Insight into the therapeutic aspects of ‘Zeta-Chain Associated Protein Kinase 70 kDa’ inhibitors: A review

Cellular Signalling 26 (2014) 2481–2492

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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig

Review

Insight into the therapeutic aspects of ‘Zeta-Chain Associated Protein Kinase 70 kDa’ inhibitors: A review Maninder Kaur, Manjinder Singh, Om Silakari ⁎ Molecular Modeling Lab (MML), Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab 147002, India

a r t i c l e

i n f o

Article history: Received 6 June 2014 Accepted 27 June 2014 Available online 15 July 2014 Keywords: Zeta-Chain Associated Protein Kinase 70 kDa T-cells Spleen tyrosine kinase Chronic lymphocytic leukemia

a b s t r a c t Zeta-Chain Associated Protein Kinase 70 kDa (ZAP-70), a member of Syk family (non-receptor protein tyrosine kinase family), has an imperative function in the immune cell signaling in T cells. Its role in T-cell development has been established by the severe combined immune deficiency syndrome in ZAP-70 deficient humans. Moreover, defects in T-cell activation and downstream signaling events were observed in T-cells that lack ZAP-70. Thus, the crucial role of ZAP-70 in the development and activation of T-cell and its predominant expression in T-cells make it a logical target for the treatment of pathological conditions related to abnormal T-cell responses. The present review article portrays the domain structure of ZAP-70 along with its implication in T-cell signaling. Additionally, varied ZAP-70 inhibitors published in different patents and papers have also been reviewed. © 2014 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zeta-Chain Associated Protein Kinase 70 kDa (ZAP-70) . . . . . . . . . . . . . . . 2.1. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Domain structure . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Three dimensional structure . . . . . . . . . . . . . . . . . . . . 2.2. Role of ZAP-70 in T-cell signaling . . . . . . . . . . . . . . . . . . . . . . 2.3. Therapeutic benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Severe combined immune deficiency (SCID) . . . . . . . . . . . . 2.3.2. Chronic lymphocytic leukemia (CLL) . . . . . . . . . . . . . . . . 2.4. ZAP-70 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. 4-Pyridin-5-yl-2-(3,4,5-trimethoxyphenylamino)pyrimidine derivatives 2.4.2. Imidazo[1,2-c]pyrimidine derivatives . . . . . . . . . . . . . . . . 2.4.3. 5-Benzylaminoimidazo[1,2-c]pyrimidine-8-carboxamide derivatives . 2.4.4. 1,2,4-Oxadiazole derivatives . . . . . . . . . . . . . . . . . . . . 2.5. Patent information of ZAP-70 inhibitors . . . . . . . . . . . . . . . . . . . 2.6. Assay methods for ZAP-70 inhibitory activity . . . . . . . . . . . . . . . . 3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction T-cells acting as central players in the adaptive immune system have five major types i.e. helper T-cell, cytotoxic T-cells, memory T-cells, ⁎ Corresponding author. Tel.: +91 9501542696. E-mail address: [email protected] (O. Silakari).

http://dx.doi.org/10.1016/j.cellsig.2014.06.017 0898-6568/© 2014 Elsevier Inc. All rights reserved.

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regulatory T-cells and natural killer T-cells [1]. Helper T-cells, as the name itself indicates, aid other white blood cells (WBC) in different immunological processes such as cytotoxic T-cells and macrophage activation and maturation of B-cells to memory cells and plasma cells etc. Cytotoxic T-cells in contrast have a specific function to destroy virally infected cells or tumor cells. They have also been involved in organ transplant rejection. Memory T-cells are responsible for developing

