Nonpyrogenic Molecular Adjuvants Based on norAbu-Muramyldipeptide and norAbu-Glucosaminyl Muramyldipeptide: Synthesis, Molecular Mechanisms of Action, and Biological Activities in Vitro and in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.7b00593

Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDP-Lys(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).

Full text of DOI:10.1021/acs.jmedchem.7b00593

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Article
Nonpyrogenic molecular adjuvants based on norAbu-muramyldipeptide
and norAbu-glucosaminyl muramyldipeptide: synthesis, molecular
mechanisms of action, biological activities in vitro and in vivo
Roman Effenberg, Pavlína Turánek Knötigová, Daniel Zyka, Hana #elechovská, Josef Mašek,
Eliška Bartheldyová, Frantisek Hubatka, Št#pán Koudelka, Róbert Luká#, Anna Kovalová, David
Saman, Michal K#upka, Lucia Barkocziová, Petr Kosztyu, Marek Šebela, Ladislav Drož, Michal
Hu#ko, Mária Kanásová, Andrew David Miller, Milan Raška, Miroslav Ledvina, and Jaroslav Turanek
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00593 • Publication Date (Web): 22 Aug 2017
Downloaded from http://pubs.acs.org on August 22, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Nonpyrogenic molecular adjuvants based on
norAbuꢀmuramyldipeptide and norAbuꢀ
glucosaminyl muramyldipeptide: synthesis,
molecular mechanisms of action, biological
activities in vitro and in vivo
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‡
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Roman Effenberg , Pavlína TuránekꢀKnötigová , Daniel Zyka , Hana Čelechovská , Josef
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Mašek , Eliška Bartheldyová , František Hubatka , Štěpán Koudelka , Róbert Lukáč , Anna
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Kovalová , David Šaman , Michal Křupka , Lucia Barkocziova , Petr Kosztyu , Marek Šebela ,
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Ladislav Drož , Michal Hučko , Mária Kanásová , Andrew D. Miller
, Milan Raška
,
*1
*2
Miroslav Ledvina , and Jaroslav Turánek
1.
Department of Chemistry of Natural Compounds, University of Chemistry and
Technology, Technická 5,166, 28 Prague 6, Czech Republic
2.
Department of Pharmacology and Immunotherapy, Veterinary Research Institute, v.v.i.,
Hudcova 70, 621 00 Brno, Czech Republic
3.
APIGENEX s.r.o., Poděbradská 173/5, Prague 9, 190 00, Czech Republic
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.
Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Flemingovo nám. 2, 160
00 Prague, Czech Republic
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Department of Immunology, Institute of Molecular and Translational Medicine, Faculty
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of Medicine and Dentistry, Palacky University Olomouc, Hněvotínská 3, 775 15 Olomouc,
Czech Republic
6.
Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of
Science, Palacky University Olomouc, Olomouc, Czech Republic
7.
Department of Organic Chemistry, University of Chemistry and Technology, Technická
5, 166 28 Prague 6, Czech Republic
8.
Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova
2030/8, 128 43 Prague 2, Czech Republic
9.
King's College London, Institute of Pharmaceutical Science, London SE1 9NH, United
Kingdom
10.
KP Therapeutics Ltd, Manchester M3 2ER, United Kingdom
KEYWORDS
glycopeptides; muramylglycopeptide analogues; muramyl dipeptide; NOD2; NOD1; NLRP3,
inflammasome; MDP; GMDP; molecular adjuvants; adjuvants; innate immunity; adaptive
immunity; liposomes; recombinant vaccines
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ABSTRACT
Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated NꢀL18 norAbuGMDP, NꢀB30
norAbuGMDP, norAbuMDPꢀLys(L18), norAbuMDPꢀLys(B30), norAbuGMDPꢀLys(L18),
norAbuGMDPꢀLys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized
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comprising the normuramylꢀLꢀαꢀaminobutanoyl (norAbu) structural moiety. All new analogues
show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when
tested in rabbits in vivo (s.c. and i.v. 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 s.c. with individual liposomeꢀloaded analogues causing proliferation
of bone marrowꢀderived GMꢀprogenitors cells. Importantly, vaccination nanoparticles prepared
from metallochelation liposomes, Hisꢀtagged antigen rOspA from Borrelia burgdorferi, and
lipophilic analogue norAbuMDPꢀLys(B30) as adjuvant, are shown to provoke OspAꢀspecific
antibody responses with a strong Th1ꢀbias (dominance of IgG2a response). In contrast, the
adjuvant effects of Alum or parent MDP show a strong Th2ꢀbias (dominance of IgG1 response).
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 development of new
biocompatible antigen carriers of antigens and potent adjuvants. Potent molecular adjuvants may
be derived from pathogenꢀ and dangerꢀderived molecular signatures, collectively referred to as
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pathogenꢀassociated molecular patterns (PAMPs) and dangerꢀassociated 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
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immunostimulants . One very specific and promising group of PAMP/DAMPꢀrelated adjuvants
or immunostimulants are synthetic or semisynthetic muramylglycopeptides that derive from
2
peptidoglycan (PGN) fragments of the bacterial cell wall. In 1974, Adam et al. described how
muramyldipeptide (MDP; NꢀacetylmuramoylꢀLꢀalanylꢀDꢀisoglutamine) 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 Nꢀacetyl glucosaminyl muramyldipeptide (GMDP; Nꢀacetylꢀβꢀ
Dꢀglucosaminylꢀ(1→4)ꢀ
Nꢀacetylmuramoylꢀ ꢀalanylꢀ ꢀisoglutamine), was shown to be both more immunostimulatory
L
D
3
ꢀ4
than MDP and less pyrogenic, but the side effects were still significant . Mechanistic studies
have recently suggested that MDP and GMDP are recognized by family members of the
intracellular nucleotideꢀbinding oligomerization domainꢀlike receptors (NLRs), such as NOD2.
