, and IgG2a isotypes, almost indistinguishable from results following a mock immunization with p24-hsp70 antigen (Figure 7). On the other hand, the administration of MDP 42-OspA proteoliposomes elicited the highest levels of OspA-specific antibodies in total Ig and IgG1 isotypes followed

following parameters: sequence coverage 63% (13 peptides) and corresponding probability-based MOWSE score 157. For immunization experiments, endotoxin was removed from rOspA by repeating phase extraction method with Triton X-114 to reduce levels below 2.5 EU per mg of protein.34,35 Subsequently, OspA-specific antibody responses were compared by ELISA assays in mice immunized by various formulations 7753



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Figure 6. Stimulation of GM-progenitor cells by sera of mice exposed to analogues of MDP administered by sc route. Sera were collected from mice (ICR, female) 6 h after sc application of particular preparation. Direct stimulatory effect of sera on proliferation of GM-cells in vitro was tested and expressed in number of colonies of GM-CFU. C means control cells cultivated without serum. Dunett’s Multiple Comparison Test negative control (solution for infusion − column 1) versus the other columns: * p < 0.05, ** p < 0.01, *** p < 0.001.

Table 2. Results of Pyrogenity Tests on Rabbitsa +ΔTmax °C values for free and liposomal preparations of norAbuMDP/norAbuGMDP analogues versus controls free liposomal a

control

MDP 42

norAbuMDP

norAbuGMDP

20

21

29

30

37

38

39

40

0.2

2.9 2.7

0.6 0.5

0.4 0.5

0.5 0.4

0.7 0.5

0.4 0.5

0.7 0.6

0.5 0.4

0.4 0.5

0.6 0.5

0.6 0.6

A preparation was evaluated as nonpyrogenic, if the Sum + ΔTmax < 1.1 °C (3 rabbits per group)

the formation of the caspase 1 inflammasome through their N-terminal pyrin domain.37,38 Several studies have indicated that recognition of MDP 42 by NOD2 induces multiple effector responses that enhance intercellular communications through cytokine, chemokine, and defensin production and secretion and so promoting antimicrobial function by ROS production. The interplay between Toll-like receptors (TLR) and NLRs is crucial for adaptive immunity and for the actions of those immunostimulants derived from PAMPs and DAMPs that are in preclinical and clinical studies as future adjuvants for recombinant vaccines. NOD1, NOD2, and NLRP3 inflammasome ligands37,39−44 are required for the action of PGN-derived adjuvants like desmuramylpeptides-containing diaminopimelate (lFK156, FK565) and muramylpeptides such as MDP 42.43 Activation of NOD2 by MDP 42 leads to production of precursors of IL-1β as well as precursors of other cytokines, including IL-18 and IL-33. MDP 42 also activates the NLRP3 inflammasome, a multiprotein complex that recruits and activates caspase-1 to transform interleukin precursors into their active forms. Because IL-1β and IL-18 are key cytokines that act on numerous immune cells, this makes NLRP3 agonists useful immunostimulants and vaccine components able to elicit potential Th1 associated antigen specific immune responses.37 Here, established transgenic cell-based in vitro systems were employed to test the abilities of norAbuMDP and norAbuGMDP analogues to activate NOD2 and NLRP3 (Figures 3A and 5).

by norAbuMDP-Lys(B30) 30-rOspA proteoliposomes and Alum-soluble rOspA (Figure 7). Importantly, rOspA proteoliposomes loaded with norAbuMDP-Lys(B30) 30 elicited the strongest OspA-specific responses in IgG2a, an antibody isotype associated with opsonization and complement activation functions both crucial for protecting the host from Borrelia infection. Only a modest IgG2a OspA-specific antibody response was detected after immunization with MDP 42-rOspA proteoliposomes. Of note, norAbuMDP-Lys(B30) 30-rOspA proteoliposomes administration to mice was not associated with detectable irritations or local reactions at site of administration (data not shown).

■

DISCUSSION NOD 2 and NLRP3 Activation. The relationship between innate immunity and antimicrobial pathogen effects relies on the specific host receptor-based detection of pathogen- and danger signal-derived molecular signatures PAMPs and DAMPs, respectively. PGN and PGN-derived fragments represent structures derived from PAMPs. Some fragments of PGN are recognized by intracellular receptors NOD1 and NOD2.1,36 NOD1 and NOD2 receptors have an amino terminal caspase recruitment domain (CARD) required to trigger nuclear factorκB (NF-κB) signaling. Another NLR family relative, the pyrin domain-containing proteins (NLRPs), are essential for 7754



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acting similarly on NOD2, we employed the NOD2 inhibitor GSK 717 that competes competitively with MDP 42 for binding to the nucleotide oligomerization domain (IC50 submicromolar concentration).45 Gratifyingly, GSK 717 was seen to inhibit directly stimulation of NOD2 by all our lipophilic norAbuMDP and norAbuGMDP analogues to the same extent as MDP 42 and L18 MDP 41 (Figure 3B). Data from the literature suggest that the binding of MDP 42 to NOD2 leads to the formation of an active signaling complex that contains both oligomeric ATP-NOD2 and other adapter or effector molecules, plus the binding of RIPK2 through CARD−CARD interactions. Therefore, we also tested for the effect of the RIPK2 inhibitor gefitinib, which inhibits the NOD2 signaling cascade at the level of RIPK2.46 In this case, gefitinib (10 μM) also suppressed activation of the NOD2 cascade induced by either MDP 42 or our lipophilic analogues (results not shown). Accordingly, all these data interlock to suggest that our lipophilic norAbuMDP and norAbuGMDP analogues do indeed stimulate NOD2 by a molecular mechanism comparable to if not identical with MDP 42. Examination of NLRP3 activation data revealed a similar pattern to NOD2 activation (Figure 5), although N-B30 norAbuGMDP 21 was found to be noticeably weak in NLRP3 activation while L18 norAbuMDP 40 was found to provoke levels of NLRP3 activation significantly in excess of all positive controls. Given this, we would suggest that some lipophilic norAbuMDP and norAbuGMDP analogues may induce weaker NOD2 and/or NLRP3 activations because of their reduced bioavailability to target receptors. In support of this, some lipophilic MDP analogues have been reported that possess a more metabolically stable ether-bond41 or else fluorinated hydrocarbon chains.47 In these cases, analogues were found inactive, consistent with the possibility that fatty acyl lipophilic MDP analogues may need to be hydrolyzed inside cells in order to produce a hydrophilic metabolite suitable for activation of antigen-presenting cells. On the other hand, a biotinylated MDP derivative was reported recently, with biotin conjugated via an alkyl spacer attached to C-6 that was perfectly functional in binding recombinant NOD2.46 Therefore, there is the alternative possibility that at least some lipophilic norAbuMDP and even norAbuGMDP analogues may be able to bind NOD2 without the prior need for intracellular hydrolytic release of their hydrophobic fatty acyl moieties. This suggestion is supported by some of our recent kinetic data concerning NOD2 binding and stimulation mediated by lipophilic MDP analogues (manuscript in preparation). Following on from the above, our lipophilic norAbuMDP and norAbuGMDP analogues were also studied for activity alongside MDP 41 using an in vivo GM-progenitor cell proliferation assay after formulation into neutral liposomes (Figure 6). In this instance, only administration of norAbuGMDP-Lys(L18) 37 or L18 norAbuMDP 40 was comparable in effectiveness with MDP 42 control. This test as such represents a reliable end-point assay measuring the stimulation of complex signaling processes in innate immunity (e.g., cytokine induction) that lead to stimulation of hemopoiesis in bone morrow, in particular the production of granulocytes and macrophages. Therefore, the effectiveness of lipophilic norAbuMDP and norAbuGMDP analogues is promising even though positive control MDP 42 remained still the most effective stimulator of GM-progenitor cell proliferation. Reviewing all these data together (Figures 3, 5, and 6), L18 norAbuMDP 40 and norAbuGMDP-Lys(L18) 37 are the standout analogues that were uniformly excellent immunostimulants

Figure 7. OspA-specific antibodies in mice immunized with rOspA proteoliposomes loaded with norAbuMDP-Lys(B30) 30 are prominent in stimulation of Th1 isotype IgG2a.