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memory against a specific kind of antigens whereas regulatory T-cells maintain immunological tolerance. All the subtypes of T-cells collectively functions to protect the host from different pathogenic attacks. Balanced immune response of the T-cells is crucial, as their augmented response may lead to autoimmune disease condition whereas diminished response may lead to infection and death [2,3]. Thus, T-cells should act in a precise manner in each situation. A large number of reports concerning their activation and molecular level signaling have been published in literature in past few years. In this review article, ZAP-70 is described at three aspects: structure, role in T-cell signaling and its small molecule inhibitors. T-lymphocytes are activated via integration of antigen bound Major Histocompatibility complex (MHC) embedded on the Antigen presenting cell (APC) with T-cell receptors (TCRs) present on the T-cells [4]. The TCR complex acts as a distinguishing factor that discriminates T-cells from other lymphocytes. TCR complex consists of six different polypeptide chains including α, β, γ, δ, ε and ζ chains having varied functions in the T-cell signaling. α and β chains are responsible for the binding directly with the MHC complex whereas γ, δ, ε and ζ chains, collectively forming CD3 complex, are engaged in the binding of the intracellular molecules implicated in T-cell signaling [5,6]. Followed by the activation of T-cell, the principle step i.e. the protein tyrosine phosphorylation is initiated by Lck kinase that phosphorylated the ITAMs [7]. Dually phosphorylated ITAMs encourage the migration of ZAP-70 that is subsequently stimulated by Lck. Subsequently, ZAP-70's active form causes the phosphorylation of trimeric complex of LAT/SLP-76/GAD via ITK phosphorylation [8–10]. This step is followed by the activation of PLCγ that commences two downstream pathways i.e. IP3 and DAG pathways [11]. The tyrosine phosphorylation cascade will eventually activate numerous transcription factors like NFAT and AP-1 which finally leads to the transcription of the new genes required for T-cell response [12]. As protein tyrosine phosphorylation is such a crucial step in T-cell signaling it should be monitored carefully. Thus, tyrosine kinases being the key players in immune signaling can be targeted in the development of therapies to treat various T-cell mediated diseases [13,14]. Among the contributor kinases, ZAP-70 being the upstream signaling kinase performs a master part in the T-cell activation [15]. Mutational studies of ZAP-70 reported the development of a familial type of severe combined immunodeficiency in humans defined by a selective loss of ability to develop CD8+ T cells along with a signal transduction defect in peripheral CD4 + T cells [16–19]. Additionally, mice with altered ZAP-70 gene showed abnormal activation of T-cells along with defective thymic development [20]. Given the vital implication of ZAP-70 in T cell activation, it is currently the subject of interest for pharmacological research as it may be important in both switching on and off immunoreceptor induced signaling events. 2. Zeta-Chain Associated Protein Kinase 70 kDa (ZAP-70) ZAP-70 is a 70 kDa tyrosine kinase that belongs to Syk family kinase and has been linked with the T cell receptor via its zeta-subunit. It has been identified in 1991 by Chan et al. [21]. It is found to be expressed predominantly in T-cells and natural killer (NK) cells and has been reported to play a significant role in the instigation of normal signaling, activation and development of T-cells [22]. 2.1. Structure 2.1.1. Domain structure The domain structure of ZAP-70 comprises of three domains that include two Src homology (SH2) domains present at the N-terminal and a kinase domain at the C-terminal. Additionally, two interdomains A and B are at hand that tie two SH2 domains, and SH2 domain and kinase domain respectively [23]. These five segments are collectively composed of 619 amino acids. The composition of individual segment and their organization in domain structure has been shown in Fig. 1.

SH2 domains of ZAP-70 are liable for recruiting it to the diphosphorylated immunoreceptor tyrosine-based activating motifs (ITAMs) located on the CD3 and ζ chain dimers of T cell receptors. Such interaction of ZAP-70 with ITAMs is required for the phosphorylation and activation of ZAP-70 [24,25]. It has been evidenced that conformational changes that occur in SH2 domains while their binding to ITAMs affects the activation/inhibition of kinase domain. This fact has also been supported by the NMR analysis that revealed the rigid nature of interdomain A (SH2 domain linker) when SH2 is bound to ITAMs whereas it was flexible in SH2 unbound state [26]. The successive interdomain B of ZAP-70 has been imperative for its association with other signaling molecules like c-Cbl, Vav, Lck, CrkII and PLCγ [23]. It contains three tyrosine residues i.e. Tyr292, Tyr315 and Tyr319 that are phosphorylated by Lck followed by the recruitment of other signaling molecules (Cbl, Vav, PLCγ, Lck) to the activated TCR complex. Among these tyrosine residues, Tyr292 was found to be a negative regulator whereas Tyr315 and Tyr319 were found to be positive regulators of ZAP-70 activity by mutational analysis. The negative role of Y292 was experimentally demonstrated by impaired TCR internalization and increased IL-2 and IFN-γ production in Y292F mutant of ZAP-70. On the contrary, a positive role of Tyr315 was evidenced by the reduced NFAT activity in a Syk-deficient B-cell line having Y315F mutant of ZAP-70 whereas erratic PLCγ phosphorylation, calcium mobilization, Ras activation and IL-2 production were evident in ZAP-70 Y319F mutant suggesting a positive role of Tyr319 [27–38]. Interdomain B is followed by the kinase domain or catalytic domain. It contains two tyrosine residues i.e. Tyr492 and Tyr493 implicated as negative and positive regulators of kinase activity of ZAP-70 respectively [39,40]. These tyrosine residues are either phosphorylated by Lck or by autophosphorylation. Conversely, it was found in the crystal structure of ZAP-70, activation loop can acquire an active conformation even without the phosphorylation of Tyr492 and Tyr493 [41]. Thus, some other mechanisms may possibly be involved in the regulation of catalytic activity. 2.1.2. Three dimensional structure The significant involvement of ZAP-70 in T-cell activation and immune response makes it a rational target for the immunomodulatory therapies. Several numbers of reports have been published in the literature revealing the structural organization of ZAP-70. The crystal structure of tandem SH2 domains of ZAP-70 in complex with a peptide derived from the ζ-subunit of the TCR has been determined by Hatada et al. at 1.9 Å resolution [42]. The two SH2 domains consist of first 259 residues of ZAP-70 connected by a coiled coil of α-helices. The SH2-N terminal domain and SH2-C terminal domain form upper branches of Y-geometry and the intervening 65 residues form the stem of Y-shaped geometry. Both SH2 domains have a central antiparallel β sheet flanked by two α-helices. The linker-SH2 region commences as a β strand followed by a coiled coil of two antiparallel α-helices that forms the stem of overall Y-shape. ζ-Chain establishes contact with both N terminal and C-terminal SH2 domains with head to tail binding orientation i.e. the amino terminus of the peptide is in contact with C-terminal SH2 domain. Binding of phosphorylated peptides to SH2 domain has been explained as socket and plug conformation, where phosphotyrosine (pY) and the pY + 3 residues are prongs of the plug. The C-terminal phosphotyrosine binds to the pocket formed by both SH2 domains. Folmer et al. in 2002, carried out X-ray crystal structure and NMR studies of the apo-SH2 domains, tSH2, in the absence of ITAM [26]. The authors compared the previously reported structure of SH2 domains in complex with ITAM and tSH2 domains without ITAM. It was ascertained that the secondary elements of both domains were positioned in a similar manner as compared to the published structure of ITAM complexed with SH2 domains. On the contrary, the positioning of the two domains themselves was found to be different. The Y-shaped geometry was changed to L-shape as the central