NLRs play key roles in both adaptive and innate immunity by triggering a cascade of regulatory
5ꢀ6
cytokines, chemokines, plus the production of antimicrobial peptides .
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 dipalmitoyl phosphatidylethanolamine
(
Nꢀacetylmuramoylꢀ
L
ꢀalanylꢀ
D
ꢀisoglutaminylꢀ
Lꢀalanineꢀ2ꢀ[1,2ꢀdipalmitoylꢀsnꢀglyceroꢀ3ꢀ(hydroꢀ
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xyphosphoryloxy)]ethylamide) was approved as an immunotherapeutic for combined immunoꢀ
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chemotherapy of osteosarcoma under the clinical designation Mifamurtide . Mifamurtide has
also been assayed as a coꢀadjuvant in combination with MF59ꢀadjuvant in influenza and HIVꢀ1
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vaccines , 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 stearoylꢀsubstituted lysine (Nꢀacetylmuramoylꢀ
alanylꢀ ꢀisoglutaminylꢀεꢀstearoylꢀ ꢀlysine), is currently used in clinic as a proꢀhaemopoietic
agent for treatment of leukopenia induced by chemotherapy and/or radiotherapy of cancers
The most common adverse reaction in cancer patients treated with Romurtide is fever
Lꢀ
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.
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accompanied by chill . Otherwise, GMDP is the active component of a preparation known as
Likopid, registered in Russia and postꢀSoviet Republics as an immunotherapeutic with broad
applicability, e.g. treatment of infectious diseases, and prevention of infections in patients postꢀ
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trauma, postꢀoperative, postꢀchemotherapy and postꢀradiotherapy
Romurtide, the extension of the GMDP peptide chain with stearoylꢀsubstituted lysine, to give Nꢀ
acetylꢀβꢀ ꢀglucosaminylꢀ(1→4)ꢀNꢀacetylmuramoylꢀ ꢀalanylꢀ ꢀisoglutaminylꢀεꢀstearoylꢀ
lysine, led to significantly increased systemic toxicity .
. Contrary to the case with
D
L
D
Lꢀ
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. This research was initiated by the finding that Nꢀacetylnormuramoylꢀ
aminobutanoylꢀ ꢀisoglutamine (norAbuMDP) is less pyrogenic and possesses a higher
immunoadjuvant activity in comparison with parent MDP
changes in GMDP, i.e., replacement of muramic acid to normuramic acid, and of
ꢀαꢀamininobutanoic acid, we thus obtained a nonpyrogenic and highly immunostimulatory
Lꢀαꢀ
D
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6ꢀ17
. By making the same structural
ꢀalanine with
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norAbuGMDP (Nꢀacetylꢀβꢀ
D
ꢀglucosaminylꢀ(1→4)ꢀNꢀacetylnormuramoylꢀLꢀαꢀaminobutanoylꢀDꢀ
1
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isoglutamine) . Following this, we demonstrated that the introduction of bulky lipophilic
residues into norAbuMDP or norAbuGMDP enhanced their immunomodulatory activities, whilst
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19ꢀ22
retaining the favorable pharmacological parameters of the parent structures
. 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
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residue in order to modify their immunopharmacological parameters
. Here we now outline
the synthetic pathways for the preparation of these novel norAbuMDP and norAbuGMDP
muramylglycopeptide analogues and characterisation 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.
RESULTS
Chemistry
In order to prepare target norAbuMDP and norAbuGMDP muramylglycopeptide analogues
comprising the normuramylꢀLꢀαꢀaminobutanoyl (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 solidꢀphase synthesis. All building blocks were designed with
orthogonal protection, thus allowing for subsequent regioselective acylation of downstreamꢀ
protected muramyl glycopeptide analogue intermediates with selected fatty acids (e.g. stearic
acid [L18] or 2ꢀtetradecylhexadecanoic acid [B30]).
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The precursors of protected normuramic or glucosaminyl normuramic acid building blocks
were NꢀacetylꢀDꢀglucosamine (GlcNAc) derivatives with 3ꢀOH 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
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typically involved three main steps, starting from appropriate protected 3ꢀOꢀallylꢀDꢀGlcNAc
intermediates. Generally, the allyl group was first isomerized to become a propenyl group, using
Wilkinson’s catalyst [RhCl(PPh ) ], then subject to acid catalyzed cleavage, leaving the
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opportunity for attachment of a glycoloyl moiety by simple alkylation
could be conflated to one by conversion of the allyl group to a glycoloyl ether moiety by the
action of RuO , that can be generated in situ by catalytic amount of RuCl using NaIO as an
. All three steps
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oxidant under controlled conditions (Scheme 1 and 2) . Bearing in mind that RuO is very
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powerful oxidizing agent reaction conditions were carefully adjusted for selective allyl group
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release in the presence of other protecting groups .
