Myristoylation confers oral bioavailability and ... - ACS Publications

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Myristoylation confers oral bioavailability and improves the bioactivity of c(MyD 4-4), a cyclic peptide inhibitor of MyD88 Shira Dishon, Adi Schumacher-Klinger, Chaim Gilon, Amnon Hoffman, and Gabriel Nussbaum Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01180 • Publication Date (Web): 12 Mar 2019 Downloaded from http://pubs.acs.org on March 16, 2019

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Molecular Pharmaceutics

Myristoylation confers oral bioavailability and improves the bioactivity of c(MyD 4-4), a cyclic peptide inhibitor of MyD88

Shira Dishon1, Adi Schumacher-Klinger2, Chaim Gilon3, Amnon Hoffman2 and Gabriel Nussbaum1*

1The

Institute of Dental Sciences, Hebrew University- Hadassah Faculty of Dental

Medicine, Jerusalem, Israel 2The

Institute for Drug Research, Hebrew University- Hadassah Faculty of Medicine,

Jerusalem, Israel 3The

Institute of Chemistry, Hebrew University, Jerusalem, Israel

*Corresponding author: Gabriel Nussbaum, MD PhD, Faculty of Dental Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel. Tel: +972-2-6758581, Fax: +972-2-6758561, Email: [email protected].

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

Keywords: MyD88, Myristoylation, c(MyD 4-4), oral bioavailability, EAE



Introduction Myeloid differentiation primary response 88 (MyD88) is a critical intracellular protein in innate immunity. MyD88 forms a multiprotein complex that transfers signals from Toll-like receptors (TLRs) and members of the IL-1 receptor superfamily to downstream kinases such as TRAF6 (TNF receptor associated factor 6).1,2 MyD88 deficiency in mice has profound effects on the immune system; MyD88-deficient mice are highly susceptible to infection, but are resistant to autoimmune disease.3-6 Targeted inhibition of MyD88 in antigen presenting cells (APCs) shifts the phenotype of activated human CD4+ T cells from Th1/Th17 (T helper) to a Th2-type response.7 Therefore, MyD88 is an attractive target to affect a shift in autoimmune disease from pathogenic autoimmunity driven by Th1 and Th17 cells, to protective autoimmunity driven by Th2 cells.8,9 In fact, we and others demonstrated that pharmacologic inhibition of MyD88 in animals significantly ameliorates disease in several immune and inflammatory disease models.7,10-12 MyD88 function is dependent on dimerization mediated through the Toll-IL1 Receptor (TIR) domain that is highly conserved through evolution.13 A highly conserved dimerization region is found between the second β-strand and the second alpha helix, termed the “BB loop”. A heptapeptide from this region, RDVLPGT, competitively inhibits MyD88 dimerization and function.14 We previously developed c(MyD 4-4), a stable cyclic peptide inhibitor of MyD88 based on the linear heptapeptide. c(MyD 44) is resistant to serum proteases and brush border membrane vesicles that rapidly degrade the linear BB loop peptide. c(MyD 4-4) is also more bioactive than the linear peptide and parenteral administration of c(MyD 4-4) ameliorates disease in the multiple sclerosis model EAE (Experimental autoimmune encephalomyelitis (EAE).15 However, in the current study we show that c(MyD 4-4) is not orally bioavailable, a


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significant drawback for drug development.16

Furthermore, since MyD88 is an

intracellular target, the permeability barrier of biological membranes is a challenge for maximizing the efficacy of the inhibitor. We therefore sought a means of improving bioactivity through enhanced intracellular penetration, and a modification that would enable oral administration of the MyD88 inhibitor. Covalent modification of peptides with fatty acids represents a known approach to enhance peptide penetration to mammalian cells.17 Myristic acid is a hydrophobic 14carbon saturated fatty acid known to insert into purified lipid bilayers through a direct hydrophobic interaction without the need of specific cell surface molecules.18 Myristoylation is a naturally occurring post-translational modification that serves to target cytoplasmic proteins to the intracellular surface of membranes. For example, the TRIF-related adaptor molecule (TRAM), a TLR4 adaptor protein, contains a putative N-terminal myristoylation site that when mutated prevent TRAM from localizing to the plasma membrane.19 Therefore, we hypothesized that addition of a myristoyl group (MyR-) to the N terminus of c(MyD 4-4) would improve bioactivity and potentially enable oral administration of c(MyD 4-4). Our objectives were to compare the effects of myristoylation on the ability of c(MyD 4-4) to inhibit MyD88 dimerization and block human and mouse macrophage TLR2 and TLR4 stimulation. Furthermore, we aimed to determine if myristoylation of c(MyD 4-4) will convert the compound to become orally bioavailable, and whether oral administration of MyR-c(MyD 4-4) will ameliorate disease in the mouse multiple sclerosis model EAE.



