A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation

<div><p>The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.</p></div

Ugi compounds activate guinea pig TLR4/MD2-HEK transfectants.

<p>HEK293 cells underwent transient co-transfection with expression vectors for guinea pig TLR4, MD2, and CD14 along with SEAP reporter plasmid. Transfectants were stimulated with TLR4 agonist compounds at 10⁻⁵ to 10⁻¹⁰ M for 20 h. The plates were developed with the addition of DDAO phosphate as substrate and then read on a plate reader at 620 nm. Data were analyzed as % of the maximum 1 μM Ultrapure (0111:B4) LPS activation signal.</p

Secreted cytokine profile of rat PBMCs stimulated by AZ618.

<p>PBMCs were isolated from three naïve Sprague-Dawley rats. TLR4 agonists were used at 1 and 0.2 μg/ml doses to stimulate human PBMCs and murine splenocytes for 24 hrs. SNs were analyzed via rat MILLIPLEX kits. Data are expressed as the means of 3 rats at the 1 μg/ml dose of TLR4 agonist and are representative of 2 studies. Raw data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164632#pone.0164632.s005" target="_blank">S5 Fig</a>.</p

Species-specific activation of IL-6, TNF-α, and chemokine expression in primary cells.

<p>PBMCs were isolated from peripheral blood from human healthy volunteers, cynomolgus macaques, Hartley guinea pigs, ferrets, New Zealand white rabbits, Sprague-Dawley rats, and cotton rats, and splenocytes were isolated from BALB/c mice, <i>n</i> = 3 for each species, except <i>n</i> = 2 for cotton rats. TLR4 agonists were used at 1 μg/ml to stimulate primary cells of all species for 24 hrs. For human, cyno, mouse, and rat, SNs were analyzed via human, NHP, mouse, and rat MILLIPLEX kits. For guinea pig, ferret, rabbit, and cotton rat, cell lysates were analyzed via Affymetrix QuantiGene Plex kits. No one chemokine analyte was available in all 8 species-specific assays, so data were collected for any one of three different chemokines, MCP-1, MIP-1β, and RANTES. Data are expressed as mean compound % difference from the mean MPL value for that species and cytokine with associated 90% confidence interval (CI) listed below. A compound is considered significantly higher than MPL if the lower bound of the CI is higher than 0% (p value < 0.05); such values are in bold. Increasing shades of green and purple indicate increasingly higher and lower respective values than values achieved by MPL stimulation. Raw data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164632#pone.0164632.s006" target="_blank">S6 Fig</a>.</p

Ugi compounds preferentially activate TLR4-HEK transfectants expressing human MD2.

<p>A-B) HEK293 cells transfected with human TLR4/MD2 or mouse TLR4/MD2 (InVivogen) were stimulated with TLR4 agonists including MPL, PHAD, and Ugi compounds at 1 μg/ml to 4 ng/ml and with LPS at 25 ng/ml to 64 pg/ml for 24 h. SNs were analyzed for SEAP content by the QUANTI-Blue assay (InVivogen) and analysis at 650 nm by spectrophotometer. AUC (area under the curve) values were calculated as in Methods. No AUC value was calculated for LPS since it was utilized with a different dose range. C) HEK transfectants with cis- or trans-species expression of TLR4, MD2, and CD14 were stimulated with Ugi compounds in a dose response range of 9 μM to 1 nM or with LPS (boxes) at 75 ng/ml to 5 pg/ml for 48 h. Data was obtained as Vmax calculated over the initial linear portion of the kinetic absorbance measurements. The AUC (area under the curve) value for the dose response curve of Vmax vs log₁₀ (molar units) was calculated for each compound using Trapezoidal rule.</p

The Ugi compound-induced cytokine profile is similar to MPL and LPS in human PBMCs but not mouse splenocytes.

<p>PBMCs were isolated from three healthy volunteers, while murine splenocytes were isolated from three naïve BALB/c mice. TLR4 agonists were used at 1 and 0.2 μg/ml doses to stimulate human PBMCs and murine splenocytes for 24 hrs. SNs were analyzed via human and mouse MILLIPLEX kits. Data are expressed as the means of 3 donors/mice at the 1 μg/ml dose of TLR4 agonist and are representative of 2 studies. Raw data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164632#pone.0164632.s003" target="_blank">S3 Fig</a>.</p

Representative docking models.

<p>Ugi ligand is green, other key residues—as labeled on the Fig The rest of MD-2 is yellow backbone trace with the flexible loop region shown as sticks. A) Docking mode where Ugi compound is predicted to interact with MD-2 residues Tyr131 and Phe126 (best-scored model for AZ617 docked into human receptor). B) Alternative docking mode where Ugi compound is deep inside the binding pocket away from Tyr131 and dimerization interface (best-scored model for AZ617 and the mouse receptor). C) Comparison of the docked Ugi compound and crystallographically solved Neoseptin-3 ligand in complex with the mouse receptor (model 7 from AZ161-mouse docking). MD-2 coordinates from both complexes were superimposed. AZ161—green sticks, Neoseptin-3—orange sticks.</p

Ugi reaction products with variation of side-chains.

<p>*LogD represents the lipophilicity whereby D is the octanol/water distribution coefficient at pH7.4; whereby LogD 1–3 is considered optimal for drug-like properties. **Human TLR-4 NFκB SEAP reporter assay; protocol as described in Methods (N.B. pEC₅₀ = –Log₁₀(EC₅₀), thus values of 6 and 9 represent EC₅₀ values of 1 μM and 1 nM (0.001 μM) respectively. ***NA = not available.</p

AZ618 demonstrates in vivo activity in hTLR4/MD2 KIKO mice.

<p>C57BL/6 mice (6–12 per group) were immunized IP with 50 μg MPL or AZ618 and then were bled at 2 h via retro-orbital route and at 6 h via cardiac puncture. Serum was analyzed by multiplex Millipore kits for cytokine content. Presented are individual results with a bar representing the group geometric mean with SD. Open circles represent wild-type C57BL/6 mice; X’s represent animals deficient for TLR4 and MD2; red circles represent TLR4/MD2 -/- mice transgenic for human TLR4/MD2. Statistical comparisons are between C57BL/6 w.t. and hTLR4/MD2 KIKO groups. Statistical test was one-way ANOVA performed with family-wise error rate to adjust multiple comparisons; ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p