Initially, allyl derivative 1 was subject to benzylation to give benzyl 2ꢀacetamidoꢀ3ꢀOꢀallylꢀ
4
,6ꢀdiꢀOꢀbenzylꢀ2ꢀdeoxyꢀαꢀ
hydride in DMF according to published procedure
with NaIO and RuCl in CCl ꢀMeCNꢀH O at 0 °C (Scheme 1) . In early trial runs with these
Dꢀglucopyranoside 2 by treatment with benzyl bromide and sodium
1
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. Glucopyranoside 2 was then treated
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reactions conditions, a range of reaction products were observed being diol 3, aldehyde 4, cyclic
byꢀproduct 5 and the desired protected normuramic acid 6 intermediate. The formation of
undesired cyclic byꢀproduct 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
NaIO , thereby giving the protected normuramic acid 6 intermediate in high overall yield.
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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 aboveꢀ
mentioned aldehyde 4 (prepared by reaction of diol 3 with Pb(OAc) ) in toluene, which resulted
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in the compound 5 as the only isolated product. The diol 3 and acid 6 were fully characterized
after their conversion to the diacetate 7 and methyl ester 8, respectively.
Scheme 1. Synthesis of protected normuramyl building block by oxidative cleavage of 3ꢀOꢀallyl
protective group
a
Reagents and conditions. (a) BnBr, NaH, DMF, r.t., 24h, 89%. (b) NaIO , RuCl , CCl ꢀMeCNꢀ
4
3
4
H O, 0 °C, Method A or Method B. (c) Ac O, Py, DMAP, DCM, r.t., 24h, 94%. (d) Pb(OAc) ,
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CHCl , r.t., 2h, 63%. (e) toluene, reflux, 9h, 52%. (f) CH N , Et O, CH Cl ꢀMeOH, 0 °C, 86%
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In a corresponding manner, amine 9 and its acetyl derivative 10 were prepared as described
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previously . Nꢀ2,2,2ꢀtrichloroethoxycarbonyl (Troc) derivative 11 was prepared by reaction of
amine 9 with trichloroethoxycarbonyl chloride in pyridine. Thereafter, 10 and 11 were used to
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prepare intermediate glucosaminyl normuramic acid building blocks 12 and 13 (Scheme 2) ,
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then converted to their respective methyl esters 14 and 15, by reaction with diazomethane in
order to allow complete characterization of these compounds.
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9
0
Scheme 2. Synthesis of protected glucosaminyl normuramyl building block by allyl oxidative
cleavage
a
Reagents and conditions. (a) for 10: Ac O, DCM, pyridine, r.t., 73%, for 11: TrocCl, CH Cl ,
2
2
2
pyridine, DMAP, r.t., 74%. (b) NaIO , RuCl , CCl ꢀMeCNꢀH O, 0 °C, 57% for 12 or r.t., 64%
4
3
4
2
for 13. (c) CH N , Et O, CH Cl ꢀMeOH, 0 °C, 93% for 14 and 91% for 15.
2
2
2
2
2
The introduction of Troc protection into intermediate glucosaminyl normuramic acid building
block 13 was intended to enable selective fatty acid acylisation of the glucosaminyl moiety
following selective deprotection, following by global deprotection to yield novel norAbuGMDP
muramylglycopeptide analogues (Scheme 3).
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60
Scheme 3. Synthesis of norAbuGMDP muramylglycopeptide analogues
Reagents and conditions. (a) DCC, HOBt, DCM, DMF, DIPEA, r.t., 12h, 60%. (b) Zn, AcOH,
a
r.t., 6h, 79%. (c) for 18: CH (CH ) COCl or for 19: [CH (CH ) ] CHCOCl; DMAP, DMF,
3
2 16
3
2 13 2
DIPEA, 60 °C, 5h/10h, 69% (18) or 71% (19). (d) H , Pd/C, r.t., 15h, 75% (20) and 69% (21).
2
The method of preparation of two norAbuGMDP muramylglycopeptide analogues was initiated
by coupling of intermediate glucosaminyl normuramic acid building block 13 with a
corresponding Abuꢀdipeptide building block by means of DCC in the presence of HOBt giving a
protected glucosaminyl normuramyl glycopeptide intermediate 16. Following NꢀTroc protecting
2
9ꢀ30
group removal under mild conditions involving Zn in AcOH
, the resulting free amino
functional group of 17 could be acylated with DMAP and DIPEA in DMF using stearoyl (L18)
chloride or 2ꢀtetradecylhexadecanoyl (B30) chloride giving Nꢀacyl derivatives 18 and 19
respectively. Final catalytic hydrogenolysis to remove all benzylꢀprotecting groups yielded the
desired norAbuGMDP muramylglycopeptide analogues 20 and 21. We observed that the Troc
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6
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
0
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0
20
reported prepared by a different synthetic route previously reported .
The method of preparation of two novel norAbuMDP muramylglycopeptide analogues was
initiated from protected normuramyl building block 6 by DCC/HOBt mediated coupling of a
protected NꢀAbuꢀtripeptide building block anchored on a solid phase chlorotrityl (Barlos) resin
that could be cleaved at an appropriate time using mild AcOHꢀTFEꢀCH Cl conditions (Scheme
2
2
6, 31
4
)
.
