<i>Mycobacterium tuberculosis</i> IMPDH in Complexes with Substrates, Products and Antitubercular Compounds

<div><p>Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD⁺ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of <i>Mtb</i>IMPDH2 in the apo form and in complex with the product XMP and substrate NAD⁺ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of <i>Mtb</i>IMPDH2-targeted antibiotics.</p></div

Data collection and refinement statistics.

<p>ASU, Asymmetric Unit,</p><p>^<i>a</i>Values in parentheses correspond to the highest-resolution shell.</p><p>^<i>b</i><i>R</i>_<i>merge</i> = Σ_<i>hkΣi</i>|<i>I</i>_<i>i</i>(<i>hkl</i>)– 〈<i>I(hkl)</i>〉|/Σ_<i>hkl</i>Σ_<i>i</i>|〈<i>I</i>_<i>i</i><i>(hkl)</i>〉|, where <i>I</i>_<i>i</i>(hkl) is the intensity for the <i>i</i>th measurement of an equivalent reflection with indices <i>h</i>, <i>k</i>, and <i>l</i>.</p><p>^<i>c</i><i>R</i>_<i>work</i> = Σ_<i>hkl</i>||<i>F</i>_<i>obs</i>|—|<i>F</i>_<i>calc</i>||/ Σ_<i>hkl</i> |<i>F</i>_<i>obs</i>|, where <i>F</i>_<i>obs</i> and <i>F</i>_<i>calc</i> are observed and calculated structure factors, respectively. <i>R</i>_<i>free</i> is calculated analogously for the test reflections, which were randomly selected and excluded from the refinement.</p><p>^<i>d</i>Ligands include all atoms excluding protein and water atoms.</p><p>Data collection and refinement statistics.</p

Active site flap in apo <i>Mtb</i>IMPDH2ΔCBS and cofactor orientation in <i>Mtb</i>IMPDH2ΔCBS•XMP•NAD⁺ complex.

<p>(A) Overlay of apo <i>Mtb</i>IMPDH2ΔCBS and <i>Mtb</i>IMPDH2ΔCBS•IMP•<b>P41</b> structures with a flap residue K454 in the apo form clashing with the linker position of <b>P41</b>, indicating that these two elements occupy the same space in the active site. For <i>Mtb</i>IMPDH2ΔCBS•IMP•<b>P41</b>, only residues (lines) and <b>P41</b> (sticks) are shown; color code for <i>Mtb</i>IMPDH2ΔCBS•IMP•<b>P41</b> as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138976#pone.0138976.g005" target="_blank">Fig 5A</a>. For the apo structure, chains A (lime) and C (gray) are shown in a cartoon representation and residues corresponding to these involved in inhibitor binding are shown as lines. A prime denotes a residue from the adjacent monomer. (B) Top view of the active site showing XMP interactions. Chain A (slate blue) and symmetry-generated adjacent chain (violet) are shown. Residues are represented as lines. XMP (pale yellow) and NAD⁺ (green) are shown as sticks. (C) Side view of the active site detailing NAD⁺ binding. Color code and depiction as in panel (B). For panels (B) and (C) 2m<i>Fo</i>-D<i>Fc</i> electron density maps contoured at the 2 σ level for XMP (pale yellow) and 1.5 σ level for NAD⁺ (green) are shown on the right. Atoms discussed in text are labeled. (D) Cofactor position in superimposed structures <i>Mtb</i>IMPDH2ΔCBS•XMP•NAD⁺ and <i>Vc</i>IMPDHΔCBS•XMP•NAD⁺. Only ligands (depicted as sticks) and the interacting residues (represented as lines) are shown. Residues are labeled according to <i>Mtb</i>IMPDH2 numbering with <i>Vc</i>IMPDH numbering in parentheses. Color code is as follows: for the <i>Mtb</i> structure as in panel (A); for the <i>V</i>c structure: chain A (light orange), symmetry-generated adjacent chain (brown), NAD⁺ (orange), XMP and selected hydrogen bonds are omitted for clarity. (E) Overlay of the cofactor position in <i>Mtb</i>IMPDH2ΔCBS•XMP•NAD⁺ and the ternary complex of hIMPDH2 with NAD⁺ and substrate analog, CPR (hIMPDH2•CPR•NAD⁺; PDB code 1NFB). Residues are labeled according to <i>Mtb</i>IMPDH2 numbering with hIMPDH2 numbering in parentheses. Color code is as follows: for the <i>Mtb</i> structure as in panel (B); for the human structure: chain A (light gray), symmetry-generated adjacent chain (dark gray), NAD⁺ (gray), CPR is omitted for clarity. Localization of the eukaryotic A^E-subsite and the bacterial A^B-subsite is indicated. For all panels (where applicable): a prime denotes a residue from the adjacent monomer. Water molecules are shown as red spheres. Hydrogen bonds are depicted as red dashed lines.</p

Mechanism and inhibitors of <i>Mtb</i>IMPDH2.

<p>(A) Reactions catalyzed by IMPDH. <i>Mtb</i>IMPDH2 numbering is shown. (B) The <i>anti</i>- and <i>syn</i>- conformations of nicotinamide ring in NADH. (C) <i>Cp</i>IMPDH inhibitors, for which crystal structures of <i>Mtb</i>IMPDH2•IMP•<b>I</b> were obtained. Portions of inhibitors that bind in the nicotinamide-subsite (left side) and the A^B-subsite (right side) are indicated. Stereochemistry is denoted where applicable. NAD⁺ is shown for comparison.</p

<i>Mtb</i>IMPDH2ΔCBS kinetic parameters.

