Development of 2-(4-pyridyl)-benzimidazoles as PKN2 chemical tools to probe cancer

Kinases are signalling proteins which have proven to be successful targets for the treatment of a variety of diseases, predominantly in cancers. However, only a small proportion of kinases (<20%) have been investigated for their therapeutic viability, likely due to the lack of available chemical tools across the kinome. In this work we describe initial efforts in the development of a selective chemical tool for protein kinase N2 (PKN2), a relatively unexplored kinase of interest in several types of cancer. The most successful compound, 5, has a measured IC50 of 0.064 μM against PKN2, with ca. 17-fold selectivity over close homologue, PKN1

Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2

Development of dihydropyrrolopyridinone-based PKN2/PRK2 chemical tools to enable drug discovery

The Protein Kinase N proteins (PKN1, PKN2 and PKN3) are Rho GTPase effectors. They are involved in several biological processes such as cytoskeleton organization, cell mobility, adhesion, and cell cycle. Recently PKNs have been reported as essential for survival in several tumor cell lines, including prostate and breast cancer. Here, we report the development of dihydropyrrolopyridinone-based inhibitors for PKN2 and its closest homologue, PKN1, and their associated structure–activity relationship (SAR). Our studies identified a range of molecules with high potency exemplified by compound 8 with Ki = 8 nM for PKN2 and 14x selectivity over PKN1. Membrane permeability and target engagement for PKN2 were assessed by a NanoBRET cellular assay. Importantly, good selectivity across the wider human kinome and other kinase family members was achieved. These compounds provide strong starting points for lead optimization to PKN1/2 development compounds

PRMT inhibition induces a viral mimicry response in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here we identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), which has antitumor growth activity in TNBC. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses before and after MS023 treatment is a functional biomarker and determinant of response, and these observations extend to a panel of human-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA, which is derived, at least in part, from inverted repeat Alu elements. Together, our results represent a shift in understanding the antitumor mechanism of type I PRMT inhibitors and provide a rationale and biomarker approach for the clinical development of type I PRMT inhibitors

The miR-35-41 Family of MicroRNAs Regulates RNAi Sensitivity in Caenorhabditis elegans

RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) to direct silencing of specific genes through transcriptional and post-transcriptional mechanisms. The siRNA guides can originate from exogenous (exo–RNAi) or natural endogenous (endo–RNAi) sources of double-stranded RNA (dsRNA). In Caenorhabditis elegans, inactivation of genes that function in the endo–RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Here we show that members of another small RNA pathway, the mir-35-41 cluster of microRNAs (miRNAs) can regulate RNAi. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene, previously shown to regulate RNAi responsiveness. Genome-wide microarray analyses show that targets of endo–siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir-35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of miR-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs

The embryonic mir-35 microRNA cluster regulates development and RNAi efficiency in C. elegans

MicroRNAs (miRNAs) are 22 nucleotide small RNAs that regulate gene expression by pairing with partial complementarity to target mRNAs. While loss of specific miRNAs can result in distinct phenotypic abnormalities, very few miRNA genes have been shown to be essential for viability. In this dissertation, I demonstrate that the mir-35 gene in Caenorabditis elegans has a vital role in embryogenesis. This gene encodes a cluster of 7 paralogous miRNAs, mir-35-41, that are highly expressed in C. elegans embryos. I show that loss of the mir-35-41 cluster in the genetic mutant mir-35(gk262), results in embryonic lethality, with defects in cytokinesis during the first embryonic cellular divisions and delays in cell cycle progression during subsequent embryonic cell divisions. Consistent with a role for mir-35 miRNAs in early embryogenesis, the inviability of mir-35(gk262) worms was rescued maternally, suggesting that deposition of the miRNA gene products is sufficient for embryogenesis. Supporting this idea, the precursor and mature forms of mir-35 are detectable in female worms that produce only oocytes. Additionally, accumulation of mature mir-35 miRNA correlated with the production of embryos. Taken together, my results suggest that RNA products expressed by the mir- 35 gene are deposited in oocytes; following fertilization, increased maturation and synthesis of mir-35-41 miRNAs are then available to control early embryonic events, including cytokinesis and cell cycle progression. In the second part of my dissertation, I investigated a surprising link between the mir-35 gene and the RNAi pathway. While performing RNA interference (RNAi) experiments in mir-35(gk262) worms, I observed strong RNAi hypersensitivity of the strain. The hypersensitivity is dependent on the canonical RNAi pathway and is similar in levels to the described lin-35 mutant. Additionally, microarrays indicated overlap in gene regulatory pathways for mir-35 and lin-35. I found that LIN-35 protein levels are significantly reduced in mir-35(gk262) embryos, indicating that the mir-35-41 miRNAs positively regulate accumulation of LIN-35 protein. Although the regulation is probably indirect, the decreased level of LIN-35 likely explains the RNAi hypersensitive phenotype of mir-35 mutant worms. Importantly, lin-35 encodes the worm homolog of the human Rb retinoblastoma gene. Another connection between the mir-35 gene and the RNAi pathway is the unexpected finding that nonspecific dsRNA affects the viability of mir-35(gk262) mutants. In contrast to almost complete lethality observed in the absence of mir-35-41 miRNAs at the restrictive temperature, I found that the introduction of non-specific dsRNA could partially rescue this lethality. Since the rescue is dependent on RNAi pathway genes, my results indicate that mir-35-41 is required for embryonic viability in a pathway that can be compensated by the initiation of RNAi. In conclusion, my research demonstrates that the mir-35-41 miRNAs are important for embryonic viability and regulate the efficiency of RNAi in C. elegans. Although the RNAi hypersensitivity of mir-35 mutants may be largely through down-regulation of LIN-35 in these mutants, the mir-35-41 miRNAs regulate lin-35 and other genes in parallel pathways important for embryogenesis. In support of this model, embryos with loss of lin-35 alone are viable, with loss of mir-35 alone have reduced viability and with loss of both genes are inviable. My work establishes a new regulator of lin-35 and demonstrates novel connections between RNAi pathway genes and embryonic viabilit