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Fig. 1. Domain structure of ZAP-70 kinase.

β-sheet of C-terminal SH2 domain rotated 50° forming an angle of 110° with the central β-sheet of N-terminal SH2 domain. The interSH2 domain that formed a coiled coil became unwound, with residues 155–157 being stretched to a random-coil conformation. The exhaustive crystallographic study revealed that helical region linking two SH2 domains was found more flexible in the absence of ITAM. It has also been evidenced in the NMR data as no signal was observed for the helical region owing to broadened peaks due to conformational exchange. Only upon the binding to ITAM, helical region was stabilized into rigid conformation and possibly this conformational change activates the kinase domain in ZAP-70.

In 2004, Jin et al. solved the X-ray crystal structure of catalytic subunit (327–606 amino acids) of ZAP-70 as a complex with staurosporine at 2.3 Å resolution [43]. The staurosporine binds in such an orientation that NH and carbonyl ‘O’ of the lactam ring of staurosporine form hydrogen bonding interactions with Glu415 and Ala417 respectively. The methylamino nitrogen of glycosidic ring constitutes a hydrogen bond with the carbonyl of Arg465 of the catalytic loop similar to ribose moiety of ATP. One indole ring resides in the gatekeeper pocket whereas the other indole ring was found to fill the lipophilic pocket. The glycosidic oxygen forms a CH–O interaction with Cα of Gly345. Additionally indolylcarbazole moiety was found to display prevalent van der Waals

Fig. 2. The ligand-interaction diagram of staurosporine complexed with kinase domain of ZAP-70.

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interactions with Leu344, Gly345, Val352, Ala367, Lys369, Val399, Met414, Glu415 and Met416 from N-terminal lobe and Ala417, Gly420, Pro421, Arg465, Asn466, Leu468, Ser478 and Asp479 from C-terminal lobe (Fig. 2). This information about intermolecular interactions of this complex can be further explored for the designing of new ZAP-70 inhibitors resembling staurosporine. 2.2. Role of ZAP-70 in T-cell signaling In the resting state of T-cells, ZAP-70 is distributed throughout the cytoplasm which upon the activation of T-cells by antigens and ITAM phosphorylation gets recruited to the membrane. The mobility of ZAP-70 was rapid initially but decreases as it gets associated with the plasma membrane. The recruitment of ZAP-70 has been reported to be regulated by Lck activity and phosphorylation of ITAM (Fig. 3). When the active APCs consisting of antigen bounded to MHC (I or II) interacts with TCRs, it triggers the TCRs and starts the T-cell signaling cascade. The interaction of APC with T-cell receptors dephosphorylates the inhibitory amino acid residue tyrosine 505 of Lck, bound to CD4+ and CD8+, which activates ZAP-70 kinase. The activated Lck migrates towards TCR/CD3 complex, which further phosphorylates the ITAMs