All tested compounds including the parent norAbu-MTP were able to activate RAW cells expressing NOD2 and NLRP3 indicator cells. As expected, NOD1 reporter cells were not activated by target lipophilic analogues of norAbu-MDP or norAbu-GMDP (N-L18 norAbuGMDP 20, N-B30 norAbuGMDP 21, norAbuMDP-Lys(L18) 29, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, norAbuGMDP-Lys(B30) 38, B30 norAbuMDP 39, and L18 norAbuMDP 40), presumably owing to the absence of diaminopimelic acid moieties in their molecule. On closer examination (Figure 3), NOD2 activation mediated by B30 norAbuMDP 39, N-L18 norAbuGMDP 20, L18 norAbuMDP 40, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, or norAbuGMDPLys(B30) 38 was comparable if not better than activation mediated by the positive controls L18 MDP 41, MDP 42, N-glycolyl-MDP 43, or norAbuMTP 44. N-B30 norAbuGMDP 21 and norAbuMDP-Lys(L18) 29 both mediated NOD2 activation to levels intermediate between positive controls and the negative control desmuramyl derivative adamantyl dipeptide (AdDP) 45. The implication from a structure−activity perspective is that our lipophilic norAbuMDP and norAbuGMDP analogues are as essentially bioactive in terms of NOD2 mediated immunostimulation as parent MDP structures. At the molecular level, NOD2 is thought to undergo a conformational change upon binding MDP 42 to enable ATP binding, oligomerization, and recruitment of the serine/ threonine protein kinase RIPK2. To be completely sure that our lipophilic analogues of norAbuMDP and norAbuGMDP were 7755



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Figure 8. (A) Schematic presentation of immune reactions toward lipophilic norAbuMDP or norAbuGMDP analogues prepared in liposome formulations. (B) Schematic description of possible pathways at molecular level leading to activation of NOD2 receptor. Our lipophilic norAbuMDP and norAbuGMDP analogues are either free (micellar) or in liposome formulation. Liposomal formulations or micellar structures enter cells via endocytosis, while individual solution free analogues may penetrate freely through cell membranes. Inside the cell, hydrophobic fatty acyl moieties may be removed by esterase or amidase enzymes to enable interactions of the muramyl glycopeptide (MDP, norAbuMDP, norAbuGMDP) with NOD2 or NLRP3. However, such hydrolysis may not be needed for effective NOD2 and/or NLRP3 interactions. Competitive inhibitor GSK 717 inhibits MDP analogue mediated NOD2 stimulation while RIPK2 inhibitor Gefitinib blocks transmission of the signal via the kinase cascade. This process culminates in the production of signaling molecules (e.g., cytokines, chemokines, antibacterial peptides), thereby triggering polarized immune reactions.

Pyrogenicity. The pyrogenicity of MDP and GMDP structures is generally associated with their affinity for NLRP3 which forms part of the multiprotein inflammasome complex that mediates the activation of caspase-1 and promotes secretion of the proinflammatory cytokines IL-18, IL-33, and IL-1β, which act predominantly as endogenous pyrogens.42,49 In addition, MDP 42 is known to stimulate production of prostaglandins, superoxides, collagenases, nitric oxides, β-defensins, and chemokines such as RANTES and CXCL5.40,50 However, in a study published by the group of Luis Chedid, when minute amounts of MDP 42 were applied directly to the brain by intracisternal injection, elevated fever was observed without the apparent release of endogenous pyrogens into plasma or into cerebrospinal

in all assays. Having said this, in vivo immunostimulation experiments performed with rOspA antigen were carried out using norAbuMDP-Lys(B30) 30 as adjuvant even though this norAbuMDP/norAbuGMDP analogue was only capable of middling activation of NOD2 or NLRP3 and stimulation of GM-progenitor cells. Nevertheless, data with analogue 30 were also impressively positive (Figure 7), indicating that our latest lipophilic norAbuMDP/norAbuGMDP analogues might be uniformly excellent immunostimulants for the stimulation of innate and adaptive immune responses, with the possible exception of N-B30 norAbuGMDP 21 that was only poorly soluble and a weak immunostimulant (Figures 3, 5, and 6).48 7756



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fluid. Moreover, indomethacin-inhibited hyperthermia was also produced following intracerebroventricular administration of MDP 42, suggesting that MDP-mediated pyrogenicity might be linked to the activation of prostaglandin synthesis. Such findings argue in favor of a direct effect of MDP 42 on the thermoregulatory hypothalamic centers as well as indirect induction of fever by stimulating the production of endogenous pyrogens like IL-1β.51 Such a direct effect requires MDP 42 to penetrate through the hematoencephalic barrier to centers of thermoregulation in the rostral hypothalamus. In comparison with MDP 42, our lipophilic norAbuMDP and norAbuGMDP analogues were found to be clearly nonpyrogenic when administered as free (micellar) compounds and in liposome formulations (Table 2). In both instances, analogues will encounter immune cells (e.g., dendritic cells) at the sites of administration or in draining lymph nodes. After being internalized by endocytosis or phagocytosis, analogues are most likely to be experience intracellular release, enabling NOD2 and NLRP3 interactions. Activation of NOD2 and NLRP3 should lead to induction of various cytokines, including proinflammatory cytokines IL-1β and IL-18.52 Indeed, we have noted recently that lipophilic norAbuMDP and norAbuGMDP analogues are able to induce production of endogenous pyrogens IL-1β, IL-18, IL-6, and IFN-α, etc. in common with MDP 42 (manuscript in preparation). However, MDP 42 is clearly substantially more pyrogenic. Why? In our view, the difference might arise from the differential level of access and behavior of MDP 42 with respect to the centers of thermoregulation in the hypothalamus. In other words, although NOD2 and/or NLRP3 stimulation leads to proinflammatory cytokine production, we would suggest that this is fundamentally insufficient to cause systemic pyrogenic reactions. Instead, the activation of receptor(s) in the centers of thermoregulation in the hypothalamus should also be required. Accordingly, we would now like to suggest that MDP 42 is fundamentally pyrogenic because of its access to key receptors on the vascular network supplying centers for thermoregulation in the anterior hypothalamus.53 The differences in pyrogenicities of MDP 42 and our norAbuMDP/norAbuGMDP analogues might therefore be related to structural differences between MDP 42 and our analogues that decrease analogue affinities toward receptors of the thermoregulatory center in the anterior hypothalamus.53 For example, the presence of bulky fatty acyl (stearoyl and α-branched acyl residue) moieties in our norAbuMDP and norAbuGMDP analogues might fundamentally inhibit bioavailability to such hypothalamic receptors and even impair receptor interactions on the grounds of steric hindrance. Such a hypothesis would certainly seem to explain the general lack of pyrogenicity exhibited by all of our lipophilic analogues N-L18 norAbuGMDP 20, N-B30 norAbuGMDP 21, norAbuMDPLys(L18) 29, norAbuMDP-Lys(B30) 30, norAbuGMDP-Lys(L18) 37, norAbuGMDP-Lys(B30) 38, B30 norAbuMDP 39, and L18 norAbuMDP 40 in comparison to MDP 42. In addition, the implication that MDP/GMDP-based compounds might exhibit a significant mechanistic dichotomy between NOD2/NLRP3 stimulation on the one hand and receptor activation in hypothalamic centers of thermoregulation on the other hand could be of great importance with respect to the development of further nonpyrogenic MDP/GMDP-based immunotherapeutics and adjuvants. The physiological effect of our norAbuMDP and norAbuGMDP analogues at various organ, cellular, and molecular levels is summarized (Figure 8). Adjuvant Activities. Several nontoxic MDP derivatives have been identified from more than a thousand known synthetic

