Targeted covalent inhibitors (TCIs) are designed to bind poorly conserved amino acids by means of reactive groups, the so-called warheads. Currently, targeting noncatalytic cysteine residues with acrylamides and other α,β-unsaturated carbonyl compounds is the predominant strategy in TCI development. The recent ascent of covalent drugs has stimulated considerable efforts to characterize alternative warheads for the covalent-reversible and irreversible engagement of noncatalytic cysteine residues as well as other amino acids. This Perspective article provides an overview of warheadsbeyond α,β-unsaturated amidesrecently used in the design of targeted covalent ligands. Promising reactive groups that have not yet demonstrated their utility in TCI development are also highlighted. Special emphasis is placed on the discussion of reactivity and of case studies illustrating applications in medicinal chemistry and chemical biology.

The anti-inflammatory drug licofelone [ϭML3000; 2-[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] acetic acid], currently undergoing phase III trials for osteoarthritis, inhibits the prostaglandin (PG) and leukotriene biosynthetic pathway. Licofelone was reported to suppress the formation of PGE 2 in various cell-based test systems, but the underlying molecular mechanisms are not entirely clear. Here, we examined the direct interference of licofelone with enzymes participating in PGE 2 biosynthesis, that is, cyclooxygenase (COX)-1 and COX-2 as well as microsomal PGE 2 synthase (mPGES)-1. Licofelone concentration-dependently inhibited isolated COX-1 (IC 50 ϭ 0.8 M), whereas isolated COX-2 was less affected (IC 50 Ͼ 30 M). However, licofelone efficiently blocked the conversion of PGH 2 to PGE 2 mediated by mPGES-1 (IC 50 ϭ 6 M) derived from microsomes of interleukin-1␤-treated A549 cells, being about equipotent to 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid (MK-886), a well recognized mPGES-1 inhibitor. In intact interleukin-1␤-treated A549 cells, licofelone potently (IC 50 Ͻ 1 M) blocked formation of PGE 2 in response to calcimycin (A23187) plus exogenous arachidonic acid, but the concomitant generation of 6-keto PGF 1␣ , used as a biomarker for COX-2 activity, was not inhibited. We conclude that licofelone suppresses inflammatory PGE 2 formation preferentially by inhibiting mPGES-1 at concentrations that do not affect COX-2, implying an attractive and thus far unique molecular pharmacological dynamics as inhibitor of COX-1, the 5-lipoxygenase pathway, and of mPGES-1.Prostaglandins (PGs) and leukotrienes are powerful bioactive lipid mediators that are involved not only in numerous homeostatic biological functions but also in inflammation (Funk, 2001). The biosynthesis of PGs is initialized by COX isoenzymes, namely, COX-1, a constitutively expressed enzyme in numerous cell types thought to provide PGs mainly for physiological functions; and COX-2, an inducible isoform in inflammatory cells, primarily producing PGs relevant for inflammation, fever, and pain (Hawkey, 1999). After conversion of arachidonic acid to PGH 2 by COX enzymes, PGH 2 is subsequently isomerized by three different PGE 2 synthases to PGE 2 . Whereas the cytosolic PGE 2 synthase (cPGES) and the membrane-bound PGE 2 synthase (mPGES)-2 are constitutive enzymes, the mPGES-1 is an inducible isoform (Samuelsson et al., 2007). Cotransfection experiments of COX-1/2 with PGES isoenzymes imply that select molecular interactions between COX and PGES isoenzymes cause preferential functional coupling (Murakami et al., 2000;Samuelsson et al., 2007). Thus, cPGES uses PGH 2 produced by COX-1, whereas mPGES-1 receives PGH 2 from COX-2. PGE 2 plays a major role in the pathophysiology of inflammation, pain, and pyresis, but it also regulates physiological functions in the gastrointestinal tract, the kidney, and in the immune and nervous system (Smith, 1989). The nonsteroidal anti-inflamm...

Drugs that function through covalent bond formation represent a considerable fraction of our repository of effective medicines but safety concerns and the complexity of developing covalent inhibitors has rendered covalent targeting a less attractive strategy for rational drug design. The recent approval of four covalent kinase inhibitors and the development of highly potent covalent kinase probes with exceptional selectivity has raised significant interest in industry and academic research and validated the concept of covalent kinase targeting for clinical applications. The abundance of cysteines at diverse positions in and around the kinase active site suggests that a large fraction of kinases can be targeted by covalent inhibitors. Herein, we review recent developments of this rapidly growing area in kinase drug development and highlight the unique opportunities and challenges of this strategy.

In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA-mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14-dependent activation of Mek-Erk and Atf2 signaling. Elevated Mapk14-Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p-Mapk14-expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC.

Previous studies have shown that activation of p38 mitogen-activating kinase (MAPK) in spinal microglia participates in the generation of inflammatory and neuropathic pain in various rodent models. However, these studies focused on male mice to avoid confounding effects of the estrous cycle of females. Recent studies have shown that some spinal pro-inflammatory signaling such as Toll-like receptor 4-mediated signaling contributes to pain hypersensitivity only in male mice. In this study we investigated the distinct role of spinal p38 in inflammatory and neuropathic pain using a highly selective p38 inhibitor skepinone. Intrathecal injection of skepinone prevented formalin induced inflammatory pain in male but not female mice. Furthermore, intrathecal skepinone reduced chronic constriction injury (CCI) induced neuropathic pain (mechanical allodynia) in male mice on CCI-day 7 but not CCI-day 21. This male-dependent inhibition of neuropathic pain also occurred in rats following intrathecal skepinone. Nerve injury induced spinal p38 activation (phosphorylation) in CX3CR1-GFP+ microglia on CCI-day 7, and this activation was more prominent in male mice. In contrast, CCI induced comparable microgliosis and expression of the microglial markers CX3CR1 and IBA-1 in both sexes. Notably, intraperitoneal or local perineural administration of skepinone inhibited CCI-induced mechanical allodynia in both sexes of mice. Finally, skepinone only reduced the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in lamina IIo neurons of spinal cord slices of males 7 days post CCI. Therefore, the sex-specific p38 activation and signaling is confined to the spinal cord in inflammatory and neuropathic pain conditions.

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Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)-in a strip format-that generate force under auxotonic stretch conditions. 10-15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.

MYC oncoproteins are involved in the genesis and maintenance of the majority of human tumors but are considered undruggable. By using a direct in vivo shRNA screen, we show that liver cancer cells that have mutations in the gene encoding the tumor suppressor protein p53 (Trp53 in mice and TP53 in humans) and that are driven by the oncoprotein NRAS become addicted to MYC stabilization via a mechanism mediated by aurora kinase A (AURKA). This MYC stabilization enables the tumor cells to overcome a latent G2/M cell cycle arrest that is mediated by AURKA and the tumor suppressor protein p19(ARF). MYC directly binds to AURKA, and inhibition of this protein-protein interaction by conformation-changing AURKA inhibitors results in subsequent MYC degradation and cell death. These conformation-changing AURKA inhibitors, with one of them currently being tested in early clinical trials, suppressed tumor growth and prolonged survival in mice bearing Trp53-deficient, NRAS-driven MYC-expressing hepatocellular carcinomas (HCCs). TP53-mutated human HCCs revealed increased AURKA expression and a positive correlation between AURKA and MYC expression. In xenograft models, mice bearing TP53-mutated or TP53-deleted human HCCs were hypersensitive to treatment with conformation-changing AURKA inhibitors, thus suggesting a therapeutic strategy for this subgroup of human HCCs.