of the TCR/CD3 complex and this phosphorylation encourages the recruitment of ZAP-70. The tandem SH2 domains of the ZAP-70 interact with the doubly phosphorylated ITAMs in such a position that it releases autoinhibited state of ZAP-70 by exposing tyrosines (Tyr315 and Tyr319) that are involved in aromatic–aromatic interactions to connect the SH2 linker to the kinase region, for phosphorylation by Lck. Subsequently, tyrosines (Tyr492 and Tyr 493) in the activation loop of the ZAP-70 kinase domain are phosphorylated either by Lck or by ZAP-70 itself to further promote its catalytic activity. The activated ZAP-70, in turn phosphorylates a transmembrane protein linker of activated T-cells (LAT) that acts as a linker between initial TCR signal and the downstream events of T-cell signaling cascade [44–46]. Activated LAT binds to the SH2 domain containing leukocyte protein of 76 kDa (SLP-76) via GRB2-related adapter protein-2 (GADS) constituting a trimeric complex SLP-76/GADS/LAT which (complex) is further phosphorylated and activated by ZAP-70 [47]. Thus, in addition to its catalytic activity ZAP-70 can also act as rostrum for recruiting further signaling molecules to activated TCR complex. The activated trimeric complex SLP-76/GADS/LAT alleviates the migration of auto-inhibited ITK from the cytoplasm to the membrane where it binds through its PH domain. Subsequently, the interaction of ITK with SLP-76/GADS/ZAP-70

Fig. 3. ZAP-70 regulated T-cell signaling: TCR — T-cell receptor; ZAP-70 — Zeta-Chain Associated Protein Kinase 70 kDa; Lck — lymphocyte specific protein tyrosine kinase; ITAMs — intracellular tyrosine activation motifs; LAT — linker of activated T-cells; GADS; SLP76 — SH2 domain containing leukocyte phosphoprotein of 76 kDa; ITK— interleukin-II inducible T-cell kinase; PLCγ1 — Phospholipase C γ1; PIP2 — phosphatidyl inositol 4,5-biphosphate; IP3 — inositol triphosphate; DAG — diacyl glycerol; NFAT — nuclear factor of activated T-cells; PKC-θ — protein kinase C theta; IKK — IκB kinase; RasGRP — Ras guanyl releasing protein; JNK — jun-N-terminal kinase; c-Jun; c-Fos; AP-1 — activating protein-1.

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complex completely activates ITK by the trans-phosphorylation of tyrosine (Y511; present in the activation loop of ITK) by Lck enzyme and auto-phosphorylation of tyrosine 180 residue in SH3 domain by ITK itself [48]. The activated ITK then interacts with the SH2 domain of PLCγ1 lipase and activates the lipase by phosphorylating tyrosine 775 and 783 amino acid residues [49–51]. Activated PLCγ1 subsequently hydrolyzes the membrane bound PIP2 into two secondary messengers: inositol triphosphate (IP3) and diacyl glycerol (DAG) that prompts two different pathways i.e. IP3 pathway and DAG pathway respectively. IP3 mediated pathway: IP3 is a small polar molecule that is released into the cytosol, where it directs the release of Ca(II) from intracellular stores. IP3 accumulates rapidly and transiently, and subsequently binds to its intracellular receptor, IP3R, located in the endoplasmic reticulum mobilizing Ca(II) from internal stores [52]. The cytoplasmic calcium activates calmodulin. The activated Ca(II)/calmodulin complex further binds to and stimulates calcium dependent serine threonine phosphatase, i.e. calcineurin. Activated calcineurin activates cytoplasmic NFAT (a transcription factor) via dephosphorylation. The activated cNFAT then translocates into the nucleus and governs the expression of pro-inflammatory cytokines such as interleukin 2 (IL-2) that ultimately promotes the proliferation and differentiation of T-cells [53]. DAG mediated pathway: DAG, on the other hand, triggers Protein kinase C-θ (PKC-θ), to stimulate the inhibitor of kappa B kinase (IKK) induced release of activated NF-κB that translocates to the nucleus to instigate a transcriptional role leading to growth and differentiation of T-cells [54]. DAG also initiates mitogen-activated protein kinases 1 and 2 (MEK1 and MEK2)/Mitogen-activated protein kinases 3 and 1 (ERK1 and ERK2) cascade that, in turn, activates several transcription factors including AP-1 that plays a vital role in transcription, ultimately leading to immune response (T-cell proliferation and differentiation) [55,56]. 2.3. Therapeutic benefits 2.3.1. Severe combined immune deficiency (SCID) ZAP70 deficiency is a kind of rare autosomal recessive form of severe combined immune deficiency (SCID) that is characterized by deficit of CD8+ T cells along with a regular number of circulating CD4+ T cells that are though unresponsive to T-cell-receptor (TCR) mediated stimulation in vitro [57]. Loss of function or expression of ZAP70 leads to an unusual form of SCID in humans, revealing the critical role of human ZAP70 in both mature T-cell signaling and differentiation of thymic precursors. Thus far, the ZAP70 mutations occurring in human SCID have resided mostly in the kinase domain, although the loss of transcription and one mutation in the N-terminal SH2 domain that resulted in a rapid degradation of ZAP70 protein has also been reported [23,58–61]. This rare autosomal recessive form of SCID indicates the absolute requirement of ZAP70 for the development of CD8+ T cells. The importance of ZAP70 in thymocyte development was further demonstrated in an animal model where mice developed rheumatoid arthritis (RA), resembling human RA, caused by a point mutation in ZAP70. As a result of this mutation, an altered signal transduction from the TCR causes a change in thymic selection leading to a positive selection of autoimmune T cells that should otherwise be negatively selected against [62,63]. Altogether, reports on human ZAP70 immunodeficiency have confirmed the crucial role of ZAP70 in TCR signaling during thymocyte development and in peripheral T-cell function. 2.3.2. Chronic lymphocytic leukemia (CLL) The protein tyrosine kinase ZAP70, normally expressed in T cells and a subset of B cells, is solely expressed in poor prognosis chronic lymphocytic leukemia and implicated in enhanced B cell receptor signaling [64]. As a result, the expression of this protein provides an ideal prognostic marker for chronic lymphocytic leukemia. ZAP70 was first recognized as a potential surrogate for IgVH mutational status as a result of gene