compounds derived from MDP structures. These include adamantylamine dipeptide (AdDP) 45, L18 MDP 41, MDPLys(L18), murabutide (an ester derivate), threonyl-MDP, and GMDP.54−59 The adjuvant effects of MDP 42 were shown to be mediated in a NOD2 receptor dependent manner using NOD2deficient mice that were unable to mount a normal humoral immune response after immunization with MDP 42 and antigen.60 Thereafter, the adjuvant effects of MDP were also found to depend on the context of administration.2,61 For instance, when hydrophilic derivatives of MDP 42 were administrated in saline solution, mainly humoral immune responses were generated.60,62,63 However, when administered in conjunction with lipophilic carrier systems such as liposomes, oil-in-water emulsions, or in the form of lipophilic analogues, then a strong cellular immune responses could be developed.64 Here, lipophilic norAbuMDP analogue, norAbuMDP-Lys(B30) 30, was selected for immunization experiments using the antigen rOspA. Importantly, even though rOspA is only poorly immunogenic, analogue 30 was able to provoke a substantial adjuvant effect comparable with MDP 42 and in excess of alum reflected by the specific induced IgG titer (Figure 7). These results are the more remarkable given that analogue 30 was not even the most effective immunostimulant studied here, as observed and noted above (Figures 3, 5, and 6). The Th1 versus Th2 polarization of immune responses are typically demonstrated by differences between IgG1 and IgG2a antibody titers. In this case, norAbuMDP-Lys(B30) 30 formulated in rOspA metallochelation proteoliposomes was found to induce primarily IgG2a antibodies typical of the Th1 immune response. Intriguingly, MDP 42 tended toward IgG1 antibody titer production typical of the Th2 immune response, which may result from the fact that MDP 42 lacks a lipophilic hydrocarbon chain. Contrary to norAbuMDP-Lys(B30) 30, which is firmly associated with liposomes, MDP 42 is continuously released from liposomes destabilized by extracellular proteins in the tissue fluid. Therefore, activity of MDP is a superposition of free MDP released from liposomes and MDP associated with liposomes. This mechanism seems to be reflected by data in Figure 6. In our previously reported studies, the adjuvant effects of norAbuMDP-Lys(B30) 30 were evaluated in vivo in experimental mice after formulation into rOspA metallochelation proteo-liposomes with two different recombinant antigens, namely OspC from Borrelia burgdorferi and heat shock protein hsp90 from Candida albicans.35,48 In both studies, norAbuMDPLys(B30) 30 also predominantly provoked Th1-biased antigenspecific immune responses in comparison to MDP 42, alum, and PET GEL. In accordance with these findings, Th1-biased antibody responses were also detected when L18 norAbuMDP 40 and norAbuMDP-lys(L18) 29 were used as adjuvants in association with hsp90 and OspC antigens.35,48 Th1 Candida albicans hsp90-specific antibodies would appear to act against Candida by opsonization, neutralization of extracellular virulence factors, inhibition of Candida adherence to host tissues, inhibition of the yeast-to-mycelium transition, and by direct fungicidal activity.65 Similarly, Th1 Borrelia burgdorferi OspC− antibodies would appear to act by opsonization and complement activation.66,67 The importance of these observations cannot be underestimated because the majority of currently used adjuvants invoke primarily Th2-biased immune responses that are insufficient as antipathogen responses. Therefore, most of our lipophilic norAbuMDP and norAbuGMDP analogues of the type reported here and studied mechanistically would appear to be potent adjuvants that can be used and administered free 7757



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size 5 μm (Merck, Darmstadt, Germany). Molecular sieves 4A (Fluka) were activated at 1.32 Pa and 240 °C for 24 h. Solvents were evaporated using rotary vacuum evaporator. Analytical samples were dried at 6.5 Pa and 25 °C for 8 h. Carrier used 2-chlorotrityl resin 100−200 mesh (BACHEM, substitution 1.87 mmol/g). The purity of all lipophilic norAbuMDP and norAbuGMDP was found to be ≥95%, as established by Waters UPLC Acquty/MS Micromass ZQ, equipped with column BEH Shield RP18 1.7 μm 50 mm × 2.1 mm and using acetonitrile− water as solvent system. General Procedure for Synthesis of Analogues 20, 21, 29, 30, 37, and 38: Hydrogenolytic O-Debenzylation of Precursor Intermediates 18, 19, 27, 28, 35, and 36. Benzyl protected 18 (365 mg, 0.25 mmol), 19 (407 mg, 0.25 mmol), 27 (89 mg, mmol), 28, (332 mg, 0.25 mmol), 35 (385 mg, 0.25 mmol), or 36 (407 mg, 0.25 mmol) were hydrogenolyzed in glacial AcOH (30 mL) over 10% Pd/C (300 mg) at room temperature for 15 h. In each case, post reaction, the vessel was flushed with argon and the catalyst removed by filtration and washed with AcOH (3 × 30 mL). Thereafter, the filtrate was lyophilized and the solid residue purified by HPLC on a LiChrosorb RP-18 column using a linear gradient of methanol in water (80% → 100%). Lyophilization of homogeneous fractions from acetic acid afforded an α/β-anomeric mixture of analogues 20, 21, 29, 30, 37, or 38. The synthetic details and analytical data/characterization of intermediates 2−8, 11−19, 22−28, and 31−36 and target analogues 20, 21, 29, 30, 37, and 38 are given in the Supporting Information. Formulation of Lipophilic Fatty Acyl Analogues As Micelles and in Liposomes. Preparation of Micelles. Formulation of tested compounds were done as described previously.48 In brief, stearoyl- and B-30-modified analogues except N-B30 norAbuGMDP 21 were dissolved in ethanol (96%, medical grade) and diluted by water. N-B30 norAbuGMDP 21 was only partially soluble in ethanol; therefore, it was dissolved in a minimal amount of DMSO and consequently diluted by water. Stock solution: 1−2 mg of a particular compound was solubilized in 50 μL of ethanol and then quickly diluted by 1−2 mL of water pro injectione (or PBS, isotonic NaCl solution, etc.). Final solution needed clarification owing to the formation of small micelles. The final concentration of ethanol was 1.15 °C (three animals per group), then the liposomal formulation of the administered analogue or control compound was judged to be pyrogenic. Thereafter, body temperature was monitored for a further 24 h to detect a possible delayed pyrogenic effect. Activation of NOD2 and NOD1 Receptors in Vitro to Assess the Selectivity of Tested Analogues toward NOD2. Both 264.7 RAW-Blue and 293 HEK-Blue hNOD1 reporter cell lines (InvivoGen, San Diego, USA) were used to study the interaction and activation of NOD2 and NOD1 receptors, respectively, as mediated by administration of norAbuMDP or norAbuGMDP analogues and positive or negative controls. Experiments were carried out strictly according to methods recommended by manufacturer. MDP 42 and L18 MDP 41 were used as positive controls for stimulation of NOD2. Diaminopimelic acid derivatives (C12-iE-DAP 46, Tri-DAP 47, M-Tri-DAP 48) and PGN were used as positive controls for stimulation of NOD1 receptor. All controls were obtained from InvivoGen. The concentration of all compounds under evaluation was 2 μM. A multiplate reader Synergy II (BioTek) was used to evaluate cellular responses to administered compounds by detection at A650. RAW-Blue cells are derived from RAW 264.7 macrophages. They stably express a secreted embryonic alkaline phosphatase (SEAP) gene inducible by NF-κB and AP-1 transcription factors. RAW-Blue cell line express all TLRs (with the exception of TLR5) as well as RIG-I, MDA-5, NOD1, and NOD2, expression of TLR3 and NOD1 being very low. The presence of specific agonists of these receptors induces signaling pathways leading to the activation of NF-κB and AP-1. Upon TLR, NOD, or dectin-1 stimulation, RAW-Blue cells activate NF-κB and/or AP-1 leading to the secretion of SEAP. The HEK-Blue hNOD1 cell lines was designed for studying the stimulation of human NOD1 (hNOD1) by monitoring the activation of NF-κB. HEK-Blue hNOD1 cells were obtained by cotransfection of the hNOD1 gene and an optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene into HEK293 cells. Stimulation with a NOD1 ligand activates NF-κB and AP-1 which induce the production of SEAP. SEAP activity can be assessed using the alkaline phosphatase detection medium, QUANTI-Blue. Inhibition of NOD2 Receptor Activation by Tested Analogues. RAW-Blue cells were used as described above. Specificity of MDP and its analogues toward NOD2 was studied by application of the NOD2 inhibitor GSK717 (Merck). The cells were treated by the inhibitor (0.8 μM) 2 h prior addition of the tested analogues. Thereafter, cells were exposed for 24 h to equimolar concentration of tested compounds (10 μM) and induction of reporter gene was measured by color enzymatic reaction at 650 nm. As positive controls, MDP 42 and L18 MDP 41 were administered without the inhibitor GSK717. Activation of NLRP3 (Inflamasome). Experiments were performed according to manufacturer’s instructions. In brief, THP1-Null cells (derived from THP-1 human monocytic cells) produce IL-1β upon stimulation with inflammasome inducers, such as ATP. THP1-Null cells are designed for the study of inflammasome activation as they express high levels of NLRP3, ASC, and pro-caspase 1. To become susceptible to inflammasome inducers, these cells must be induced by stimuli commonly used for induction such as lipopolysaccharide (LPS). Stimulation by LPS induces the production of pro-IL-1β, the immature form of IL-1β. Subsequent stimulation with inflammasome inducers (ATP is a standard) leads to caspase-1 activation and pro-IL-1β cleavage to IL-1β followed by protein secretion. Mature IL-1β can be detected by

