Inhibition of Inosine-5′-monophosphate Dehydrogenase from <i>Bacillus anthracis</i>: Mechanism Revealed by Pre-Steady-State Kinetics

Inosine-5′-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). The enzyme is an emerging target for antimicrobial therapy. The small molecule inhibitor <b>A110</b> has been identified as a potent and selective inhibitor of IMPDHs from a variety of pathogenic microorganisms. A recent X-ray crystallographic study reported that the inhibitor binds to the NAD⁺ cofactor site and forms a ternary complex with IMP. Here we report a pre-steady-state stopped-flow kinetic investigation of IMPDH from <i>Bacillus anthracis</i> designed to assess the kinetic significance of the crystallographic results. Stopped-flow kinetic experiments defined nine microscopic rate constants and two equilibrium constants that characterize both the catalytic cycle and details of the inhibition mechanism. In combination with steady-state initial rate studies, the results show that the inhibitor binds with high affinity (<i>K</i>_d ≈ 50 nM) predominantly to the covalent intermediate on the reaction pathway. Only a weak binding interaction (<i>K</i>_d ≈ 1 μM) is observed between the inhibitor and E·IMP. Thus, the E·IMP·<b>A110</b> ternary complex, observed by X-ray crystallography, is largely kinetically irrelevant

Novel Bivalent Ligands for D2/D3 Dopamine Receptors: Significant Cooperative Gain in D2 Affinity and Potency

This report describes development of a series of novel bivalent molecules with a pharmacophore derived from the D2/D3 agonist 5-OH-DPAT. The spacer length in the bivalent compounds had a pronounced influence on affinity for D2 receptors. A 23-fold increase of D2 affinity was observed at a spacer length of 9 or 10 (compounds <b>11d</b> and <b>14b</b>) as compared to monovalent 5-OH-DPAT (<i>K</i>_i; 2.5 and 2.0 vs 59 nM for <b>11d</b> and <b>14b</b> vs 5-OH-DPAT, respectively). The functional potency of <b>11d</b> and <b>14b</b> indicated a 24- and 94-fold increase in potency at the D2 receptor as compared to 5-OH-DPAT (EC₅₀; 1.7 and 0.44 vs 41 nM for <b>11d</b> and <b>14b</b> vs 5-OH-DPAT, respectively). These are the most potent bivalent agonists for the D2 receptor known to date. This synergism is consonant with cooperative interaction at the two orthosteric binding sites in the homodimeric receptor

<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

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

<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

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

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