Discovery of 5‑Amino‑<i>N</i>‑(1<i>H</i>‑pyrazol-4-yl)pyrazolo[1,5‑<i>a</i>]pyrimidine-3-carboxamide Inhibitors of IRAK4

Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential signal transducer downstream of the IL-1R and TLR superfamily, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of 5-amino-<i>N</i>-(1<i>H</i>-pyrazol-4-yl)­pyrazolo­[1,5-<i>a</i>]­pyrimidine-3-carboxamides was developed via sequential modifications to the 5-position of the pyrazolopyrimidine ring and the 3-position of the pyrazole ring. Replacement of substituents responsible for poor permeability and improvement of physical properties guided by cLogD led to the identification of IRAK4 inhibitors with excellent potency, kinase selectivity, and pharmacokinetic properties suitable for oral dosing

Whole-genome sequencing of L638^R mutants.

<p>Heat map summary of all non-synonymous mutations identified by Illumina-based whole-genome sequencing (100X genome coverage) of L638^R mutants in MRSE CLB26329 (A) or MRSA COL (B). Red, non-synonymous mutation; grey, no change versus parental genome sequence; yellow, non-synonymous mutations in genes other than <i>mnaA</i>. Genome position, base pair change, and resulting amino acid residue substitution are highlighted. Note: with only one exception (<i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i>), no additional non-synonymous mutations besides the indicated <i>mnaA</i> mutation were identified in each of the drug resistant strains examined.</p

Genetic inactivation of <i>mnaA</i> in methicillin-resistant <i>Staphylococci</i> restores β-lactam susceptibility.

<p>Genetic inactivation of <i>mnaA</i> in methicillin-resistant <i>Staphylococci</i> restores β-lactam susceptibility.</p

Mapping of MnaA LOF mutations into the MnaA crystal structure reveal key residues for substrate binding site stability and charge.

<p>(A) Overall MRSA COL MnaA crystal structure. The molecular surface is shown in grey. The protein is represented as a cartoon. In all figures one monomer is consistently colored in orange and the other in cyan, and the bound UDP molecules are shown as sticks, methyl groups colored in light blue. Nitrogen, oxygen and phosphor atoms are in blue, red or orange, respectively. (B,C,D,E) Comparison with the <i>M</i>. <i>jannaschii</i> structure in “opened” form (PDB 3NEQ) or “closed” form (PDB 3NES). Both structures are represented as ribbons, one monomer at a time, and UDP as sticks. (B) and (C) compares the opened form, in grey, with each monomer, while the superposition is with the closed form, in (D) and (E), drawn in purple. The RMS deviation in Cα positions are 1.6Å for 262 atoms, 1.6Å for 256 atoms, 1.5Å for 321 atoms, and 1.3Å for 336 atoms, for the superpositions in cartoon (B), (C), (D) and (E), respectively. (F) Mapping MRSE LOF mutants. Eight mutation sites are mapped onto the X-ray crystal structure of UDP bound MRSA COL MnaA. The allosteric site ligand UDP-GlcNAc was taken from the structure of UDP-GlcNAc bound <i>B</i>. <i>anthracis</i> 2-epimerase (PDB ID 3BEO). UDP and UDP-GlcNAc are displayed as thin lines with the carbon atoms colored in light blue. One monomer of MnaA dimer is colored in cyan and the other in white. The mutation sites are highlighted in stick. The carbon atoms of the wild-type residues are colored in yellow; those of the mutant residues are in green. (G) Mapping MRSA <i>mnaA</i> LOF mutants. LOF mutations isolated in MRSA COL MnaA are highlighted. All coloring as in (C), but for simplicity, only the original sequence is shown.</p

MnaA loss of function mutants in MRSA and MRSE fail to produce WTA.

<p>WTA extraction and SDS PAGE analysis from L638^R MRSE CLB26329 (A) and MRSA COL (B) mutants. Note, wild-type MRSA WTA polymers appear as a ladder of discretely sized bands whereas a more diffuse staining of MRSE WTA polymer is observed. WTA material was normalized to cell biomass prior to loading. Wild-type copies of <i>cap5P</i>, <i>mnaA</i>_<i>Sa</i>, and <i>mnaA</i>_<i>Se</i>, as well as the empty vector introduced into these strains for complementation studies are indicated. The <i>tarO</i> and <i>tarA</i> deletion mutants serve as a control for complete impairment of WTA polymer production.</p

MRSA and MRSE MnaA LOF mutants are highly susceptible to imipenem in a murine thigh infection.