cell-based assay using HEK-Blue IL-1β cells. HEK-Blue IL-1β cells respond specifically to IL-1β. Binding of IL-1β to its receptor IL-1R on the surface of HEK-Blue allowing sensitive specific detection of bioactive IL-1β via colorimetric assay of enzyme activity of expressed reporter gene SEAP. Detection of SEAP in the supernatant of HEK-Blue IL-1β cells can be readily assessed using QUANTI-Blue, a SEAP detection medium. QUANTI-Blue turns blue in the presence of SEAP which can be easily quantified using a spectrophotometer. in Vivo Stimulation of Innate Immunity: Induction of GM-CSF Stimulatory Activity in Mice Sera. ICR mice (female, age of 3 months, four per group, obtained from the Laboratory Animal Breeding and Experimental Facility, Faculty of Medicine, Masaryk University, Building Z, Complex University Campus at Bohunice, Kamenice 753/5, Brno) were stimulated by sc administration of preparations for testing (volume of 200 μL, 100 nmol per dose) 6 h prior to blood sampling. The stimulation of granulocyte−monocyte precursors was assayed by the counting of colonies (CFC) grown after cultivation, as described previously.70,71 These experiments here were conducted according to principles enunciated in the Guide for the Care and Use of Laboratory Animals issued by the Czech Society for Laboratory Animal Science. All experiments were approved by the Ethics Committee of Veterinary Research Institute, Brno. Preparation and Purification of rOspA. Borrelia burgdorferi rOspA was expressed from recombinant pET28 plasmid constructed using OspA cDNA (GenBank accession no. X63412.1), with a C-terminal His-tag and as a nonlipidated protein (lacking first 18 amino acids). rOspA was expressed in BL21(DE3) Escherichia coli and purified under native conditions using Ni-NTA agarose as described previously.68 Endotoxin (LPS) was removed by repeating the phase extraction method using Triton X-114 detergent at working concentration 1%.72 The concentration of LPS was monitored by the gel-clot assay using Limulus amoebocyte lysate (Associates of Cape Cod, USA). All steps were repeated until the endotoxin level of rOspA preparation was below 2.5 EU/mg of rOspA. Characterization of rOspA by SDS-PAGE and MALDI-TOF MS. The purity of rOspA was analyzed using 12% T/3% C SDS-PAGE followed by staining with Coomassie Brilliant Blue R-250. Furthermore, protein identity was confirmed by peptide mass fingerprinting of SDS-PAGE-resolved samples on a Microflex LRF20 MALDI-TOF mass spectrometer (Bruker Daltonik, Bremen, Germany) as described previously.73 Proteins were identified by searching experimental peptide mass lists against the NCBInr protein sequence database using the program Mascot (Matrix Science, London, UK). Preparation of rOspA-Based Formulations for Immunization Experiments. Each vaccine dose contained 20 μg of rOspA. Vaccination nanoparticle formulations were prepared by adding 140 μg of rOspA antigen in PBS to 300 μg of preformed metallochelation liposomes (per one immunization group), prepared by the method of hydration of a lipid film followed by extrusion through 0.2 μm polycarbonate filters (Mini-Extruder, Avanti Polar Lipids) as described previously.68,69,74 Metallochelation liposomes were formulated as follows: EPC/POPG/DOGS-NTA-Ni/adjuvant 71:19:5:5 m/m/m/m and comprised norAbuMDP-Lys(B30) 30 or MDP 42 as adjuvants (reference standard Invivogen). All lipids were purchased from Avanti Polar Lipids. USA. A nonliposomal-soluble rOspA-based formulation was prepared by mixing 140 μg rOspA with 87.5 μL of alum (aluminum hydroxide, Bioveta, Ivanovice na Hané, Czech Republic) or with 87.5 μL of sterile nonpyrogenic PBS. The composition of each particular formulation combination is specified (Table 3). As a control for subsequent OspA-specific serum antibody response, sera were taken from a group of mice immunized specifically with 20 μg of a recombinant His-tagged fusion protein p24-hsp70, prepared as described previously.75 Induction of OspA-Specific Immune Response in Experimental Mice. All immunization experiments were performed on 6−8-week old female BALB/c mice (purchased from Biotest, Konarovice, Czech Republic). All animals were free of known pathogens at the time of the experiment. A standard pellet diet with water was given ad libitum. The research was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals issued by the Czech Society for Laboratory Animal Science. 7759



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Table 3. Composition of Experimental Vaccines composition per one dose (50 μL) formulation

rOspA (20 μg)

DOGS-NTA-Ni lip

AlOH

norAbuMDP-Lys(B30)

MDP

rOspA alum + rOspA DOGS-Ni liposomes + rOspA + norAbuMDP-Lys(B30) 30 DOGS-Ni liposomes + rOspA + MDP 42 control

+ + + + p24-hsp70 (20 μg)

− − + + −

− + − − −

− − + − −

− − − + −

preparation of experimental liposomal vaccine. Pavlı ́na Turánek Knötigová, Josef Mašek: pyrogenicity test. Michal Křupka, Lucia Barkocziova, Petr Kosztyu, Josef Mašek, Marek Šebela: preparation and characterization of rOspA antigen. Pavlı ́na Turánek Knötigová, Hana Č elechovská: in vitro assays of NOD1, NOD2, and NLRP3 stimulation. Pavlı ́na Turánek Knötigová, Michal Křupka: in vivo experiments (immunization experiments, stimulation activity of sera). R.E. and P.T.K. contributed equally.