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expression profiling with cDNA microarrays comparing the two subtypes of CLL. Out of the 240 genes found to be differentially expressed between the two types, ZAP70 was identified as the most prominent among these genes with expression levels 5-fold higher in patients with unmutated IgVH genes as in those with mutated genes [63,65]. 2.4. ZAP-70 inhibitors A number of ZAP-70 inhibitors have been compiled in the literature, having diverse chemical structures that can be classified as 4-pyridin-5-yl-2-(3,4,5-trimethoxyphenylamino)pyrimidine, imidazo[1,2-c]pyrimidine, 5-benzylaminoimidazo[1,2-c]pyrimidine-8carboxamide, and 1,2,4-oxadiazole derivatives on the basis of their parent scaffolds. 2.4.1. 4-Pyridin-5-yl-2-(3,4,5-trimethoxyphenylamino)pyrimidine derivatives Since there was no specific ZAP-70 inhibitor till 1999, Moffat et al. provided lead structures by designing the compounds with known inhibitor scaffolds, deduced from ATP competitive kinase inhibitors. This led to the identification of a series of 2-phenylaminopyrimidines as potent and selective inhibitors of ZAP-70 [66]. The compound 1 (Table 1) a 2-phenylaminopyrimidine derivative was considered as a lead compound for optimizing ZAP-70 inhibitory activity. Introduction of the 2-aminoethylamino group at the 2nd position of the pyridine ring resulted in a 10 fold increased inhibitory activity (2; Table 1). Further increase in the potency was observed by incorporating piperazine ring at the 2nd position of pyridine (3; Table 1). A small group like methyl substituted at piperazine ring yielded a compound with increased IC50 of 46 nM (4; Table 1) whereas large substituents like ethyl resulted in decreased IC50 of 4300 nM (5; Table 1). The charged ammonium species in all the above compounds was found to be important as it occupies cation binding site (Fig. 4). It can be evidenced from decreased activity of morpholino substituted pyridine derivatives (6; Table 1). Additionally substitution of methyl at piperazine ring results in increased potency and its S conformer was found to be more potent as compared to its R conformer (7, 8; Table 1). Most active compound of the series was obtained by incorporating ethyl substituted piperazine ring with IC50 of 8 nM (9; Table 1). It also showed excellent selectivity over PKC, Lck, EGFr and csk. 2.4.2. Imidazo[1,2-c]pyrimidine derivatives In 2008, Hirabayashi et al. developed a series of imidazo[1,2-c] pyrimidine derivatives as ZAP-70 inhibitors in order to improve oral effectiveness of the inhibitors as compared to initially developed 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-c]pyrimidine derivatives [67]. Among all the synthesized compounds, compound 10 (Table 1) showed a strong ZAP-70 inhibitory activity in addition to invivo curtailment of passive cutaneous anaphylaxis reaction and IL-2 production in mouse model. The substitutions were done at the 5th position of imidazo[1,2-c]pyrimidine skeleton and aniline groups at the 7th position of the imidazo[1,2-c]pyrimidine skeleton. The amino and 3-aminopropylamino groups at the 5th position of the basic scaffold give the derivatives (11, 12; Table 1) with weak ZAP-70 inhibitory activity whereas substitution of hydroxyethylamino and 2aminoethylamino groups (13, 14; Table 1) resulted in improved ZAP-70 inhibitory activity. Several substituted cyclohexylamino groups were also analyzed for their effect on the ZAP-70 inhibitory activity as they have an ability to fit in the sugar pocket. The 2-methylcyclohexylamino derivative (15; Table 1) showed a moderate inhibitory activity against ZAP-70 whereas the 2-hydroxycyclohexylamino derivative (16; Table 1) exhibited a good ZAP-70 inhibitory activity. Interestingly, in the case of cyclohexyldiamino derivatives, the trans-cyclohexyldiamino derivative (17; Table 1) completely lost the inhibitory activity whereas the ciscyclohexyldiamino derivative (10) exhibited an excellent activity. The above SAR depicts that amino group and its R configuration in the