Experimental Section Cell culture THP-1, HEK 293T and RAW 264.7 cell lines were obtained from the ATCC (VA, USA). RAW 264.7 NFkB-Luc reporter cells were previously described.15 Cell lines were grown in RPMI or DMEM (Sigma, Rehovot, Israel) supplemented with Fetal calf serum, L-glutamine, sodium pyruvate, penicillin and streptomycin (Biological Industries, Israel) at 37° C and 5% CO2.

Reagents/Peptides The linear MyD88 inhibitory peptide, RDVLPGT (MyDI), and a control inhibitory peptide, PTDLVRG (MyDI-sc), were synthesized in our university and purified by HPLC. MyDI with the addition of an N-terminal myristoyl group (MyR-MyDI) was synthesized by GenScript (NJ, USA). c(MyD 4-4) and MyR-c(MyD 4-4) were synthesized by Ontores Biotechnologies Co. Ltd, China. Escherichia coli LPS was obtained from Sigma, and Pam3CSK4 from Invitrogen (San Diego, CA, USA).

PK sample preparation All procedures were performed on 275–300 g male Wistar rats (Envigo, Jerusalem, Israel) in the SPF unit of the Hebrew University, which is an AAALAC-approved unit. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC). Animals were anesthetized by 1 mL/kg of 9:1 ketamine:xylazine solution i.p., and temperature was controlled using a 37°C heated surface (Harvard Apparatus Inc., Holliston, MA). For blood sampling, each animal had an indwelling cannula in the right jugular vein. Animals were transferred to cages to recover overnight (12–18 h). c(MyD 4-4) was dissolved in water and administered 0.5 mg/kg (0.3 mg/mL)


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i.v. and blood was sampled 5 min pre-dose, and 3, 15, 30, and 60 min post-dose. MyRc(MyD 4-4) was dissolved in DMSO and the concentrated stock was dissolved in water and administered at 2.5 mg/kg (1.5 mg/mL) i.v. Blood was sampled 5 min pre-dose, and 3, 7, 15, 20, 30, 60, 90 and 120 min post-dose. For oral bioavailability, compounds were administered by gavage as follows: MyR-c(MyD 4-4) at 6 mg/kg (1.2 mg/mL, n=4) and c(MyD 4-4) at 5 mg/kg (1 mg/mL, n=3). Blood was sampled 5 min pre-dose, and 10, 20, 30, 45 min and 1, 1.5, 2, 3,4 and 6 hr post-dose for MyR- c(MyD 4-4) and 5 min pre-dose, 5, 15 and 30 min and 1, 1.5, 2 and 3 hr post-dose for c(MyD 4-4). Matched volumes of saline were administered after each blood sampling in order to avoid dehydration. Blood samples were collected sequentially at the indicated times into test tubes containing heparin. Tubes were centrifuged (5322 g, 10 min) and plasma samples were separated and kept at −20°C until analyzed.

Determination of PK parameters The trapezoidal rule with extrapolation to infinity was used to calculate Area under the plasma concentration−time curve (AUC) by dividing the last measured concentration by the elimination rate constant (kel). Linear regression analysis of the last points on the logarithmic plot of plasma concentration versus time was used to determine kel values. . Pharmacokinetic parameters such as peak serum concentration (Cmax), clearance (CL), volume of distribution (Vd), and absolute bioavailability (F) were calculated using noncompartmental analysis.

All the parameters were calculated for each rat

individually. Absolute bioavailability values were calculated using the following 𝐴𝑈𝐶𝑃𝑂 × 𝐷𝑜𝑠𝑒𝐼𝑉

formula: = 𝐴𝑈𝐶 × 𝐼𝑉

𝐷𝑜𝑠𝑒𝑃𝑂

.

