Histone demethylases LSD1 JMJC Lysine demethylase A B S T R A C TReversible histone methylation has emerged in the last few years as an important mechanism of epigenetic regulation. Histone methyltransferases and demethylases have been identified as contributing factors in the development of several diseases, especially cancer. Therefore, they have been postulated to be new drug targets with high therapeutic potential. Here, we review histone demethylases with a special focus on their potential role in oncology drug discovery. We present an overview over the different classes of enzymes, their biochemistry, selected data on their role in physiology and already available inhibitors.ª 2012 Federation of European Biochemical Societies.Published by Elsevier B.V. All rights reserved. IntroductionHistone methylation had long been thought to be an irreversible process but since (Metzger et al., 2005Shi et al., 2004) it is known that histones, but also other proteins (Huang et al., 2007a;Nicholson and Chen, 2009), are also subject to active enzymatic demethylation (Agger et al., 2008). Reversible histone methylation has been shown to be involved in gene regulation and hence is interesting as a target for therapeutic intervention (Shi, 2007;Yoshimi and Kurokawa, 2011). Very rapidly inhibitors of these enzymes were identified and already show promise for drug development (Lohse et al., 2011a;Spannhoff et al., 2009a). Here, we present an overview over the different classes of histone demethylases, their biochemistry, selected evidence for their role in oncogenesis and inhibitor studies. Reversible histone methylationMethylation of histones occurs posttranslationally both on lysines as well as arginines (Trievel, 2004). Methyltransferases use the cofactor S-adenosyl methionine (SAM) to transfer a methyl group onto the basic side chains of these amino acids within proteins.

Lysine demethylases play an important role in epigenetic regulation and thus in the development of diseases like cancer or neurodegenerative disorders. As the lysine specific demethylase 1 (LSD1/KDM1) has been strongly connected to androgen and estrogen dependent gene expression, it serves as a promising target for the therapy of hormone dependent cancer. Here, we report on the discovery of new small molecule inhibitors of LSD1 containing a propargylamine warhead, starting out from lysine containing substrate analogues. Based on these substrate mimicking inhibitors we were able to increase potency by a combination of similarity-based virtual screening and subsequent synthetic optimization resulting in more druglike LSD1 inhibitors that lead to histone hypermethylation in breast cancer cells.

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Protein kinases play crucial roles in several cell transformation processes and are validated drug targets for many human diseases, including cancer. Nevertheless, most tumors have eluded the effects of inhibition of a single kinase by activating resistance mechanisms and/or alternative pathways and escape mechanisms. In recent years, multitarget approaches directed toward inhibition of kinases and targets of different families have received increasing attention. In particular, co-targeting kinases and bromodomain epigenetic reader proteins has rapidly emerged as a promising approach to cancer drug development. In this manuscript, we will review the recent discoveries that led to the identification and optimization of dual kinase/bromodomain inhibitors. We will analyze and compare the structural features required for dual inhibition and comment on the potential of this approach in anticancer drug discovery. Moreover, we will introduce computational approaches useful for the identification of dual kinase/bromodomain inhibitors and generate ad hoc pharmacophore and docking models.

Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.