Scheme 4. Synthesis of norAbuMDP muramylglycopeptide analogues using Barlos resin
a
Reagents and conditions. (a) CH Cl , DIPEA, r.t., 1h; MeOH, r.t., 10min; wash CH Cl , DMF,
2
2
2
2
MeOH, Et O, 1.56 mmol/dry gram. (b) 5% piperidine in DMFꢀDCM (1:1) 5min, r.t., 20%
2
piperidine in DMF 20min, r.t.; wash DMF; Fmocꢀ
D
ꢀiGlnꢀOH, HOBt, HBTU, DIPEA, DMF, r.t.,
ꢀαꢀAbuꢀOH, HBTU,
9
0min. (c) 5% piperidine in DMFꢀCH Cl (1:1) 25min; wash DMF; Fmocꢀ
L
2
2
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DIPEA, DMF, r.t., 60min. (d) 20% piperidine in DMF 20min, r.t. (e) HBTU, DIPEA, DMF, r.t.,
2
0h (double coupling). (f) 2% N H .H O, DMF, 10min, r.t. (g) For 25: CH (CH ) COOH or for
2 4 2 3 2 16
2
6: [CH (CH ) ] CHCOOH; HBTU, DMF, DIPEA, r.t., 6h/6h, 70% (25) or 87% (26). (h) For
3 2 13 2
27: AcOHꢀTFEꢀCH Cl (1:1:8, v/v/v; 60 ml) for 1h, r.t., 35%; For 28: AcOHꢀTFEꢀCH Cl
2
2
2
2
(
1:1:8; 60 ml) for 1h, r.t., 33%. (i) H , Pd/C, r.t., 24h/24h, 39% (29) and 35% (30).
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 5. Synthesis of norAbuGMDP muramylglycopeptide analogues using Barlos resin
Reagents and conditions. (a) HBTU, DIPEA, DMF, r.t., 3.5h; +DMAP r.t. 1h. (b) 2%
a
N H .H O, DMF, 10min, r.t. (c) For 33: CH (CH ) COOH or for 34: [CH (CH ) ] CHCOOH;
2
4
2
3
2 16
3
2 13 2
HBTU, DMF, DIPEA, r.t., 6h/6h, 33 or 34. (d) For 35: AcOH ꢀ TFE – CH Cl (1:1:8, v/v/v; 60
2
2
ml) for 1h, r.t., 41%; For 36: AcOH ꢀ TFE ꢀ CH Cl (1:1:8; 60 ml) for 1h, r.t., 32%. (e) H , Pd/C,
2
2
2
r.t., 48h/24h, 78% (37) and 37% (38).
The protected NꢀAbuꢀtripeptide building block attached to resin was prepared according to
2
3ꢀ24, 32
standard Fmoc solid phase synthesis protocols with standard orthogonal protecting groups
,
culminating in the free αꢀamino ꢀAbuꢀ ꢀisoGlnꢀ ꢀLys(Dde)ꢀOꢀ2ꢀchlorotrityl conjugate 22. This
L
D
L
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6
conjugate was then coupled with protected normuramic acid 6 intermediate employing “swelling
3
3
volume” method , resulting in the formation of protected resinꢀbound normuramylglycopeptide
3. Thereafter, Dde deprotection with 2% hydrazine solution in DMF yielded resinꢀbound
2
0
1
2
3
4
5
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7
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0
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0
intermediate 24 that was acylated directly using stearic (L18) acid or 2ꢀtetradecylhexadecanoic
(B30) acid with HBTU and DIPEA in DMF, resulting in Nꢀacyl derivatives 25 and 26
respectively. These were released from resin using AcOHꢀTFEꢀCH Cl (1:1:8, v/v/v) to give
2
2
protected and acylated normuramylglycopeptides 27 and 28 respectively. Finally, the desired
norAbuMDP muramylglycopeptide analogues 29 and 30 were revealed following catalytic
hydrogenolysis of remaining Oꢀbenzyl protecting groups.
Following this, two novel norAbuGMDP muramylglycopeptide analogues were prepared in a
comparable manner. Firstly, protected glucosaminyl normuramyl building block 12 was coupled
by DCC/HOBt mediated coupling to the same protected NꢀAbuꢀtripeptide building block
attached to resin (as above), resulting in the formation of protected resinꢀbound glucosaminyl
normuramylglycopeptide 31 (Scheme 5). Thereafter, Dde deprotection with 2% hydrazine
solution in DMF yielded resinꢀbound intermediate 32 that was then acylated directly using
stearic (L18) acid or 2ꢀtetradecylhexadecanoic (B30) acid with HBTU and DIPEA in DMF,
resulting in Nꢀacyl derivatives 33 and 34 respectively. These were released from resin using
AcOHꢀTFEꢀCH Cl
(1:1:8, v/v/v) to give protected and acylated glucosaminyl
2
2
normuramylglycopeptides 35 and 36 respectively. Finally, the desired norAbuGMDP
muramylglycopeptide analogues 37 and 38 were revealed following catalytic hydrogenolysis of
remaining Oꢀbenzyl protecting groups.