PK sample analysis



Plasma samples were spiked with metoprolol (1.5 µg/mL) as an internal standard. Acetonitrile (ACN) was added to each sample (2:1) and samples were vortexed and centrifuged (14,635g, 10 min) followed by evaporation to dryness (Vacuum Evaporation System, Labconco, Kansas City, MO, USA). The tubes were reconstituted with 80 µL of mobile phase, centrifuged (14,635 g 10 min) and transferred to HPLC vials. The amount of the compounds was determined using high- performance liquid chromatography mass spectrometry (HPLC-MS) on a Waters 2695 Separation Module, equipped with Micromass ZQ detector. For c(MyD 4-4) a Kinetex® 2.6 µm HILIC 100Å, 100 x 2.1 mm column (Phenomenex®, Torrance, CA, USA) was used at an isocratic mobile phase, acetonitrile:water:ammonium acetate buffer 50 mM (70:10:20, v/v/v). For MyR- c(MyD 4-4) a Luna® (Phenomenex®) 3 µm C8 100Å, 100 x 2.0 mm column, and isocratic mobile phase ACN:water supplemented with 0.1% formic acid (50:50, v/v), flow rate of 0.2 mL/min at 25°C. The detection masses (m/z) were 1151.9 for MyR- c(MyD 4-4) and 565.76 for c(MyD 4-4). The limit of quantification was 10 ng/mL.

CLogP analysis Calculated logarithms of water–octanol partition coefficients (ClogP values) for c(MyD 4-4) and MyR- c(MyD 4-4) were obtained from the ClogP tool in CHEMDRAW ULTRA version 12.0.3.1216 (CambridgeSoft, Cambridge, MA).

Cytokine analysis hTNF-α, mIL-17, and mINF-γ protein levels were measured by ELISA (human/mouse OptEIA, BD Biosciences, CA, USA) according to the company’s instructions.


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Luciferase Assay Luciferase levels were measured using the Promega Bright Glo™ luciferase assay (Promega, Madison, WI, USA), in an Infinite 200 Pro plate reader (Tecan, Männedorf, Switzerland). Data were obtained as relative luminescence units (RLU).

Western blot and Co-immunoprecipitation Plasmid pCMV-HA-MyD88 (full length) was a gift from Bruce Beutler (Addgene plasmid # 12287) 20 and plasmid pCMV-Flag-1 MyD88 (full length) was obtained from Dundee university (Dundee, U.K). Plasmids were co-transfected using TurboFect (ThermoFisher Scientific, MA, USA) to 293T cells. 48 hr following transfection, cells were treated with the various compounds for 3 hr, stimulated for 20 minutes with 20 ng/mL IL-1β (ProSpec, Rehovot, Israel), and then lysed in RIPA buffer. Lysates were immunoprecipitated with anti-HA magnetic beads (ThermoFisher Scientific, MA, USA) and analyzed by WB as described previously.15

Flow cytometry HEK 293T cells were seeded in six well plates for 3 hr and then FITC-labeled MyDI or MyR-MyDI were added for 30 minutes. Cells were analyzed using a BD ACCURI C6 cytometer (BD Biosciences, San Jose, CA), and data were analyzed using FCS Express software (De Novo Software, Glendale, CA).

Mice C57BL/6 (B6) mice (female, 8-14 weeks) were purchased from Envigo. - Experimental protocols were approved by the Hebrew University-Hadassah IACUC and experiments were conducted in the AAALAC approved SPF unit of our university. .



Induction of EAE and treatment Mice were immunized subcutaneously with 100µg MOG35-55 emulsified in CFA containing an additional 300µg M. tuberculosis (Mt) H37RA (BD Difco, NJ, USA). Pertussis Toxin (PTX, List Biological Laboratories, CA, USA) was injected intraperitoneally on the day of immunization and again after two days. MyR-c(MyD 44) was administered by gavage vs. equal volume DMSO/PBS on days 0, 2, and 4. Some animals were sacrificed on day 9 and single cell suspensions from the popliteal, inguinal and axillary lymph nodes were prepared. Cells were cultured in 96 well plates (0.5x106 per well) for 72 hr with or without increasing concentrations of MOG35-55 peptide. In other animals, EAE was scored on a scale of 0-6: 0, no impairment; 1, limp tail; 2, limp tail and hind limb paresis; 3, ≥1 hind limb paralysis; 4, full hind limb and hind body paralysis; 5, hind body paralysis and front limb paresis; 6, death.