Immunization experiments were approved by the Ethics Committee of the Faculty of Medicine and Dentistry, Palacky University in Olomouc, Czech Republic, and Czech Ministry of Education, Youth, and Sport. Mice (five per group) were immunized by id administration using rOspA alone, rOspA with alum, and rOspA-proteoliposomes loaded with norAbuMDP-Lys(B30) 30 or MDP 42. Prime-boost doses are specified (Table 3). In each case, the prime dose administration was followed after 14 days later by boost administration. OspA-Specific Serum Antibody Response Determination by ELISA. All assays were performed in duplicates. For all ELISA, rOspA was removed of all tags using recombinant enterokinase as described recently.75 ELISA assays were performed as specified in details elsewhere.75 In brief, ELISA wells were coated with 100 ng/well of nontagged rOspA. Sera pooled from mice in individual groups obtained 14 days after the boost immunization were applied in duplicates to ELISA plates at serial dilution (1:1000 to 1:32000). Plates were then incubated, washed, and analyzed for levels of OspA specific IgG + IgM + IgA (total Ig), antimouse IgG1, or antimouse IgG2a. The results were expressed as the mean OD490 absorbance ± SD determined at 1:8000 dilution which corresponds to the linear portion of titration curves obtained with adjuvant + OspA-immunized mice. OD absorbance measurements were made using Genesis Lite Software (version 3.03, Life Sciences, Basingstoke, UK). Statistical Analyses. The program Prism version 5.0 (GraphPad, USA) was used for statistical analyses and preparation of graphs.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the following grants: the Ministry of Education, Youth and Sports OPVVV PO1 project “FIT” (Pharmacology, Immunotherapy, nanoToxicology) CZ.02.1.01/ 0.0/0.0/15_003/0000495 (J.T.); Project Centre of Excellence for Nanotoxicology CENATOX GAP503/12/G147 (J.T.); Technological Agency of Czech Republic TA02010760−Development of anticancer immunotherapeutics of new generation (M.L., L.D., A.F.); the Ministry of Education, Youth and Sports CZ.1.07/ 2.3.00/20.0164 and grant number LO1304 (J.T. and M.R., P.K., M.K.); the Ministry of Health CZ AZV-Č R 15-32198A (M.R. and J.T.); the project MZE0002716202 RO0517 of the Czech Ministry of Agriculture (J.T.); financial support from specific university research (MSMT No 20-SVV/2017) (M.L.).

ASSOCIATED CONTENT

* Supporting Information S

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00593. Synthesis details and analytical data/characterization of intermediate compounds 2−8, 11−19, 22−28, and 31−36; analytical data for target analogues 20, 21, 29, 30, 37, and 38 (PDF) Molecular formula strings (CSV)

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DEDICATION We dedicate this paper to MUDr. Antonı ́n Vacek, CSc, who pioneered the study of γ-ray radiation effects on the immune system and haematopoiesis. His scientific work contributed to human space flight, and he was awarded by NASA and the Russian Space Agency within the programme Intercosmos. We also dedicate this paper to the memory of Prof. Antonı ́n Holý, who died in 2012 and who pioneered the field of antiviral drugs. The development of new norAbuMDP/GMDP analogues would not have been possible without his long-lasting support of the collaboration between his lab at the Institute of Chemistry and Biochemistry, Prague and the Veterinary Research Institute, Brno.

AUTHOR INFORMATION

Corresponding Authors

*For J.T.: phone, +420 533 331 311; E-mail, [email protected]. *For M.L.: phone, +420 220444283; E-mail, miroslav.ledvina@ vscht.cz. *For M.R.: E-mail, [email protected].

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ABBREVIATIONS USED B30, 2-tetradecylhexadecanoyl, [CH3(CH2)13]2CH(CO)−; Bn, benzyl; CARD, caspase recruitment domain; CSF, colonystimulating factor; DAMPs, danger-associated molecular patterns; mDAP, meso-diaminopimelic acid; Dde, 1-(4,4-dimethyl2,6-dioxocyclohexylidene)-ethyl; D-iGln, D -isoglutamine; DIPEA, N,N-diisopropylethylamine; DLS, dynamic light scattering; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DOGS-NTA-Ni, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1carboxypentyl)iminodiacetic acid)succinyl] (nickel salt); DTP-GDP, disaccharide tripeptide glyceroldipalmitate, N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate; EPC, L-α-phosphatidylcholine; FCA, Freund complete

ORCID

Jaroslav Turánek: 0000-0001-8001-4047 Author Contributions

The team responsible for design and synthetic work is led by Dr. M. Ledvina, Dr. L. Drož, Dr. J. Turánek, and Prof. A. D. Miller. The teams responsible for formulation and testing of biological activities in vitro/in vivo is led by Asst. Prof. J. Turánek and Prof. M. Raška.Roman Effenberg, Daniel Zyka, Anna Kovalová, David Šaman, Michal Hučko, and Kanásová Mária: synthesis and purification of precursors and final products. Josef Mašek, Eliška Bartheldyová, Štěpán Koudelka, Róbert Lukác,̌ František Hubatka: formulation and characterization of drugs, 7760