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Lipophilic analogues and derivatives tested in this study
Structural formulae, codes and abbreviations are present in Fig. 1, Fig. 2 and Table 1.
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12
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17
18
19
20
21
22
23
24
25
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41
42
43
44
45
46
47
48
49
50
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59
60
Figure 1. Structural formulae of norAbuMDP or norAbuGMDP analogues and controls tested
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1
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
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6
OH
OH
O
HO
HO
O
O
O
OH
OH
NH
OH
AcNH
O
0
1
2
3
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7
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HO
HO
O
O
O
O
=
O
OH
NH
B30
NH
O
AcNH
O
HN
L-Abu D-iGln(OH)
O
OH
NH2
O
N-B30 norAbuGMDP 21
N-B30 norAbuGMDP 21
OH
OH
O
O
HO
O
OH
HO
AcNH
O
OH
AcNH
O
O
=
NH
O
L-Abu D-iGln L-Lys(B30)-OH
norAbuMDP-Lys(B30) 30
HN
H
N
H
O
N
NH2
O
O
O
OH
norAbuMDP-Lys(B30) 30
Figure 2. Expanded structures of analogues 21 and 30 showing explicit linkages and
stereocenters
Reference Number Abbreviations
20
21
29
NꢀL18 norAbuGMDP
NꢀB30 norAbuGMDP
norAbuMDPꢀLys(L18)
norAbuMDPꢀLys(B30)
norAbuGMDPꢀLys(L18)
norAbuGMDPꢀLys(B30)
B30 norAbuMDP
30
3
3
3
7
8
9
40
L18 norAbuMDP
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Table 1. Codes and abbreviations of lipophilic analogues of norAbuMDP or norAbuGMDP
evaluated biologically
Characterisation of free (micellar) and liposomal formulations of lipophilic analogues of
norAbuMDP and norAbuGMDP
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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 physicalꢀchemical properties they are
surfaceꢀactive 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 6ꢀ8 nm. Bꢀ30 modified analogues were also soluble in
ethanol (96%, medical grade) and after dilution in water they formed micelles or vesicles of
bimodal size distribution (35ꢀ50 and 80ꢀ120 nm). Notably, NꢀB30 norAbuGMDP 21 was only
partially soluble in ethanol. Therefore, NꢀB30 norAbuGMDP 21 was solubilized in a minimal
amount of dimethyl sulfoxide (DMSO) and subsequently diluted by phosphateꢀbuffered saline
(PBS). The procedure with DMSO was found generally applicable to all B30 modified
analogues.
Characterization of liposomes by DLS The size distribution, zetaꢀpotential and structures of
various liposomal preparations were analysed 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 (NꢀL18 norAbuGMDP 20, NꢀB30
norAbuGMDP 21, norAbuMDPꢀLys(L18) 29, norAbuMDPꢀLys(B30) 30, norAbuGMDPꢀ
Lys(L18) 37, norAbuGMDPꢀLys(B30) 38, B30 norAbuMDP 39, L18 norAbuMDP 40) (results
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6
not shown). Liposomes were prepared by lipid film hydration, freezingꢀthawing and extrusion
through polycarbonate filters, and size distributions were measured in the range 95 ꢀ120 nm with
polydispersity indexes in the range 0.09 – 0.12.
0
1
2
3
4
5
6
7
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9
0
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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 (i.v. application or modified for s.c. application) were performed with rabbits.
These tests demonstrated that both free and liposomal formulations of MDP 42 were pyrogenic.
The sum +ꢁTmax (3 rabbits per group) was shown to be 2.7 °C and 2.9 °C, respectively. In
comparison, the sum +ꢁTmax for control liposomes was only 0.2 °C. In comparison, both free and
liposomal formulations of norAbuMDP, norAbuGMDP, and their lipophilic analogues (NꢀL18
norAbuGMDP 20, NꢀB30 norAbuGMDP 21, norAbuMDPꢀLys(L18) 29, norAbuMDPꢀLys(B30)
30, norAbuGMDPꢀLys(L18) 37, norAbuGMDPꢀLys(B30) 38, B30 norAbuMDP 39, and L18
norAbuMDP 40), exhibited values of +ꢁTmax only within the range 0.4 – 0.6 °C. Accordingly
since pyrogenicity is defined by the situation if the sum +ꢁTmax ˃ 1.15 °C (according to the Ph.
Eur. Chapter 2.6.14), then free (micellar) and liposomal formulations of norAbuMDP,
norAbuGMDP and all their lipophilic analogues were here declared nonꢀpyrogenic. In addition,
no other adverse or toxic effects were observed (e.g. weight lost, skin irritation and necrosis at
site of application, behavioural changes), over a period of 2 months in rabbits post administration
of either norAbuMDP, norAbuGMDP or their lipophilic analogues.