Maintenance and differentiation of neural stem cells

The adult mammalian brain contains self-renewable, multipotent neural stem cells (NSCs) that are responsible for neurogenesis and plasticity in specific regions of the adult brain. Extracellular matrix, vasculature, glial cells, and other neurons are components of the niche where NSCs are located. This surrounding environment is the source of extrinsic signals that instruct NSCs to either self-renew or differentiate. Additionally, factors such as the intracellular epigenetics state and retrotransposition events can influence the decision of NSC`s fate into neurons or glia. Extrinsic and intrinsic factors form an intricate signaling network, which is not completely understood. These factors altogether reflect a few of the key players characterized so far in the new field of NSC research and are covered in this review. (C) 2010 John Wiley & Sons, Inc. WIREs Syst Biol Med 2011 3 107-114 DOI:10.1002/wsbm:10

Structural Characterization of Maize SIRK1 Kinase Domain Reveals an Unusual Architecture of the Activation Segment

Kinases are primary regulators of plant metabolism and excellent targets for plant breeding. However, most kinases, including the abundant receptor-like kinases (RLK), have no assigned role. SIRK1 is a leucine-rich repeat receptor-like kinase (LRR-RLK), the largest family of RLK. In Arabidopsis thaliana, SIRK1 (AtSIRK1) is phosphorylated after sucrose is resupplied to sucrose-starved seedlings and it modulates the sugar response by phosphorylating several substrates. In maize, the ZmSIRK1 expression is altered in response to drought stress. In neither Arabidopsis nor in maize has the function of SIRK1 been completely elucidated. As a first step toward the biochemical characterization of ZmSIRK1, we obtained its recombinant kinase domain, demonstrated that it binds AMP-PNP, a non-hydrolysable ATP-analog, and solved the structure of ZmSIRK1- AMP-PNP co-crystal. The ZmSIRK1 crystal structure revealed a unique conformation for the activation segment. In an attempt to find inhibitors for ZmSIRK1, we screened a focused small molecule library and identified six compounds that stabilized ZmSIRK1 against thermal melt. ITC analysis confirmed that three of these compounds bound to ZmSIRK1 with low micromolar affinity. Solving the 3D structure of ZmSIRK1-AMP-PNP co-crystal provided information on the molecular mechanism of ZmSIRK1 activity. Furthermore, the identification of small molecules that bind this kinase can serve as initial backbone for development of new potent and selective ZmSIRK1 antagonists

Decreased LIN-35/Rb contributes to the RNAi hypersensitivity of <i>mir-35-41(gk262)</i> worms.

<p>(A) Northern blot analyses of <i>lin-35/Rb</i> mRNA levels in WT, <i>mir-35-41(gk262)</i> and <i>lin-35(n745)</i> embryos. After normalization to actin mRNA the average and standard deviation from 3 independent experiments was calculated with wild type levels set to one. (B) LIN-35 protein is decreased in <i>mir-35-41(gk262)</i> embryos compared to wild type, as shown by western blotting. After normalization to tubulin the average and standard deviation from 4 independent experiments was calculated with wild type levels set to one (Student's t-test *p<4×10⁻⁵). (C) PAGE Northern blot analysis of RNA from wild type and <i>lin-35(n475)</i> embryos shows similar levels of pre- and mature miR-35 expression. The rRNAs are shown as loading controls. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002536#s2" target="_blank">Results</a> are representative of 3 independent experiments. (D) The indicated worm strains were grown on bacteria expressing <i>unc-22</i> dsRNA. <i>Ex[lin-35; sur-5::GFP]</i> is an extrachromosomal array that expresses <i>lin-35</i> in GFP positive (+) worms. <i>Int[sur-5::GFP]</i> is an integrated array that expresses GFP in all worms and <i>Ex[myo-2::GFP]</i> is an extrachromosomal array that expresses GFP in worms that inherit the array. Phenotype was scored as percent of twitching or paralyzed worms after 28 h of exposure to RNAi from the L4 to adult stage. Error bars represent the standard error of the mean (s.e.m) for at least two independent experiments.</p

Mis-regulation of the E01G4.5 endo–siRNA target in <i>mir-35-41</i> mutants.

<p>RT-qPCR of E01G4.5 pre-mRNA (A) and mature mRNA (B) normalized to 18S rRNA and compared to wild type levels (mean ± s.e.m., n = 3, *, P<0.05).</p