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Horiuchi, A.; Furuse, K.; Ito, M.; Nagai, K.; Ogura, T.; Kozuru, M.; Hara, N.; Hara, K.; Ichimaru, M.; Takatsuki, K. Restorative activity of muroctasin on leukopenia associated with anticancer treatment. Arzneim.-Forsch. 1988, 38−2 (7A), 1070−1074. (13) Ichihara, N.; Kanazawa, R.; Sasaki, S.; Ono, K.; Otani, T.; Yamaguchi, F.; Une, T. Phase I study and clinical pharmacological study of mutoctasin. Arzneim.-Forsch. 1988, 38 (7A), 1043−1069. (14) Aston, R. Muramyl Compounds for Treatment of Septic Shock. WO9310148A1, 1993. (15) Aston, R.; Maitchouk, I.; Andronova, T. M. Antiviral Hexopyranose Peptide Derivatives Using Muramyl Peptide Compound, e.g. GMDP or Murabutide, Especially for Treating Herpetic Stromal Keratitis. WO9609063A1, 1996. (16) Baschang, G. D.; Hartmann, A. D.; Stanek, J. D.; Sele, A. Glucosaminderivate und ein Verfahren zu Deren Herstellung. DE2655500A1, 1977. (17) Jones, G. H.; Moffatt, J. G.; Nestor, J. J. J. Neue Immunologische Adjuvansverbindungen und Verfahren zur Herstellung Derselben. DE2718010A1, 1977. (18) Farkaš, J.; Ledvina, M.; Brokeš, J.; Ježek, J.; Zajíček, J.; Zaoral, M. The Synthesis of O-(2-acetamido-2-deoxy-b-D-glucopyranosyl)(1®4)-N-acetylnormuramoyl-L-a-aminobutanoyl-D-isoglutamine. Carbohydr. Res. 1987, 163 (1), 63−72. (19) Ledvina, M.; Ježek, J.; Šaman, D.; Hříbalová, V. Synthesis and immunomodulating activity of lipophilic analogs of N-acetylnormuramoyl-L-2-aminobutanoyl-D-isoglutamine. Collect. Czech. Chem. Commun. 1998, 63 (4), 590−598. (20) Ledvina, M.; Šaman, D.; Ježek, J. Synthsis of O-(2-deoxy-2stearoylamino-b-D-glucopyranosyl)-(1®4)-N-acetylnormuramoyl-L-aaminobutanoyl-D-isoglutamine, a lipopholic disaccharide analog of MDP. Collect. Czech. Chem. Commun. 1992, 57 (3), 579−589. (21) Ledvina, M.; Zyka, D.; Ježek, J.; Trnka, T.; Šaman, D. New effective synthesis of (N-acetyl- and N-stearoyl-2-amino-2- deoxy-b-Dglucopyranosyl)-(1®4)-muramoyl-L-2- aminobutanoyl-D-isoglutamine, analogs of GMDP with immunopotentiating activity. Collect. Czech. Chem. Commun. 1998, 63 (4), 577−589. (22) Ledvina, M.; Ježek, J.; Šaman, D.; Vaisar, T.; Hříbalová, V. Synthesis of O-[2-acetamido-2-deoxy-6-stearoyl- and −6-O-(2-tetradecylhexadecanoyl)-b-D-glucopyranosyl]-(1®4)-N-acetylnormuramoylL-a-aminobutanoyl-D-isoglutamine, lipophilic disaccharide analogs of MDP. Carbohydr. Res. 1994, 251, 269−284. (23) Hipler, K.; Miller, A. D.; Turanek, J.; Ledvina, M. Normuramyl Glycopeptide Compounds. US 08653049, 2014. (24) Hipler, K.; Miller, A. D.; Turanek, J.; Ledvina, M. Compound Derived from Muramyldipeptide. EP 13150255.1, 2015. (25) Durette, P. L.; Shen, T. Y. Immunologically Active Peptidyl Disaccharides and Methods of Preparation. US4391800, 1983. (26) Carlsen, P. H. J.; Katsuki, T.; Martin, V. S.; Sharpless, K. B. A Greatly improved procedure for ruthenium tetraoxide catalyzed oxidations of organic-compounds. J. Org. Chem. 1981, 46 (19), 3936− 3938. (27) Tamura, J.; Koike, S.; Shimadate, T. A new oxidative conversion of carbohydrate benzyl ethers to benzoyl esters with Rucl3-Naio4. J. Carbohydr. Chem. 1992, 11 (4), 531−535. (28) Zyka, D. Synthesis of Oligosaccharides Derived from the Saccharide Part of Fragments of Peptidoglycan of the Bacterial Cell Wall and Preparation of Modified Muramyl Glycopeptides. Ph.D. Thesis. Charles University, Faculty of Science: Prague, Czech Republic, 2000. (29) Imoto, M.; Yoshimura, H.; Shimamoto, T.; Sakaguchi, N.; Kusumoto, S.; Shiba, T. Total synthesis of Escherichia-coli lipid-A, the endotoxically active principle of cell-surface lipopolysaccharide. Bull. Chem. Soc. Jpn. 1987, 60 (6), 2205−2214. (30) Windholz, T. B.; Johnston, D. B. Trichloroethoxycarbonyl - A generally applicable protecting group. Tetrahedron Lett. 1967, 8 (27), 2555−2557. (31) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. 2-Chlorotrityl chloride resin - studies on anchoring of Fmoc-amino acids and peptide cleavage. Int. J. Pept. Protein Res. 1991, 37 (6), 513−520.

adjuvant; FIA, Freund incomplete adjuvant; GlcN, glucosamine, 2-amino-2-deoxy-glucose; GM-CSF, granulocyte macrophage colony-stimulating factor; GMDP, glucosaminylmuramyl dipeptide, N-acetyl-D-glucosaminyl-β-(1→4)-N-acetylmuramyl-Lalanyl- D -isoglutamine; HBTU, 2-(1H-benzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate; HEK, human embryonic kidney cell line; HOBt, 1-hydroxybenzotriazole; hsp70, heat shock protein 70; L-Abu, (L-β-Abu-OH) L-2aminobutanoic acid; LPS, lipopolysaccharide; MDP, muramyl dipeptide, N-acetyl-muramyl-L-alanyl-D-isoglutamine; MPLA, monophosphoryl lipid A; MTP, muramyl tripeptide; NLR, (NOD)-like receptors; NOD, nucleotide-binding and oligomerization domain; Palm, palmitoyl, CH3(CH2)14(CO)−; PAMPs, pathogen-associated molecular patterns; PGN, peptidoglycan; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1′-rac-glycerol); rOspA, recombinant outer surface protein A; rOspC, recombinant outer surface protein C; SEAP, embryonic alkaline phosphatase; Ste, stearoyl, CH3(CH2)16(CO)−; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; THP-1, human monocytic cell line; TLR, Toll-like receptor; Troc, 2,2,2trichloroethoxycarbonyl; normuramic acid, trivial name for 2-amino-3-O-carboxymethyl-2-deoxy-D-glucopyranose