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Table 1 Molecular structures and biological activity values of different classes of ZAP-70 inhibitors. Compound no.

Structure

1.9

ZAP70-2

0.125

ZAP70-4

IC50 (μM)

Imidazo[1,2-c]pyrimidine derivatives ZAP70-10

0.23

ZAP70-11

10.0

ZAP70-12

12.4

ZAP70-13

1.9

ZAP70-14

3.5

ZAP70-15

3.3

ZAP70-16

1.8

0.046

4.3

ZAP70-6

0.424

ZAP70-7

0.011

0.396

ZAP70-17

ZAP70-9

Structure

0.054

ZAP70-5

ZAP70-8

Compound no.

IC50 (μM)

4-Pyridin-5-yl-2-(3,4,5-trimethoxyphenylamino)pyrimidine derivatives ZAP70-1

ZAP70-3

Table 1 (continued)

0.008

N10

M. Kaur et al. / Cellular Signalling 26 (2014) 2481–2492 Table 1 (continued) Compound no.

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Table 1 (continued)

Structure

IC50 (μM)

ZAP70-18

1.7

ZAP70-19

4.1

Compound no.

Structure

IC50 (μM)

ZAP70-26

0.41

ZAP70-27

0.93

ZAP70-28

0.22

ZAP70-29

0.23

ZAP70-30

0.15

ZAP70-31

0.53

ZAP70-32

0.91

ZAP70-33

0.25

0.73

ZAP70-20

0.48

ZAP70-21

ZAP70-22

0.60

ZAP70-23

0.041

ZAP70-24

0.16

5-Benzylaminoimidazo[1,2-c]pyrimidine-8-carboxamide derivatives ZAP70-25

0.40

(continued on next page) (continued on next page)

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Table 1 (continued) Compound no.

Structure

Table 1 (continued) IC50 (μM)

5-Benzylaminoimidazo[1,2-c]pyrimidine-8-carboxamide derivatives ZAP70-34

0.15

ZAP70-35

0.19

ZAP70-36

0.18

ZAP70-37

1,2,4-Oxadiazole derivatives ZAP70-38

ZAP70-39

Compound no.

Structure

IC50 (μM)

ZAP70-42

14

ZAP70-43

8

ZAP70-44

55

ZAP70-45

6

ZAP70-46

5

ZAP70-47

7

ZAP70-48

5

ZAP70-49

3

0.088

N500

65

ZAP70-40

36

ZAP70-41

94

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Table 1 (continued) Compound no.

Structure

IC50 (μM)

ZAP70-50

2

ZAP70-51

3

ZAP70-52

1

Fig. 5. The ligand interaction diagram of compound 10 of imidazo[1,2-c]pyrimidine series with ZAP-70.

cyclohexylamino ring are important for the ZAP-70 inhibitory activity. Additionally, SAR was predicted for anilino group at the 7th position. The meta- and para-substituted aniline derivatives were tested and it was observed that meta-substituted aniline derivatives were favorable as compared to para-substituted aniline derivatives for the ZAP-70 inhibitory activity (18, 19; Table 1). Further increase in the inhibitory activity was observed in the case of dimeta-substituted aniline derivatives (20, 21; Table 1). Among the disubstituted aniline derivatives, the 3,5-dimethoxyaniline derivative was found to possess the highest ZAP-70 inhibitory activity (10). Additionally, ZAP-70 inhibitory activity was augmented with the incorporation of phenyl ring at the 2nd position of imidazo[1,2-c]pyrimidine scaffold (22, 23, 24; Table 1) but they showed lower Caco-2 permeability as compared to compound 10. The above discussed SAR has been supported with the docking study of the highest active compound carried out by the authors (10). NH group and the carbonyl group of 8-carbamoyl were found to exhibit hydrogen bonding interactions with the carbonyl group of Glu415 and the NH group of Ala 417, respectively. Additionally, NH group of aniline