<p>Immune-suppressed CD-1 mice (5 per group) were challenged intramuscularly with the parental MRSA COL strain, MRSA <i>Δcap5P</i>, or MRSA <i>Δcap5P mnaA</i>_<i>Sa</i> LOF mutants (A) or with the parental MRSE strain versus <i>mnaA</i>_<i>Se</i>, <i>tarO</i>_<i>Se</i> and <i>tarA</i>_<i>Se</i> LOF mutants (B) and treated three times daily (TID) with imipenem (IPM). Thighs were harvested at 24hrs, homogenized and plated to determine CFU per thigh. (A) Restored efficacy of IPM (10 mg kg^-1) against MRSA <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>P12L</i>, <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>Y194</i>*, and <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i>. Following IPM treatment, bacterial burden amongst mice infected with <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>P12L</i>, <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>Y194</i>*, and <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i> is reduced approximately 2–3 log at 24 hrs versus those infected with MRSA COL or <i>Δcap5P</i> controls. * p<0.01 versus parent at 24 hr; $ p<0.05 versus respective 24 hr vehicle. (B) Restored efficacy of IPM (2.5 mg kg^-1) against MRSE <i>mnaA</i>, <i>tarO</i>, and <i>tarA</i> LOF mutants. Reduction in bacterial burden of mice infected with the <i>mnaA</i>_<i>Se</i>^<i>G171D</i> is comparable to those infected with <i>tarO</i>_<i>Se</i>^<i>G84</i>* or <i>tarA</i>_<i>Se</i>^<i>G129R</i> mutants, yielding an approximate 3 log reduction in 24 hr IPM treatment versus the wild-type control. Note, as MRSE CLB26329 is more susceptible to IPM than MRSA COL, its dose was reduced to 4-fold versus the MRSA efficacy study (A).</p

Tunicamycin inhibits MnaA in a concentration-dependent manner.

<p>Representative electropherograms of the MnaA-catalyzed conversion of UDP-ManNAc to UDP-GlcNAc in the absence of tunicamycin (A), in the presence of 100 μM (B) and 200 μM tunicamycin (C). Substrate concentration is 100 μM. Peaks: <b>1</b> UDP-ManNAc; <b>2</b> UDP-GlcNAc.</p

Biophysical studies demonstrate MnaA and Cap5P bind tunicamycin.

<p>(A, D) 600 MHz 1H NMR spectra of 15 μM tunicamycin. (B, E) 1H NMR STD spectra of 15 μM tunicamycin without 2-epimerase. (C) 1H NMR STD spectra of 15 μM tunicamycin in presence of 5 μM MnaA. (F) 1H NMR STD spectra of 15 μM tunicamycin in presence of 5 μM Cap5P. Saturation of the protein was achieved with a Gaussian pulse cascade resulting in a total saturation time of 3s. The protein resonances were saturated at 100 Hz and the off resonance was set to -120 ppm. Tunicamycin-specific peaks in NMR STD spectra were only obtained in the presence of MnaA or Cap5P. (G) Structure of tunicamycin.</p

WTA biosynthesis pathway.

<p>WTA is sequentially synthesized by a series of Tar enzymes on a bactoprenyl phosphate carrier on the inner leaflet of the cell membrane and eventually transported to the outer leaflet where it is cross-linked to peptidoglycan. See inset for details. Non-essential early steps in WTA biosynthesis are shown as green arrows, late stage conditionally essential steps are shown as purple arrows. Note, MnaA is highlighted by a dashed bidirectional green arrow, highlighting its novel functional role as an epimerase that interconverts UDP-GlcNAc and UDP-ManNAc, thus providing substrates for TarO and TarA, respectively. L638 is a Staphylococcal-specific TarG inhibitor [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005585#ppat.1005585.ref033" target="_blank">33</a>]. Schematic has been adapted from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005585#ppat.1005585.ref034" target="_blank">34</a>].</p

WTA is required for biofilm formation in methicillin resistant <i>Staphylococci</i>.

<p>For total biofilm quantification, biofilms were grown in triplicates for 24 hours in 96-well plates with or without indicated sub-MIC concentrations of WTA inhibitors for MRSA COL (A,C,E) and MRSE CLB26329 (B,D) strains. Genetic complementation of described mutants was performed using plasmid-based copies of wild-type <i>cap5P</i> (p<i>cap5P</i>), <i>mnaA</i>_<i>Sa</i> (p<i>mnaA</i>_<i>Sa</i>), and <i>mnaA</i>_<i>Se</i> (p<i>mnaA</i>_<i>Se</i>) as indicated. Biofilms were stained with safranin and dissolved in glacial acetic acid before OD₅₆₄ was measured. Bars represent mean OD, error bars represent standard deviation. For Epi fluorescence microscopy, biofilms of MRSA (C) and MRSE (D) were grown as above in black clear bottom plates and stained with <i>Bac</i>Light Green fluorescent stain. Z-stacks were obtained at 60x magnification. Scale bar = 10 μm.</p