<p>^aReference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138976#pone.0138976.ref016" target="_blank">16</a>]</p><p>^bReference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138976#pone.0138976.ref033" target="_blank">33</a>]</p><p>^cReference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138976#pone.0138976.ref034" target="_blank">34</a>]</p><p>^dReference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138976#pone.0138976.ref035" target="_blank">35</a>]</p><p>^eReference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138976#pone.0138976.ref036" target="_blank">36</a>]</p><p><i>Mtb</i>IMPDH2ΔCBS kinetic parameters.</p

Binding of mycophenolic adenine nucleotide derivative MAD1 in <i>Mtb</i>IMPDH2ΔCBS complex.

<p>(A) <i>Mtb</i>IMPDH2ΔCBS•IMP•<b>MAD1</b> complex. Chain A (slate) and symmetry-generated adjacent chain (violet) are shown in a cartoon representation. Residues involved in inhibitor binding are shown as lines. A prime denotes a residue from the adjacent monomer. Molecules of IMP (light gray) and <b>MAD1</b> (yellow) are shown as sticks. Hydrogen bonds are shown as red dashed lines. 2m<i>F</i>_<i>o</i> –D<i>F</i>_<i>c</i> electron density map contoured at the 1 σ level for <b>MAD1</b> is shown on the right. Atoms discussed in text are labeled. (B) Overlay of <i>Mtb</i>IMPDH2ΔCBS•IMP•<b>MAD1</b> and <i>Mtb</i>IMPDH2ΔCBS•XMP•NAD⁺. Only the ligands (depicted as sticks) and the interacting residues (represented as lines) are shown. Color code for residues as in panel (A) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138976#pone.0138976.g003" target="_blank">Fig 3</a>, IMP (gray), <b>MAD1</b> (yellow), XMP (pale yellow), NAD⁺ (green). (C) Distinctive binding mode of MAD derivatives in bacterial and human IMPDHs. Overlay of <i>Mtb</i>IMPDH2ΔCBS•IMP•<b>MAD1</b> and hIMPDH2•RVP•C2-MAD. Only the inhibitors (depicted as sticks) and the interacting residues (represented as lines) are shown. Residues are labeled according to <i>Mtb</i>IMPDH2ΔCBS numbering with hIMPDH2 numbering in parenthesis. IMP and RVP are omitted for clarity. Color code: for <i>Mtb</i>IMPDH2ΔCBS•IMP•<b>MAD1</b> as in panel (A); for hIMPDH2, chain A (pale green), symmetry-generated adjacent chain (dark green), <b>C2-MAD</b> (teal).</p

Binding of <i>Cp</i>IMPDH-selective inhibitors P41 and Q67.

<p>(A) <i>Mtb</i>IMPDH2ΔCBS•IMP•<b>P41</b> complex. (B) <i>Mtb</i>IMPDH2ΔCBS•IMP•<b>Q67</b> complex. Chain A (slate) and symmetry-generated adjacent chain (violet) are shown in a cartoon representation. Residues are shown as lines. A prime denotes a residue from the adjacent monomer. Molecules of IMP (light gray), <b>P41</b> (magenta), and <b>Q67</b> (orange) are shown as sticks. Water molecules are depicted as red spheres. Potential hydrogen and halogen bonds are shown as red dashed lines. For panels A and B 2m<i>F</i>_<i>o</i> –D<i>F</i>_<i>c</i> electron density map contoured at the 1 σ level for each inhibitor is shown on the right. Atoms discussed in text are labeled. (C) Overlay of three <i>Mtb</i>IMPDH2ΔCBS inhibitor complexes. IMP is omitted for clarity. Color designations as follows: for <b>MAD1</b> as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138976#pone.0138976.g004" target="_blank">Fig 4A</a>; for <b>P41</b> and <b>Q67</b> as in panels (A) and (B), respectively.</p

Structures of <i>Cp</i>IMPDH inhibitors with antitubercular activity.

<p>Structures of <i>Cp</i>IMPDH inhibitors with antitubercular activity.</p

Antitubercular activity of <i>Cp</i>IMPDH inhibitors.

<p>^a. Average and standard deviation of three independent determinations unless otherwise noted.</p><p>^b. n = 1.</p><p>^c. n = 2.</p><p>^d. <i>K</i>_{<i>i</i>,<i>app</i>} of 1500 nM was determined for inhibition of wild-type <i>Mtb</i>IMPDH2 reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138976#pone.0138976.ref016" target="_blank">16</a>]</p><p>^<i>e</i>. MIC determined after two weeks.</p><p>The values of <i>K</i>_{<i>i</i>,<i>app</i>} for inhibition of <i>Mtb</i>IMPDH2ΔCBS.</p

<i>Bacillus anthracis</i> Inosine 5′-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion‑, Substrate‑, and Product-Bound Complexes

Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of <i>Bacillus anthracis</i> IMPDH, in a phosphate ion-bound (termed “apo”) form and in complex with its substrate, inosine 5′-monophosphate (IMP), and product, xanthosine 5′-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known <i>Cryptosporidium parvum</i> IMPDH inhibitors was examined for the <i>B. anthracis</i> enzyme and compared with those of three bacterial IMPDHs from <i>Campylobacter jejuni</i>, <i>Clostridium perfringens</i>, and <i>Vibrio cholerae</i>. The structures contribute to the characterization of the active site and design of inhibitors that specifically target <i>B. anthracis</i> and other microbial IMPDH enzymes