Statistical analysis The data are expressed as the means ± SD of three independent experiments statistically evaluated by the 2-tailed t-test except for the EAE model where the data is expressed as mean±s.e.m. and data analysis used the two way analysis of variance “ANOVA” test. All statistical evaluation was done with Prism software (Prism v.5, GraphPad Software Inc. San Diego, USA). P-values < 0.05 were considered statistically significant.


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Results Myristoylation of the c(MyD 4-4) backbone cyclized derivative of the MyD88 BB loop peptide RDVLPGT We previously identified c(MyD 4-4), a backbone cyclized derivative of RDVLPGT, the MyD88 BB loop peptide.15 Peptide modification by addition of myristic acid is a known approach to enhance cell penetration.17 To verify that myristoylation enhances penetration of the BB loop peptide, we first compared the penetration of the linear RDVLPGT peptide (MyDI), with and without covalent addition of myristoyl at the Nterminus. Myristoylation of MyDI led to a concentration dependent increase in cell penetration (Figure 1a). We therefore next synthesized c(MyD 4-4) with (MyR-c(MyD 4-4)) and without an N-terminal myristic acid group (Figure 1b).

MyR-c(MyD 4-4) disrupts MyD88 dimerization more than c(MyD 4-4) We previously demonstrated that c(MyD 4-4) inhibits MyD88 activity by interfering with MyD88 dimerization in living cells.15 To compare activity between MyR-c(MyD 4-4) and c(MyD 4-4), HEK 293T cells were co-transfected with HA-MyD88 and FlagMyD88, treated cells with the compounds, and measured MyD88 dimerization by immunoprecipitating HA-MyD88 followed by detecting Flag-MyD88 . 48 hr after cotransfection, cells were incubated with different concentrations (0.2nM and 20nM) of MyR-c(MyD 4-4) or c(MyD 4-4), and then stimulated for 30 min with IL-1β to enhance MyD88 dimerization. MyR-c(MyD 4-4) inhibited MyD88 homodimerization in a concentration dependent manner and to a more significant extent than c(MyD 4-4) (Figure 2). Myristoylation enhances the bioactivity of c(MyD 4-4)



We next compared the ability of the MyR-c(MyD 4-4) to inhibit TLR-stimulation of human and mouse macrophages, compared to the non-myrisotylated c(MyD 4-4). All results in Figure 3 are presented as the IC50 concentration (the concentration of the inhibitor at which cytokine production or NFκB is inhibited by 50%). Murine macrophages expressing an NFκB-luciferase reporter gene were activated by the TLR4 ligand LPS (Figure 3a) after incubation with MyR-c(MyD 4-4) or c(MyD 4-4). MyRc(MyD 4-4) was significantly more inhibitory than the c(MyD 4-4) when cells were stimulated with LPS. Similarly, MyR-c(MyD 4-4) inhibited human macrophage activation in response to the TLR2 ligand Pam3CSK4 (Figure 3b) or LPS (Figure 3c), significantly more than the c(MyD 4-4) peptide. Thus, MyR-c(MyD 4-4) outperforms c(MyD 4-4), consistent with the findings of better cell penetration conferred by myristoylation.

Myristoylation enhances the oral bioavailability of c(MyD 4-4) Addition of the myristoyl moiety to c(MyD 4-4) increases the compound’s lipophilicity as reflected in the shift in cLog P from cLog P -2.8 for c(MyD 4-4) to 1.5 for MyRc(MyD 4-4). The physicochemical properties of c(MyD 4-4) and MyR-c(MyD 4-4) are therefore clearly different. The pharmacokinetic parameters of c(MyD 4-4) and MyRc(MyD 4-4) after Intravenous (IV) and oral administration are summarized in Table 1, and the effect of the increased lipophilicity of MyR-c(MyD 4-4) on pharmacokinetic properties is demonstrated in Figure 4. MyR-c(MyD 4-4) exhibits a remarkable > 50 fold improvement in oral bioavailability (50.3±16.2% vs. 0.84±0.25% for the nonmyristoylated compound, P

Myristoylation confers oral bioavailability and ... - ACS Publications

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