was found to form hydrogen bonding with the carbonyl of Ala417. The compound was positioned in a way such that the imidazole core occupies the gatekeeper residue and forms a favorable CH–π interaction with the methyl group of Val352 and 7-anilino group also forms a CH–π interaction with the methylene groups of Leu344 and Gly420 (Fig. 5). The cyclohexyldiamino group was found to reside in the sugar pocket. Moreover, the phenyl group introduced at the 2nd position gives proper fitting in the binding pocket. Thus to conclude, cyclohexyldiamino at the 5th position, phenyl ring at the 2nd position and dimethoxy substituted aniline at the 7th position of the core yielded potent ZAP-70 inhibitors. 2.4.3. 5-Benzylaminoimidazo[1,2-c]pyrimidine-8-carboxamide derivatives Impairment of the Syk gene in knockout mice leads to embryonic hemorrhage and death along with the loss of immune receptor signaling. Additionally, Syk mutation distorted the differentiation of B-cells by interrupting signaling from the pre-B cell antigen receptor complex and preventing the clonal development and mutation of pre-B cells. Thus, later in 2009, Hirabayashi et al. synthesized selective ZAP-70 inhibitors having benzylamino group substituted at the 5th position of the imidazole core [68]. Several NH linked derivatives were developed by integrating various amine units like ethylenediamino (14),

Fig. 4. The docked poses of compounds 1, 3, 4, 5 and 6 showing the importance of charged ammonium species in forming cationic binding interaction.

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Fig. 6. The ligand interaction diagram of compound 37 of 5-benzylaminoimidazo[1,2-c]pyrimidine-8-carboxamide with ZAP-70.

hydroxyethylamino (13), and benzyl (25; Table 1) at the 5th position of the basic scaffold. Among them, benzylamine substituted derivative (25) showed a maximum ZAP-70 inhibitory activity with good selectivity over closely related kinase Syk. The introduction of methyl group at benzylic carbon gives a stereospecific increment the inhibitory activity. R conformer of 26; Table 1 was found to exhibit higher activity as compared to its S conformer (27; Table 1). The benzyl ring was further optimized for improving the potency and selectivity of ZAP inhibitors. The 4-flouro (28; Table 1), 4-methoxy (29; Table 1) and 2,4-difluoro (30; Table 1) substituted analogues showed increased inhibitory activity as compared to unsubstituted benzyl derivative (25) whereas 3methoxy (31; Table 1) and 3,5-difluoro substituted derivatives (32; Table 1) exhibited a decreased ZAP-70 inhibitory activity as compared to 25. The above SAR depicts that the para-position is more favorable for substitution as compared to meta with increased ZAP-70 inhibitory activity and retained selectivity. Also the incorporation of heteroaromatic ring pyridyl at the 5th position resulted in an increased ZAP-70 inhibitory activity (33; Table 1). The para-position of the benzyl ring was further optimized by various substituents like sulphonamide, sulphone, and carboxyl groups (34, 35, 36; Table 1) resulting in an increased ZAP-70 inhibitory activity with good selectivity against Syk as compared to 25. A drastic increase in ZAP-70 inhibitory activity (IC50 = 0.088 μM) was found by substituting a π-electron unit i.e. acrylate at para-position of benzyl ring (37; Table 1) with excellent selectivity against Syk (IC50 N 10 μM). The ZAP-70 inhibitory activity and selectivity were also revealed by docking studies. The carbamoyl group of the compound 37 showed hydrogen bonding interaction with Ala417 and Glu415 whereas the methoxy group of aniline ring substituted at the 7th position displayed hydrogen bonding with Asp479 (Fig. 6). In addition to hydrogen bonding interactions, the molecule also forms a CH–π interaction between the 5-benzylamino group and Pro421. In addition, the acrylate group of the compound formed a CH–π interaction between the ethenyl group and methylene group in Arg465, and between the ethyl group of ester and guanidyl group of Arg465. These interactions were believed to be accountable for the increased ZAP-70 inhibitory activity and interactions with distinctive Pro421-Leu422-His423-Lys424 motif in ZAP-70 impart specific ZAP-70 inhibition.