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Table 2. Results of pyrogenity tests on rabbits
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+∆Tmax °C values for free and liposomal preparations of norAbuMDP/norAbuGMDP analogues versus
controls
0
1
2
3
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7
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9
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2
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9
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2
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9
0
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2
3
4
5
6
7
8
9
0
Control
MDP 42
2.9
norAbuMDP
norAbuGMDP 20 21 29 30 37 38 39 40
free
ꢀ
0.6
0.5
0.4
0.5
0.5 0.7 0.4 0.7 0.5 0.4 0.6 0.6
0.4 0.5 0.5 0.6 0.4 0.5 0.5 0.6
liposomal
0.2
2.7
A preparation was evaluated as nonpyrogenic, if the Sum +∆Tmax < 1.1 °C (3 rabbits per group)
Stimulation of innate immunity
Stimulation of NOD2 receptor Biological assay studies revealed the ability of all synthesised
analogues to stimulate NOD2 receptor in in vitro model based on RAWꢀBlue reporter cells. The
extent of stimulation was comparable to lipophilic analogue L18 MDP 41, to MDP 42, Nꢀ
glycolylꢀMDP 43, and to norAbuꢀMTP 44. Lipophilic norAbuMDP or norGMDP analogues B30
norAbuMDP 39, NꢀL18 norAbuGMDP 20, L18 norAbuMDP 40, norAbuMDPꢀLys(B30) 30,
norAbuGMDPꢀLys(L18) 37, and norAbuGMDPꢀLys(B30) 38 each exerted high levels of
receptor stimulation comparable with L18 MDP 41 if not better. NꢀB30 norAbuGMDP 21 and
norAbuMDPꢀLys(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
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6
muramylglycopeptide analogues did appear to impact at least in part on receptor stimulation as
demonstrated with NꢀB30 norAbuGMDP 21 and norAbuMDPꢀLys(L18) 29 (Fig. 3)
0
1
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9
0
Figure 3. Stimulation of NOD2 receptors by various MDP analogues
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Fig. 3 A) Stimulation of NOD2 receptor. Testing system RAWꢀBlue reporter cells NOD2
(
Invivogen) was used for assaying stimulatory activity of lipophic analogues of norAbuMDP and
norAbuGMDP (NꢀL18 norAbuGMDP 20, NꢀB30 norAbuGMDP 21, norAbuMDPꢀLys(L18) 29,
norAbuMDPꢀLys(B30) 30, norAbuGMDPꢀLys(L18) 37, norAbuGMDPꢀLys(B30) 38, B30
norAbuMDP 39, L18 norAbuMDP 40). L18 MDP 41, MDP 42, Nꢀglycolyl MDP 43 and
norAbuꢀMTP 44 were used as positive standards. Desmuramyl derivative Adamantyl dipeptide
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
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36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
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60
(
AdDP) 45 was used as negative control. Cells were exposed for 48 h to equimolar concentration
of tested compounds (10 µM) and induction of reporter gene was measured by colour enzymatic
reaction at 650 nm. ANOVA method was used to compare MDP 42 and RAWꢀBlue™ Cells to
other compounds. Statistics were calculated using GrafPad Prism version 5.0. Dunett’s Multiple
Comparison Test negative control column (RAWꢀBlue™ Cells) versus the other columns; ꢀ *p <
0
0
B) Inhibition of NOD2 receptor interaction with MDP analogues by GSK 717. Testing system
RAWꢀBlue reporter cells NOD2 (Invivogen) was used for assaying stimulatory activity of lipophic
analogues of norAbuMDP/norAbuGMDP (NꢀL18 norAbuGMDP 20, NꢀB30 norAbuGMDP 21,
norAbuMDPꢀLys(L18) 29, norAbuMDPꢀLys(B30) 30, norAbuGMDPꢀLys(L18) 37, norAbuGMDPꢀ
Lys(B30) 38, B30 norAbuMDP 39, L18 norAbuMDP 40. L18 MDP, MDP 42. The cells were treated
by the inhibitor (0.8 ꢂM) 2 h prior addition of the tested analogues. The cells were exposed for
.05; **p < 0.01; positive control column (MDP) versus the other columns
.01;
ꢀp < 0.05; ꢀꢀp <
2
4 h to equimolar concentration of tested compounds (10 µM) and induction of reporter gene
was measured by colour enzymatic reaction at 650 nm. As positive control served MDP and L18
MDP without the inhibitor GSK717. Induction of reporter gene was measured by colour enzymatic
reaction at 650 nm. ANOVA method was used to compare MDP 42 and RAWꢀBlue™ Cells to other
compounds. Statistics were calculated using GrafPad 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.
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 (C12ꢀiEꢀDAP 46, TriꢀDAP 47, MꢀTriꢀ
DAP 48) did activate NOD1 as expected, given that they are NOD1ꢀspecific ligands. Stimulation
of NOD1 by C12ꢀiEꢀDAP 46 was less effective than by TriꢀDAP 47 and MꢀTriꢀDAP 48. As
anticipated, MDP 42, norAbuꢀMTP 44 and norAbuMDP or norAbuGMDP analogues (NꢀL18
norAbuGMDP 20, NꢀB30 norAbuGMDP 21, norAbuMDPꢀLys(L18) 29, norAbuMDPꢀLys(B30)
3
0, norAbuGMDPꢀLys(L18) 37, norAbuGMDPꢀLys(B30) 38, B30 norAbuMDP 39, and L18
norAbuMDP 40) did not stimulate NOD1 to any significant degree (Fig. 4).
