Discovery of Novel Small Molecules that Activate Satellite Cell Proliferation and Enhance Repair of Damaged Muscle

Skeletal muscle progenitor stem cells (referred to as satellite cells) represent the primary pool of stem cells in adult skeletal muscle responsible for the generation of new skeletal muscle in response to injury. Satellite cells derived from aged muscle display a significant reduction in regenerative capacity to form functional muscle. This decrease in functional recovery has been attributed to a decrease in proliferative capacity of satellite cells. Hence, agents that enhance the proliferative abilities of satellite cells may hold promise as therapies for a variety of pathological settings, including repair of injured muscle and age- or disease-associated muscle wasting. Through phenotypic screening of isolated murine satellite cells, we identified a series of 2,4-diaminopyrimidines (e.g., <b>2</b>) that increased satellite cell proliferation. Importantly, compound <b>2</b> was effective in accelerating repair of damaged skeletal muscle in an <i>in vivo</i> mouse model of skeletal muscle injury. While these compounds were originally prepared as c-Jun N-terminal kinase 1 (JNK-1) inhibitors, structure–activity analyses indicated JNK-1 inhibition does not correlate with satellite cell activity. Screening against a broad panel of kinases did not result in identification of an obvious molecular target, so we conducted cell-based proteomics experiments in an attempt to identify the molecular target(s) responsible for the potentiation of the satellite cell proliferation. These data provide the foundation for future efforts to design improved small molecules as potential therapeutics for muscle repair and regeneration

Biochemical Screening of Five Protein Kinases from <i>Plasmodium falciparum</i> against 14,000 Cell-Active Compounds

<div><p>In 2010 the identities of thousands of anti-<i>Plasmodium</i> compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of <i>Plasmodium falciparum</i> calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple <i>Plasmodium</i> kinase targets without harming human cells is challenging but feasible.</p></div

A comparison of different CDPK inhibitors’ cytotoxicity to human cells.

<p>Inhibition of HepG2 cell growth at compound concentrations of 10 μM is shown for CDPK4 inhibitors in scaffolds D and G (top) and for CDPK1 inhibitors in scaffolds F and H (bottom).</p

Assessment of compound promiscuity with human kinases.

<p>Kinobeads were incubated with K562 cell extract either in the presence of vehicle (DMSO) or TCAMS compound, respectively (20 μM-0.03 μM). Protein kinases captured by the beads (140–150 kinases per experiment) were quantified following tryptic digestion, isobaric peptide tagging, and LC-MS/MS analysis. Kinases were identified as potential targets by virtue of their reduced capture in the presence of excess TCAMS compounds. Apparent dissociation constants (K_d’s) were calculated from the extent to which capture of each kinase was reduced at each compound concentration. K_d values from duplicate experiments generally agreed with each other quite well (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.s002" target="_blank">S2 Fig</a>). Colored bands indicate kinase-ligand complexes with apparent pK_d’s of ≥6, with darker shades denoting higher pK_d’s. Kinases that did not have an apparent pK_d of ≥6 for any of the compounds are not represented; only names of every other targeted kinase are shown due to space limitations. These results are summarized numerically in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.t003" target="_blank">Table 3</a>.</p

Summary of kinobead competition assays (results reflect two independent experiments).

<p>Summary of kinobead competition assays (results reflect two independent experiments).</p

Human cytotoxicity of inhibitors of 1, 2, or 3 of the <i>P</i>. <i>falciparum</i> kinases studied.

<p>Inhibition of HepG2 cell growth at compound concentrations of 10 μM were previously reported by Gamo et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.ref003" target="_blank">3</a>].</p

Clustering of <i>P</i>. <i>falciparum</i> protein kinase hits into chemical scaffolds.

<p>Inhibition of HepG2 cell growth at compound concentrations of 10 μM were previously reported by Gamo et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.ref003" target="_blank">3</a>]. For some scaffolds, target counts exceed the number of hits because some compounds hit more than one target.</p

Venn diagrams showing overlapping and non-overlapping targets of hit compounds.

<p>225 compounds had IC₅₀’s below 1 μM against at least one kinase (left); a subset of 72 compounds had IC₅₀’s below 100 nM against at least one kinase (right).</p

Novel Antitubercular 6‑Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure–Activity Relationship and Target Identification Studies

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against <i>Mycobacterium tuberculosis</i> (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome <i>bc</i>₁ complex, disruption of cell–wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylamino­pyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure–activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties

The Discovery of Novel Antimalarial Aminoxadiazoles as a Promising Nonendoperoxide Scaffold

Since the appearance of resistance to the current front-line antimalarial treatments, ACTs (artemisinin combination therapies), the discovery of novel chemical entities to treat the disease is recognized as a major global health priority. From the GSK antimalarial set, we identified an aminoxadiazole with an antiparasitic profile comparable with artemisinin (<b>1</b>), with no cross-resistance in a resistant strains panel and a potential new mode of action. A medicinal chemistry program allowed delivery of compounds such as <b>19</b> with high solubility in aqueous media, an acceptable toxicological profile, and oral efficacy. Further evaluation of the lead compounds showed that in vivo genotoxic degradants might be generated. The compounds generated during this medicinal chemistry program and others from the GSK collection were used to build a pharmacophore model which could be used in the virtual screening of compound collections and potentially identify new chemotypes that could deliver the same antiparasitic profile