De novo drug designing coupled with brute force screening and structure guided lead optimization gives highly specific inhibitor of METTL3: a potential cure for Acute Myeloid Leukaemia

Expression of METTL3, a SAM dependent methyltransferase, which deposits m6A on mRNA is linked to poor prognosis in Acute Myeloid Leukaemia and other type of cancers. Down regulation of this epitranscriptomic regulator has been found to inhibit cancer progression. Silencing the methyltransferase activity of METTL3 is a lucrative strategy to design anticancer drugs. In this study 3600 commercially available molecules were screened against METTL3 using brute force screening approach. However, none of these compounds take advantage of the unique Y-shaped binding cavity of the protein, raising the need for de novo drug designing strategies. As such, 125 branched, Y-shaped molecules were designed by “stitching” together the chemical fragments of the best inhibitors that interact strongly with the METTL3 binding pocket. This results in molecules that have the three-dimensional structure and functional groups which enable it to fit in the METTL3 cavity like fingers in a glove, having unprecedented selectivity and binding affinities. The designed compounds were further refined based on Lipinski’s rule, docking score and synthetic accessibility. The molecules faring well in these criteria were simulated for 100 ns to check the stability of the protein inhibitor complex followed by binding free energy calculation. Communicated by Ramaswamy H. Sarma</p

Intracellular cAMP levels increase within <i>M.tb.</i>-infected THP-1 human monocytic cells upon exposure to PDE-Is.

<p>The treated infected cells received 100 uM of PDE inhibitors (PDE-I 3, and 5 class) for 2 h followed by infection. UI: uninfected cells; the PDE5-I was 4-{[3′,4′-(Methylenedioxy)benzyl]amino}-6-methoxyquinazoline (MBM); and the PDE3-I was trequinsin. Results shown (mean and SD) represent two biological replicates, each with 2 technical replicates.</p

Interaction of PDE-Is with Rifampin.

<p>BALB/c mice were infected with 3.6 log₁₀CFU <i>M.tb.</i> and were treated daily by oral gavage (starting the day after infection) with 10 or 30 mg/kg cilostazol (C10 and C30, respectively), 10 mg/kg rifampin (R10), C10 plus R10, or C30 plus R10. Lung CFU counts were determined on days 14 and 28 post-infection.</p

Administration of PDE-I monotherapy to <i>M.tb.</i>-infected mice shows therapeutic benefits by several parameters.

<p>(<b>A</b>) Time-to-death analysis; BALB/c mice infected with 3.6 log₁₀CFU <i>M.tb.</i> were treated daily by oral gavage (starting the day after infection) with 10 or 30 mg/kg of cilostazol (C) or sildenafil (S). Isoniazid at 1 or 25 mg/kg (INH1 or INH25 respectively) and sham (PBS) were used as positive and negative controls, respectively. (<b>B–D</b>) BALB/c mice infected with 3.1 log₁₀CFU <i>M.tb.</i> were treated daily by oral gavage (starting the day after infection) with 10 mg/kg cilostazol (C10), sildenafil (S10) or sham. At day 28 post-infection, lung CFU counts (<b>B</b>), spleen CFU counts (<b>C</b>) and body weight (<b>D</b>) were determined.</p

Addition of PDE-Is to standard TB therapy.

<p>The addition of PDE-Is to standard TB therapy reduces bacterial load, lung pathology and time to lung sterilization, and does not negatively interact with rifampin in <i>M.tb.</i>-infected mice. BALB/c mice were infected with 3.97 log₁₀CFU <i>M.tb</i> and treatment was started 14 days post-infection. The mice were treated daily by oral gavage with standard therapy (SD, which is 2 months 10 mg/kg rifampin (R), 25 mg/kg isoniazid H), and 150 mg/kg pyrazinamide (Z), followed by 4 months of R and H), SD plus 10 mg/kg sildenafil (S10), cilostazol (C10) or both (CS10). (<b>A</b>) CFU counts in mouse lungs. (<b>B</b>) Histopathology analyses of mouse lungs (hematoxylin and eosin stained lung sections).</p

Proportion of mice with no detectable CFUs during TB standard therapy with and without PDE-I.

<p>The proportions of mice with no detectable CFUs were determined by homogenizing and plating the entire lungs from 56 to 168 days of treatment. Treatment was terminated after 168 days, and groups of mice were kept for an additional 4 months (168+112). Shading indicates lung sterilization in all mice in the group. SD: standard drug therapy (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030749#pone-0030749-g004" target="_blank">Fig. 4</a> legend for details); S: 10 mg/kg sildenafil; C: 10 mg/kg cilostazol; CS: 10 mg/kg each of sildenafil and cilostazol.</p

A. Multiplication of mouse-passaged and -unpassaged <i>M. tuberculosis</i> clinical isolate in BALB/c mice.

<p>A. Growth in lungs of the isolate following intravenous infection. B. Growth in lungs following aerosol infection of mouse-passaged clinical isolate in comparison with regularly mouse-passaged H37Rv strain (JHU) in BALB/c mice. Errors are SD.</p

Comparative lung histopathology in guinea pigs infected with <i>M. tuberculosis</i> H37Rv with different histories of in vitro and in vivo passaging.

<p>* Not passaged in mice.</p><p>** Passaged in mice.</p

Comparison of inflammatory granulomatous pathology in the lungs of guinea pigs three weeks after aerosol infection with one of two H37Rv strains (unpassaged TAMU, top, and mouse-passaged JHU, bottom).

<p>Comparison of inflammatory granulomatous pathology in the lungs of guinea pigs three weeks after aerosol infection with one of two H37Rv strains (unpassaged TAMU, top, and mouse-passaged JHU, bottom).</p

Comparison of bacterial multiplication in the lungs of C57BL/6 mice by passaged and unpassaged <i>dosR</i> deletion mutants and their parental <i>M. tuberculosis</i> H37Rv strain following aerosol infection.

<p>Errors are SD.</p