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ꢁ ꢁ
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Figure 4. Stimulation of NOD1 receptors by various MDP analogues
Testing system RAWꢀBlue reporter cells NOD1 (Invivogen) was used for assaying selectivity of
MDP 42, norAbuꢀMTP 44 and all lipophilic norAbuMDP and norAbuGMDP analogues (NꢀL18
norAbuGMDP 20, NꢀB30 norAbuGMDP 21, norAbuMDPꢀLys(L18) 29, norAbuMDPꢀLys(B30)
30, norAbuGMDPꢀLys(L18) 37, norAbuGMDPꢀLys(B30) 38, B30 norAbuMDP 39, L18
norAbuMDP 40) towards NOD1. MDP 42 and norAbuꢀMTP 44 were used as positive standards
for NOD2 stimulation as both are not recognised by NOD1. Diaminopimelic acid derivatives
C12ꢀiEꢀDAP 46, TriꢀDAP 47, MꢀTriꢀDAP 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 colour enzymatic reaction at 650 nm.
(
(
ANOVA method was used to compare MDP 42 and RAWꢀBlue™ Cells to other compounds.
Statistics were calculated using GrafPad Prism version 5.0 Dunett’s Multiple Comparison Test,
negative control column (RAWꢀBlue™ 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
Stimulation of NLRP 3 receptor MDP 42, norAbuꢀMTP 44 and norAbuMDP or norAbuGMDP
analogues (NꢀL18 norAbuGMDP 20, NꢀB30 norAbuGMDP 21, norAbuMDPꢀLys(L18) 29,
norAbuMDPꢀLys(B30) 30, norAbuGMDPꢀLys(L18) 37, norAbuGMDPꢀLys(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 HEKꢀBlue cells used
for detection of ILꢀ1β secreted by THLP1. Stimulatory activities were comparable to ATP,
which is a standard positive control for NLRP3 stimulation ((Fig. 5). All compounds tested
stimulated NLRP3 well with the exception of NꢀB30 norAbuGMDP 21 which was found to be
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only a weak NLRP3 agonist. Likewise, NꢀB30 norAbuGMDP 21 was among the weakest
agonists of NOD2 as well (Fig. 3).
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Figure 5. Stimulation of NLRP3 receptor by various MDP analogues
Testing system based on THP1ꢀNull Cells and HEKꢀBlue™ ILꢀ1β Cells (Invivogen) was used
for assaying activation of NLRP3 receptor. L18 MDP 41, MDP 42, norAbuꢀMTP 44 and all
lipophilic norAbuMDP and norAbuGMDP analogues (NꢀL18 norAbuGMDP 20, NꢀB30
norAbuGMDP 21, norAbuMDPꢀLys(L18) 29, norAbuMDPꢀLys(B30) 30, norAbuGMDPꢀ
Lys(L18) 37, norAbuGMDPꢀLys(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 HEKꢀBlueLIꢀ1β cells to cells stimulated with tested compounds.
Statistics were calculated using GrafPad Prism version 5.0. Dunett’s Multiple Comparison Test
negative control column (HEKꢀBlueLIꢀ1β Cells) versus the other columns * p < 0.05; ** p <
0
.01; *** p < 0.001
Proliferationꢀstimulating activity of sera from mice treated by s.c. administration of
various MDP analogues.
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MDP analogues formulated into liposomes were shown to induce cellular proliferation in sera of
mice after 6h (s.c. application). Only NꢀB30 norAbuGMDP 21 did not exert significant effects
within a short period (6 h) post administration. The cellular proliferation effects of L18
norAbuMDP 40 and norAbuGMDPꢀLys(L18) 37 were comparable to MDP 42. (Fig. 6).
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Figure 6. Stimulation of GMꢀprogenitor cells by sera of mice exposed to analogues of MDP
administered by s.c. route
Sera were collected from mice (ICR, female) 6 hours after s.c. application of particular
preparation. Direct stimulatory effect of sera on proliferation of GMꢀcells in vitro was tested and
expressed in number of colonies of GMꢀCFU. 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
Adjuvant activity of liposomal norAbuMDPꢀLys(B30)
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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 Th1ꢀ versus Th2ꢀ type OspAꢀspecific
antibodies in experimental mice post immunization with vaccination nanoparticles comprised of
norAbuMDPꢀLys(B30) 30 formulated as an adjuvant in rOspA proteoliposomes. AlumꢀrOspA
and MDP 42ꢀrOspA proteoliposomes were used for controls. The identity of the rOspA was
initially confirmed by MALDIꢀTOF MS. Using a 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 following parameters: sequence
coverage 63 % (13 peptides) and corresponding probabilityꢀbased MOWSE score 157. For
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immunization experiments, endotoxin was removed from rOspA by repeating phase extraction
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method with Triton Xꢀ114 to reduce levels below 2.5 EU per mg of protein, ref.
.
Subsequently, OspAꢀspecific antibody responses were compared by ELISA assays in
mice immunized by various formulations of rOspA (Table 2). Immunizations with soluble
rOspA without adjuvant elicited very low levels of OspAꢀspecific antibodies in total Ig, IgG1,
and IgG2a isotypes, almost indistinguishable from results following a mock immunization with
p24ꢀhsp70 antigen (Fig. 7). On the other hand, the administration of MDP 42ꢀOspA
proteoliposomes elicited the highest levels of OspAꢀspecific antibodies in total Ig and IgG1
isotypes followed by norAbuMDPꢀLys(B30) 30ꢀrOspA proteoliposomes and Alumꢀsoluble
rOspA (Fig. 7). Importantly, rOspA proteoliposomes loaded with norAbuMDPꢀLys(B30) 30
elicited the strongest OspAꢀspecific 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 OspAꢀspecific antibody response was detected after
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immunization with MDP 42ꢀrOspA proteoliposomes. Of note, norAbuMDPꢀLys(B30) 30ꢀrOspA
proteoliposomes administration to mice was not associated with detectable irritations or local
reactions at site of administration (data not shown).
