Inhibiting AMPylation: A Novel Screen To Identify the First Small Molecule Inhibitors of Protein AMPylation

Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from <i>Vibrio parahemolyticus</i>, catalyzes the transfer of AMP onto the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization-based high-throughput screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g., calmidazolium, GW7647, and MK886) with <i>K</i>_i’s ranging from 6 to 50 μM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator

Identification of Verrucarin A as a Potent and Selective Steroid Receptor Coactivator-3 Small Molecule Inhibitor

<div><p>Members of the steroid receptor coactivator (SRC) family are overexpressed in numerous types of cancers. In particular, steroid receptor coactivator 3 (SRC-3) has been recognized as a critical coactivator associated with tumor initiation, progression, recurrence, metastasis, and chemoresistance where it interacts with multiple nuclear receptors and other transcription factors to enhance their transcriptional activities and facilitate cross-talk between pathways that stimulate cancer progression. Because of its central role as an integrator of growth signaling pathways, development of small molecule inhibitors (SMIs) against SRCs have the potential to simultaneously disrupt multiple signal transduction networks and transcription factors involved in tumor progression. Here, high-throughput screening was performed to identify compounds able to inhibit the intrinsic transcriptional activities of the three members of the SRC family. Verrucarin A was identified as a SMI that can selectively promote the degradation of the SRC-3 protein, while affecting SRC-1 and SRC-2 to a lesser extent and having no impact on CARM-1 and p300 protein levels. Verrucarin A was cytotoxic toward multiple types of cancer cells at low nanomolar concentrations, but not toward normal liver cells. Moreover, verrucarin A was able to inhibit expression of the SRC-3 target genes MMP2 and MMP13 and attenuated cancer cell migration. We found that verrucarin A effectively sensitized cancer cells to treatment with other anti-cancer drugs. Binding studies revealed that verrucarin A does not bind directly to SRC-3, suggesting that it inhibits SRC-3 through its interaction with an upstream effector. In conclusion, unlike other SRC SMIs characterized by our laboratory that directly bind to SRCs, verrucarin A is a potent and selective SMI that blocks SRC-3 function through an indirect mechanism.</p></div

Verrucarin A selectively reduces SRC-3 protein levels while it does not reduce CARM-1 and p300 protein levels.

<p>(A-B) A549 cells were treated with verrucarin A at different concentrations (0, 10, 20, 50, 100, and 200 nM) for 24 h, then Western analysis was performed to quantitate SRC-1, SRC-2, SRC-3, CARM1, and p300 proteins.</p

Verrucarin A inhibits H1299 cell migration.

<p>H1299 cells were plated into 24-well plates and treated with verrucarin A for 18 h for wound healing assay analysis.</p

Verrucarin A reduces the transcriptional activities of SRCs in HeLa cells.

<p>(A) Chemical structure of verrucarin A. (B) Verrucarin A inhibits pBIND-SRC luciferase activity. HeLa cells were transiently cotransfected with expression vectors for pBIND-SRC-1, pBIND-SRC-2 or pBIND-SRC-3 and the GAL4-responsive pGL5 reporter plasmid before incubation with verrucarin A at different concentrations (0, 1, 2, 5, and 10 nM) for 24 h, followed by luciferase assay. Empty pBIND vector was transfected as a negative control. (C) Verrucarin A inhibits SRC coactivation of ERα. Luciferase assays were performed in HeLa cells transiently transfected with an ERE-luc reporter vector and expression vectors for ERα, and pCR3.1-SRC before incubation with 10 nM E2 and verrucarin A at different concentrations (0, 2, 5, and 10 nM) for 24 h.</p

Verrucarin A increases cancer cell chemosensitivity to other anti-cancer drugs.

<p>(A–D) A549 cells were treated with verrucarin A in combination with gefitinib, BEZ235, gemcitabine, or docetaxel. (E) T-47D cells were treated with verrucarin A in combination with tamoxifen. (F) MDA-MB-231 cells were treated with verrucarin A in combination with paclitaxel. All cells were treated for 72 h, followed by MTS assay.</p

First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating <i>M. tuberculosis</i>

Development of effective therapies to eradicate persistent, slowly replicating <i>M. tuberculosis</i> (<i>Mtb</i>) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent <i>Mtb</i> into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent <i>Mtb</i>. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38 350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type <i>Mtb</i> as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional ΔAPSR mutant, which could be restored by complementation with <i>Mtb</i> APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live <i>Mtb</i>, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between <i>Mtb</i> persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value

Identification of Small Molecules that Disrupt Signaling between ABL and Its Positive Regulator RIN1

<div><p>Constitutively active BCR-ABL kinase fusions are causative mutations in the pathogenesis of hematopoietic neoplasias including chronic myelogenous leukemia (CML). Although these fusions have been successfully targeted with kinase inhibitors, drug-resistance and relapse continue to limit long-term survival, highlighting the need for continued innovative drug discovery. We developed a time-resolved Förster resonance energy transfer (TR-FRET) -based assay to identify compounds that disrupt stimulation of the ABL kinase by blocking its ability to bind the positive regulator RIN1. This assay was used in a high throughput screen (HTS) of two small molecule libraries totaling 444,743 compounds. 708 confirmed hits were counter-screened to eliminate off-target inhibitors and reanalyzed to prioritize compounds with IC₅₀ values below 10 μM. The CML cell line K562 was then used to identify five compounds that decrease MAPK1/3 phosphorylation, which we determined to be an indicator of RIN1-dependent ABL signaling. One of these compounds is a thiadiazole, and the other four are structurally related acyl piperidine amides. Notably, these five compounds lower cellular BCR-ABL1 kinase activity by blocking a positive regulatory interaction rather than directly inhibiting ABL catalytic function.</p></div

Five compounds significantly decrease MAPK1 phosphorylation in K562 cells.

<p>DMSO or compound was added to K562 cells at a final concentration of 1% or 10 μM, respectively, and incubated for 4 hours at 37°C before being lysed and analyzed by immunoblot with anti-pMAPK1/3 and anti-MAPK1/3. Each compound was tested with six biological replicates. Band intensity was quantified using LI-COR Odyssey, and the ratio of pMAPK/MAPK signal intensity is graphed for each compound and its DMSO control. Compounds are identified by their PubChem CID number.</p

MAPK1/3 phosphorylation is RIN1 and BCR-ABL1-dependent in K562 cells.

<p>(<b>A</b>) K562 cells expressing a control vector, RIN1 shRNA or RIN1 over-expression construct were analyzed by immunoblot with anti-pMAPK1/3 and anti-MAPK1/3 antibodies (top) and anti-RIN1 and anti-tubulin antibodies (bottom). Band intensity was quantified using LI-COR Odyssey software. Phospho-MAPK1/3 signal intensities were normalized to total MAPK1/3 and RIN1 normalized to tubulin. Changes in expression or phosphorylation were calculated as fold-change compared to control vector (normalized to 1) and presented below each blot. The immunoblot shown is representative of three independent experiments. (<b>B</b>) K562 cells were treated with 1 μM imatinib (IM) for 1 and 4 hours at 37°C, then analyzed by immunoblot as in A. (<b>C</b>) K562 cells expressing control or RIN1 shRNA were analyzed by immunoblot as in A. The ratio of pMAPK/MAPK signal intensity is graphed. Results were averaged over five independent experiments. *p-value = 1x10^-4</p