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REFERENCES

(1) Miyaji, E. N.; Carvalho, E.; Oliveira, M. L. S.; Raw, I.; Ho, P. L. Trends in adjuvant development for vaccines: DAMPs and PAMPs as potential new adjuvants. Braz. J. Med. Biol. Res. 2011, 44 (6), 500−513. (2) Ellouz, F.; Adam, A.; Ciorbaru, R.; Lederer, E. Minimal structural requirements for adjuvant activity of bacterial peptidoglycan derivatives. Biochem. Biophys. Res. Commun. 1974, 59 (4), 1317−1325. (3) Dozmorov, I. M.; Kuzin, I. I.; Lutsan, N. I.; Lutsenko, G. V.; Prokhorova, A. L.; Sapozhnikov, A. M.; Andronova, T. M.; Ivanov, V. T. Study of immunomodulatory properties of N-acetylmuramyl-L-alanylD-isoglutamine and N-acetylglucosaminyl-(beta1→4)-N-acetylmuramyl-L-alanyl-D-isoglutamine. Biomed. Sci. 1991, 2 (6), 651−658. (4) Ivanov, V. T.; Andronova, T. M.; Bezrukov, M. V.; Rar, V. A.; Makarov, E. A.; Kozmin, S. A.; Astapova, M. V.; Barkova, T. I.; Nesmeyanov, V. A. Structure, design, and synthesis of immunoactive peptides. Pure Appl. Chem. 1987, 59 (3), 317−324. (5) Meshcheryakova, E.; Makarov, E.; Philpott, D.; Andronova, T.; Ivanov, V. Evidence for correlation between the intensities of adjuvant effects and NOD2 activation by monomeric, dimeric and lipophylic derivatives of N-acetylglucosaminyl-N-acetylmuramyl peptides. Vaccine 2007, 25 (23), 4515−4520. (6) Ogawa, C.; Liu, Y.; Kobayashi, K. S. Muramyl dipeptide and its derivatives: peptide adjuvant in immunological disorders and cancer therapy. Curr. Bioact. Compd. 2011, 7 (3), 180−197. (7) Anderson, P. M.; Tomaras, M.; McConnell, K. Mifamurtide in osteosarcoma-a practical review. Drugs Today 2010, 46 (5), 327−337. (8) Graham, B. S.; Keefer, M. C.; McElrath, M. J.; Gorse, G. J.; Schwartz, D. H.; Weinhold, K.; Matthews, T. J.; Esterlitz, J. R.; Sinangil, F.; Fast, P. E.; Wright, P. F.; Dolin, R.; Corey, L.; Belshe, R. B.; Clements, M. L.; Bolognesi, D. P.; Stablein, D. M.; Chernoff, D.; Duliege, A. M.; Walker, C. M. Safety and immunogenicity of a candidate HIV-1 vaccine in healthy adults: Recombinant glycoprotein (rgp) 120 - A randomized, double-blind trial. Ann. Intern. Med. 1996, 125 (4), 270−279. (9) Keitel, W.; Couch, R.; Bond, N.; Adair, S.; Vannest, G.; Dekker, C. Pilot evaluation of influenza-virus vaccine (Ivv) combined with adjuvant. Vaccine 1993, 11 (9), 909−913. (10) Azuma, I. Inducer of cytokines invivo - overview of field and romurtide experience - review. Int. J. Immunopharmacol. 1992, 14 (3), 487−496. (11) Azuma, I.; Seya, T. Development of immunoadjuvants for immunotherapy of cancer. Int. Immunopharmacol. 2001, 1 (7), 1249− 1259. (12) Tsubura, E.; Nomura, T.; Niitani, H.; Osamura, S.; Okawa, T.; Tanaka, M.; Ota, K.; Nishikawa, H.; Masaoka, T.; Fukuoka, M.; 7761



Article

(32) Liu, G.; Zhang, S. D.; Xia, S. Q.; Ding, Z. K. Solid-phase synthesis of muramyl dipeptide (MDP) derivatives using a multipin method. Bioorg. Med. Chem. Lett. 2000, 10 (12), 1361−1363. (33) Rinnova, M.; Lebl, M.; Soucek, M. Solid-phase peptide synthesis by fragment condensation: Coupling in swelling volume. Lett. Pept. Sci. 1999, 6 (1), 15−22. (34) Zachova, K.; Krupka, M.; Chamrad, I.; Belakova, J.; Horynova, M.; Weigl, E.; Sebela, M.; Raska, M. Novel modification of growth medium enables efficient E. coli expression and simple purification of an endotoxin-free recombinant murine Hsp70 protein. J. Microbiol. Biotechnol. 2009, 19 (7), 727−733. (35) Krupka, M.; Masek, J.; Barkocziova, L.; Turanek Knotigova, P.; Kulich, P.; Plockova, J.; Lukac, R.; Bartheldyova, E.; Koudelka, S.; Chaloupkova, R.; Sebela, M.; Zyka, D.; Droz, L.; Effenberg, R.; Ledvina, M.; Miller, A. D.; Turanek, J.; Raska, M. The position of His-tag in recombinant OspC and application of various adjuvants affects the intensity and quality of specific antibody response after immunization of experimental mice. PLoS One 2016, 11 (2), e0148497. (36) Fritz, J. H.; Ferrero, R. L.; Philpott, D. J.; Girardin, S. E. Nod-like proteins in immunity, inflammation and disease. Nat. Immunol. 2006, 7 (12), 1250−1257. (37) Martinon, F.; Burns, K.; Tschopp, J. The inflammasome: A molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell 2002, 10 (2), 417−426. (38) Werts, C.; Girardin, S. E.; Philpott, D. J. TIR, CARD and PYRIN: three domains for an antimicrobial triad. Cell Death Differ. 2006, 13 (5), 798−815. (39) Fritz, J. H.; Ferrero, R. L.; Philpott, D. J.; Girardin, S. E. Nod-like proteins in immunity, inflammation and disease. Nat. Immunol. 2006, 7 (12), 1250−1257. (40) Geddes, K.; Magalhaes, J. G.; Girardin, S. E. Unleashing the therapeutic potential of NOD-like receptors. Nat. Rev. Drug Discovery 2009, 8 (6), 465−479. (41) Kubasch, N.; Schmidt, R. R. Synthesis of muramyl peptides containing meso-diaminopimelic acid. Eur. J. Org. Chem. 2002, 2002 (16), 2710−2726. (42) Rathinam, V. A. K.; Vanaja, S. K.; Fitzgerald, K. A. Regulation of inflammasome signaling. Nat. Immunol. 2012, 13 (4), 333−342. (43) Ting, J. P. Y.; Duncan, J. A.; Lei, Y. How the noninflammasome NLRs function in the innate immune system. Science 2010, 327 (5963), 286−290. (44) Werts, C.; Girardin, S. E.; Philpott, D. J. TIR, CARD and PYRIN: three domains for an antimicrobial triad. Cell Death Differ. 2006, 13 (5), 798−815. (45) Mo, J. Y.; Boyle, J. P.; Howard, C. B.; Monie, T. P.; Davis, B. K.; Duncan, J. A. Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP. J. Biol. Chem. 2012, 287 (27), 23057− 23067. (46) Canning, P.; Ruan, Q.; Schwerd, T.; Hrdinka, M.; Maki, J. L.; Saleh, D.; Suebsuwong, C.; Ray, S.; Brennan, P. E.; Cuny, G. D.; Uhlig, H. H.; Gyrd-Hansen, M.; Degterev, A.; Bullock, A. N. Inflammatory signaling by NOD-RIPK2 is inhibited by clinically relevant type II kinase inhibitors. Chem. Biol. (Oxford, U. K.) 2015, 22 (9), 1174−1184. (47) Zhang, M. Z.; Xu, J. C. Synthesis of F-alkylated MDP analogs. Chin. Chem. Lett. 1996, 7 (11), 993−994. (48) Knotigova, P. T.; Zyka, D.; Masek, J.; Kovalova, A.; Krupka, M.; Bartheldyova, E.; Kulich, P.; Koudelka, S.; Lukac, R.; Kauerova, Z.; Vacek, A.; Horynova, M. S.; Kozubik, A.; Miller, A. D.; Fekete, L.; Kratochvilova, I.; Jezek, J.; Ledvina, M.; Raska, M.; Turanek, J. Molecular adjuvants based on nonpyrogenic lipophilic derivatives of norAbuMDP/ GMDP formulated in nanoliposomes: stimulation of innate and adaptive immunity. Pharm. Res. 2015, 32 (4), 1186−1199. (49) Li, S. X.; Goorha, S.; Ballou, L. R.; Blatteis, C. M. Intracerebroventricular interleukin-6, macrophage inflammatory protein-1 beta and IL-18: pyrogenic and PGE(2)-mediated? Brain Res. 2003, 992 (1), 76−84. (50) Leclerc, C. V. Synthetic immunomodulators and synthetic vaccines. Crit. Rev. Ther. Drug Carrier Syst. 1986, 8 (6), 353−406.