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Figure 7. OspAꢀspecific antibodies in mice immunized with rOspA proteoliposomes loaded with
norAbuMDPꢀLys(B30) 30 are prominent in stimulation of Th1 isotype IgG2a
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DISCUSSION
NOD 2 and NLRP3 activation The relationship between innate immunity and antiꢀmicrobial
pathogen effects relies on the specific host receptorꢀbased detection of pathogenꢀ and danger
signalꢀderived molecular signatures PAMPs and DAMPs, respectively. PGN and PGNꢀderived
fragments represent structures derived from PAMPs. Some fragments of PGN are recognised by
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intracellular receptors NOD1 and NOD2 . NOD1 and NOD2 receptors have an amino terminal
caspase recruitment domain (CARD) required to trigger nuclear factorꢀκB (NFꢀκB) signalling.
Another NLR family relative, the pyrin domainꢀcontaining proteins (NLRPs), are essential for
37ꢀ38
the formation of the caspase 1 inflammasome through their Nꢀterminal pyrin domain
.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, so promoting antimicrobial function by ROS production. The interplay
between Tollꢀlike 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
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7, 39ꢀ44
ligands
are required for the action of PGNꢀderived adjuvants like desmuramylpeptidesꢀ
43
containing diaminopimelate (lFK156, FK565) and muramylpeptides such as MDP 42 .
Activation of NOD2 by MDP 42 leads to production of precursors of ILꢀ1β as well as precursors
of other cytokines, including ILꢀ18 and ILꢀ33. MDP 42 also activates the NLRP3 inflammasome,
a multiprotein complex that recruits and activates caspaseꢀ1 to transform interleukin precursors
into their active forms. Since, ILꢀ1β and ILꢀ18 are key cytokines that act on numerous immune
cells, this makes NLRP3 agonists useful immunostimulants and vaccine components able to
37
elicit potential Th1 associated antigen specific immune responses .
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Here established transgenic cellꢀbased in vitro systems were employed to test the abilities
of norAbuMDP and norAbuGMDP analogues to activate NOD2 and NLRP3 (Figs. 3A and 5).
All tested compounds including the parent norAbuꢀMTP 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 norAbuꢀMDP or norAbuꢀGMDP (NꢀL18
norAbuGMDP 20, NꢀB30 norAbuGMDP 21, norAbuMDPꢀLys(L18) 29, norAbuMDPꢀLys(B30)
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30, norAbuGMDPꢀLys(L18) 37, norAbuGMDPꢀLys(B30) 38, B30 norAbuMDP 39, and L18
norAbuMDP 40) presumably owing to the absence of diaminopimelic acid moieties in their
molecule. On closer examination (Fig. 3), NOD2 activation mediated by B30 norAbuMDP 39,
NꢀL18 norAbuGMDP 20, L18 norAbuMDP 40, norAbuMDPꢀLys(B30) 30, norAbuGMDPꢀ
Lys(L18) 37 or norAbuGMDPꢀLys(B30) 38 was comparable if not better than activation
mediated by the positive controls L18 MDP 41, MDP 42, NꢀglycolylꢀMDP 43 or norAbuMTP
44. NꢀB30 norAbuGMDP 21 and norAbuMDPꢀLys(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 structureꢀactivity 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. In order to be completely sure that our lipophilic analogues of
norAbuMDP and norAbuGMDP were acting similarly on NOD2 we employed the NOD2
inhibitor GSK 717 that competes competitively with MDP 42 for binding to the nucleotide
45
oligomerisation domain (IC submicromolar concentration) . Gratifyingly, GSK 717 was seen
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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 (Fig. 3B). Data from the literature
suggest that the binding of MDP 42 to NOD2 leads to the formation of an active signalling
complex that contains both oligomeric ATPꢀNOD2, and other adapter or effector molecules, plus
the binding of RIPK2 through CARDꢀCARD interactions. Therefore, we also tested for the
effect of the RIPK2 inhibitor gefitinib, which inhibits the NOD2 signalling cascade at the level
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of RIPK2 . In this case, gefitinib (10 µM) also supressed 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
(Fig. 5), although NꢀB30 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
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MDP analogues have been reported that possess a more metabolically stable etherꢀbond or else
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fluorinated hydrocarbon chains . In these cases, analogues were found inactive, consistent with
the possibility that fatty acyl lipophilic MDP analogues may need to be hydrolysed inside cells in
order to produce a hydrophilic metabolite suitable for activation of antigenꢀpresenting cells. On
the other hand, a biotinylated MDP derivative was reported recently, with biotin conjugated via
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an alkyl spacer attached to Cꢀ6, that was perfectly functional in binding recombinant NOD2 .
Therefore, there is the alternative possibility that at least some lipophilic norAbuMDP and even
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