Novel inhibitors of the calcineurin/NFATc hub - alternatives to CsA and FK506?

Cell Communication and Signaling Novel inhibitors of the calcineurin/NFATc hub - alternatives to CsA and FK506? Matthias Sieber and Ria Baumgrass* 0 Address: Deutsches Rheuma-Forschungszentrum Berlin , Chariteplatz 1, D-10117 Berlin , Germany The drugs cyclosporine A (CsA) and tacrolimus (FK506) revolutionized organ transplantation. Both compounds are still widely used in the clinic as well as for basic research, even though they have dramatic side effects and modulate other pathways than calcineurin-NFATc, too. To answer the major open question - whether the adverse side effects are secondary to the actions of the drugs on the calcineurin-NFATc pathway - alternative inhibitors were developed. Ideal inhibitors should discriminate between the inhibition of (i) calcineurin and peptidyl-prolyl cis-trans isomerases (PPIases; the matchmaker proteins of CsA and FK506), (ii) calcineurin and the other Ser/Thr protein phosphatases, and (iii) NFATc and other transcription factors. In this review we summarize the current knowledge about novel inhibitors, synthesized or identified in the last decades, and focus on their mode of action, specificity, and biological effects. - Background The calcium-dependent serine/threonine (Ser/Thr) protein phosphatase calcineurin, discovered more than 30 years ago [1], is a key factor of a multitude of cell signalling processes, in particular in immune, neuronal and muscle cells. Targeting the phosphatase activity of calcineurin has revolutionized clinical transplantation. Calcineurin represents a hub of antigen specific T cell activation and differentiation (Figure 1). Inhibition of calcineurin totally blocks the adaptive immune response. Therefore, calcineurin is considered as "Achilles' heel of the immune system" [2]. regulatory subunit B. The subunit A contains a calmodulin binding site and an autoinhibitory domain, which blocks the catalytic centre of the enzyme. Binding of Ca2+ ions and calmodulin to calcineurin leads to a change of conformation and a subsequent unmasking of the active centre. Thereby, calcineurin activity is coupled to cytosolic calcium levels, which is a unique property of calcineurin among the Ser/Thr protein phosphatases [3]. Additionally, activity and localization of calcineurin is modulated by endogenous proteins, such as RCANs, Cabin1 or AKAP79. These regulatory proteins have been recently reviewed in detail [4,5]. The Ser/Thr phosphatase calcineurin Calcineurin, also named protein phosphatase 2B (PP2B), is a ubiquitously expressed cytosolic Ser/Thr protein phosphatase, highly conserved in eukaryotes. Calcineurin consists of two subunits - the enzymatic subunit A and the Calcineurin and NFATc Calcineurin has the ability to dephosphorylate a broad range of proteins [6]. Some of the most important substrates are the transcription factors of the NFATc (nuclear factor of activated T cells) family: NFATc1 to NFATc4 [7]. SFwciaghyeusmreat1ic overview about TCR-dependent signalling pathSchematic overview about TCR-dependent signalling pathways. Engagement of TCRs and costimulatory CD28 receptors promote signalling cascades of kinases and adaptor proteins (yellow). They trigger pathways resulting in the activation of the transcription factors NFATc (red), NF-B (green) and AP-1 (blue). These transcription factors cooperate with each other during the activation of several genes, e.g. IL-2. Of special interest for this review is the calcineurinNFATc pathway: IP3, generated by PLC (orange) via cleavage of PIP2, binds to the IP3 receptor (IP3R) and causes the release of Ca2+ from the endoplasmatic reticulum (ER). This Ca2+ depletion is sensed by STIM1, which is directly coupled to the ORAI CRAC channels. Influx of extracellular Ca2+ into the cytosol activates calcineurin (CaN), leading to the dephosphorylation and nuclear translocation of NFATc. DAG: diacylglycerol; IP3: inositol-1,4,5-trisphosphate; PIP2: phosphatidylinositol-4,5-bisphosphate; PKC: protein kinase C theta; PLC: phospholipase C gamma. NFATc regulates the expression of many genes by binding to DNA as dimers or in cooperation with other transcription factors. Among the regulated genes are cytokines such as IL-2, IL-4 and IFN or surface proteins such as CD40L and CD95L [8-10]. NFATc controls the expression of the endogenous calcineurin inhibitory protein RCAN1-4, thereby forming a negative feedback loop for the calcineurin-NFATc signalling [11]. NFATc at 13 serine residues in the regulatory region [15], leading to its nuclear translocation by exposure of the nuclear localization sequences [16,17]. Concerted rephosphorylation of NFATc leads to its translocation into cytosol and abrogation of NFATc transcriptional activity [18,19]. NFATc is not only dephosphorylated by calcineurin but additionally interacts with calcineurin via two motifs binding at regions distinct from the catalytic centre of calcineurin. These motifs are named calcineurin binding region (CNBR)1 and CNBR2 or PxIxIT and LxVP according to their consensus sequences, respectively [20]. The PxIxIT region of NFATc binds even to inactive calcineurin and is responsible for basal NFATc-calcineurin interaction [21,22]. The LxVP motif interacts just with activated calcineurin, because its binding site at calcineurin is masked by the autoinhibitory domain [23,24]. Interaction of both NFATc motifs with calcineurin directs the regulatory region of NFATc into close vicinity to the active centre of calcineurin. This enables targeted dephosphorylation of specific NFATc serine residues by calcineurin. The PxIxIT calcineurin-binding motif of NFATc is shared by several other peptides and proteins binding to calcineurin. This motif may serve as a general calcineurin interface [25,26]. Calcineurin not only modulates the activity of NFATc but also several other transcription factors such as NF-B, AP1, and Elk1 [27-33]. In addition, calcineurin interferes with other signalling pathways such as TGF--dependent signalling and the MAPK cascade [33,34]. However, it is widely unknown, which components of these pathways are substrates or interaction partners of calcineurin and to which extent their dephosphorylation modulates the respective signalling. In summary, calcineurin is unique in three aspects. First, it is the only Ca2+-dependent Ser/Thr protein phosphatase. Second, to date, only calcineurin is known to activate the NFATc transcription factors thereby controlling the expression of a broad range of genes. Third, the inhibition of calcineurin activity is so far the only effective therapeutic strategy to suppress the activation of memory CD4+ and CD8+ T cells [35,36]. The "classical" drugs targeting calcineurin activity and subsequently inhibiting NFATc activation are cyclosporin A (CsA) and FK506 (tacrolimus). Inhibitors of calcineurin are indispensable for treatment of transplantation patients and represent a valuable tool for basic research. Calcineurin seems to be the only protein phosphatase that dephosph (...truncated)