(51) Riveau, G.; Masek, K.; Parant, M.; Chedid, L. Central pyrogenic activity of muramyl dipeptide. J. Exp. Med. 1980, 152 (4), 869−877. (52) Dinarello, C. A. Cytokines as endogenous pyrogens. J. Infect. Dis. 1999, 179, S294−S304. (53) Dinarello, C. A. Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed. J. Endotoxin Res. 2004, 10 (4), 201−222. (54) Balitsky, K. P.; Umansky, V. Y.; Tarakhovsky, A. M.; Andronova, T. M.; Ivanov, V. T. Glucosaminylmuramyl dipeptide-induced changes in murine macrophage metabolism. Int. J. Immunopharmacol. 1989, 11 (5), 429−434. (55) Eppstein, D. A.; Byars, N. E.; Allison, A. C. New adjuvants for vaccines containing purified protein antigens. Adv. Drug Delivery Rev. 1989, 4 (2), 233−253. (56) Chedid, L. A.; Parant, M. A.; Audibert, F. M.; Riveau, G. J.; Parant, F. J.; Lederer, E.; Choay, J. P.; Lefrancier, P. L. Biological-activity of a new synthetic muramyl peptide adjuvant devoid of pyrogenicity. Infect. Immun. 1982, 35 (2), 417−424. (57) Ishihara, C.; Yamamoto, K.; Hamada, N.; Azuma, I. Effect of stearoyl-N-acetylmuramyl-l-alanyl-D-isoglutamine on host resistance to Corynebacterium kutscheri infection in cortisone-treated mice. Vaccine 1984, 2 (4), 261−264. (58) Masek, K.; Seifert, J.; Flegel, M.; Krojidlo, M.; Kolinsky, J. The immunomodulatory property of a novel synthetic compound adamantylamide dipeptide. Methods Find. Exp. Clin. Pharmacol. 1984, 6 (11), 667−669. (59) Matsumoto, K.; Otani, T.; Une, T.; Osada, Y.; Ogawa, H.; Azuma, I. Stimulation of nonspecific resistance to infection induced by muramyl dipeptide analogs substituted in the gamma-carboxyl group and evaluation of N-alpha-muramyl dipeptide-N-epsilon-stearoyllysine. Infect. Immun. 1983, 39 (3), 1029−1040. (60) Magalhaes, J. G.; Fritz, J. H.; Le Bourhis, L.; Sellge, G.; Travassos, L. H.; Selvanantham, T.; Girardin, S. E.; Gommerman, J. L.; Philpott, D. J. Nod2-dependent Th2 polarization of antigen-specific immunity. J. Immunol. 2008, 181 (11), 7925−7935. (61) Kotani, S.; Watanabe, Y.; Kinoshita, F.; Shimono, T.; Morisaki, I.; Shiba, T.; Kusumoto, S.; Tarumi, Y.; Ikenaka, K. Immunoadjuvant activities of synthetic N-acetylmuramyl peptides or N-acetylmuramyl amino acids. Biken J. 1975, 18 (2), 105−111. (62) Audibert, F.; Chedid, L.; Lefrancier, P.; Choay, J. Distinctive adjuvanticity of synthetic analogs of mycobacterial water-soluble components. Cell. Immunol. 1976, 21 (2), 243−249. (63) Kobayashi, K. S.; Chamaillard, M.; Ogura, Y.; Henegariu, O.; Inohara, N.; Nunez, G.; Flavell, R. A. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 2005, 307 (5710), 731−734. (64) Parant, M. A.; Audibert, F. M.; Chedid, L. A.; Level, M. R.; Lefrancier, P. L.; Choay, J. P.; Lederer, E. Immunostimulant activities of a lipophilic muramyl dipeptide derivative and of desmuramyl peptidolipid analogs. Infect. Immun. 1980, 27 (3), 826−831. (65) Raška, M.; Běláková; Křupka, M.; Weigl, E. Candidiasis - do we need to fight or to tolerate the Candida fungus. Folia Microbiol. 2007, 52, 297−312. (66) Klaus, G. G. B.; Pepys, M. B.; Kitajima, K.; Askonas, B. A. Activation of mouse complement by different classes of mouse antibody. Immunology 1979, 38 (4), 687−695. (67) Krupka, M.; Zachova, K.; Weigl, E.; Raska, M. Prevention of lyme disease: promising research or sisyphean task? Arch. Immunol. Ther. Exp. 2011, 59 (4), 261−275. (68) Krupka, M.; Masek, J.; Bartheldyova, E.; Knotigova, P. T.; Plockova, J.; Korvasova, Z.; Skrabalova, M.; Koudelka, S.; Kulich, P.; Zachova, K.; Czernekova, L.; Strouhal, O.; Horynova, M.; Sebela, M.; Miller, A. D.; Ledvina, M.; Raska, M.; Turanek, J. Enhancement of immune response towards non-lipidized Borrelia burgdorferi recombinant OspC antigen by binding onto the surface of metallochelating nanoliposomes with entrapped lipophilic derivatives of norAbuMDP. J. Controlled Release 2012, 160 (2), 374−381. (69) Masek, J.; Bartheldyova, E.; Korvasova, Z.; Skrabalova, M.; Koudelka, S.; Kulich, P.; Kratochvilova, I.; Miller, A. D.; Ledvina, M.; 7762



Article

Raska, M.; Turanek, J. Immobilization of histidine-tagged proteins on monodisperse metallochelation liposomes: Preparation and study of their structure. Anal. Biochem. 2011, 408 (1), 95−104. (70) Kasna, A.; Turanek, J.; Vacek, A.; Zaluska, D.; Knotigova, P.; Masek, K. Restoration of femoral GM-CFC progenitors in sublethally irradiated mice of various ages treated with liposomal adamantylamide dipeptide. Int. Immunopharmacol. 2004, 4 (8), 1099−1106. (71) Turánek, J.; Záluská, D.; Hofer, M.; Vacek, A.; Ledvina, M.; Ježek, J. Stimulation of haemopoiesis and protection of mice against radiation injury by synthetic analogues of muramyldipeptide incorporated in liposomes. Int. J. Immunopharmacol. 1997, 19 (9−10), 611−617. (72) Zachova, K.; Krupka, M.; Chamrad, I.; Belakova, J.; Horynova, M.; Weigl, E.; Sebela, M.; Raska, M. Novel modification of growth medium enables efficient E. coli expression and simple purification of an endotoxin-free recombinant murine hsp70 protein. J. Microbiol. Biotechnol. 2009, 19 (7), 727−733. (73) Kowalska, M.; Galuszka, P.; Frebortova, J.; Sebela, M.; Beres, T.; Hluska, T.; Smehilova, M.; Bilyeu, K. D.; Frebort, I. Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: heterologous expression, purification and properties. Phytochemistry 2010, 71 (17−18), 1970−1978. (74) Mašek, K.; Bartheldyova, E.; Turánek-Knotigová, P.; Škrabalová, M.; Korvasová, Z.; Plocková, Z.; Koudelka, S.; Škodová, P.; Kulich, P.; Křupka, M.; Zachová, K.; Czerneková, L.; Horynová, M.; Kratochvilova, I.; Miller, A. D.; Zyka, D.; Michálek, J.; Vrbková, J.; Šebela, M.; Ledvina, M.; Raška, M.; Turánek, J. Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged antigens: Preparation, structural study and immune response towards rHsp90. J. Controlled Release 2011, 151, 193−201. (75) Krupka, M.; Zachova, K.; Cahlikova, R.; Vrbkova, J.; Novak, Z.; Sebela, M.; Weigl, E.; Raska, M. Endotoxin-minimized HIV-1 p24 fused to murine hsp70 activates dendritic cells, facilitates endocytosis and p24specific Th1 response in mice. Immunol. Lett. 2015, 166 (1), 36−44.

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