2.4.4. 1,2,4-Oxadiazole derivatives ZAP-70 binds to immunoreceptor tyrosine activated motifs (ITAMs) via SH2 domains and activates various downstream pathways; thereby leading to T-cell proliferation. Thus, it can be beneficial to directly target SH2 domains from triggering the early intracellular signaling. Vu et al. in 1999 developed a series of 1,2,4-oxadiazole as nonpeptidic SH2 inhibitors of ZAP-70 starting from an initial Src SH2 lead [69,70]. The SAR was explored by substituting various groups at pY + 1, pY + 3 and Nacetyl cap (pY refers to phosphotyrosine). Initially, pY + 1 amino acid was kept as L-glutamine and different groups were explored for pY + 3 position. A compound (38; Table 1) with benzyl group at this position was a good Src SH2 inhibitor and also showed modest affinity towards ZAP-70 and Syk. It was observed that with slight increase in the size of pY + 3 substituent, affinity towards the Src decreased and affinity towards ZAP-70 increased (39,40; Table 1). The increase in ZAP-70 inhibitory activity was detected by the replacement of p-methyl benzyl with p-chloro benzyl that also retained the selectivity over Syk and Src kinases. The ZAP-70 affinity was observed to decrease in m-chloro benzyl substituted derivative (41; Table 1) whereas an increase in ZAP-70 affinity was noticed in the case of m,p-dichloro benzyl substituted derivative (42; Table 1). A considerable increase in the ZAP-70 affinity was observed when m,p-dichlorobenzyl ring was replaced with 2-naphthalene ring (43; Table 1). On the contrary, a decrease in the ZAP-70 inhibitory activity was obtained in 1-naphthalene ring substituted oxadiazole derivative (44; Table 1). The increased activity of 42 and 43 may be due to increased hydrophobic contacts as hypothesized on the basis of ZAP-70 crystal structure. The selectivity over Syk was intact throughout the series. The high selectivity of compounds was depicted due to different pY + 3 pockets. The Syk pY + 3 pocket is smaller due to the presence of Tyr 244 whereas in ZAP-70 Leu 239 is present that increases the size of pY + 3 pocket that can accommodate larger groups like naphthyl and di-substituted benzyl. The pY + 1 was explored by using various starting amino acids including L-Ala, L-Abu, L-Leu, L-Asp and L-Trp. No improvement was spectated in the affinity except L-Ala that results in the compounds with 2fold increased affinity. The similar pattern of SAR was observed in the compounds with methyl substituted at N of pY + 1 position (45, 46,

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47; Table 1). Also, L-Ser was incorporated as a starting material that showed additional interactions in molecular modeling studies. The compounds thus obtained exhibited slightly more affinity towards ZAP-70 while selectivity over Syk was retained. Molecular modeling studies also depicted that a longer hydrophobic chain can be substituted at pY + 1 position. Various analogues were developed using L-homophenylalanine as the starting amino acid and obtained compounds were slightly more potent with decreased (48, 49, 50; Table 1) Syk selectivity as compared to L-Ala or L-Ser derived compounds. Another molecular modeling study suggested that the incorporation of an aromatic group at N-terminal of tyrosine would be beneficial as it offers π–cation interactions with Arg amino acid residue. Various groups were incorporated as N-cap on several compounds derived from L-Ala and L-Ser contributing approx. The compound with N-phenyl-acetyl group showed a 2 fold increase in activity as compared to derivative with N-acetyl group (51 vs 47; Table 1). Among all these derivatives, the compound derived from L-ser amino acid and naphthyl group at N terminal was observed to possess the highest ZAP-70 binding affinity with IC50 of 1 μM (52; Table 1) and highly selective over Syk and Src kinases. Thus to conclude, the naphthyl urea and indole nucleus can be considered a chiral replacements for the tyrosine group with slightly decreased ZAP-70 affinity.

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spectrophotometric assay was employed where ADP generated by ZAP-70 kinase was converted to ATP by the action of pyruvate kinase (PK), with a simultaneous production of pyruvate from phosphoenolpyruvate (PEP). Subsequently, pyruvate was reduced to lactate by lactate dehydrogenase by oxidizing NADH and NADH depletion was observed at 340 nm using a microplate reader (Spectra Max 250, Molecular Device) at 30 °C for 20 min. 3. Conclusions ZAP-70, a zeta chain associated tyrosine kinase has a well established role in T-cell development and activation; confirmed by different mutational and deficient cell line studies. Thus, it is an attractive therapeutic target for pharmacological intervention in T-cell mediated diseases. A number of ZAP-70 inhibitors have been described for their therapeutic potential in the autoimmune diseases and organ transplant rejection in the literature. But these compounds have been limited to the enzymatic assay profiling. Despite its illustrated physiological role, not even a single ZAP-70 inhibitor has made its way to clinical trials. The development of clinically beneficial ZAP-70 inhibitors needs to be investigated for the treatment of autoimmune and organ transplant rejection.

2.5. Patent information of ZAP-70 inhibitors In 2004, Novartis Ag and Novartis Pharma GmbH filed a patent claiming the pyrimidine derivatives i.e. 2,4, di(hetero)arylaminopyrimidine as ZAP-70 inhibitors that was granted in 2011 (EP1664035A1) [71]. Later, they filed more patent applications accounting for similar pyrimidine derivatives (WO2005026158A1, US8283356B2, US7671063B2, US8431589B2) [72–75]. In 2010, Cellzome Ltd. filed a patent stating some pyrimidine derivatives as ZAP-70 inhibitors for the treatment or prophylaxis of immunological, inflammatory, autoimmune, allergic disorders, and immunologicallymediated diseases [76]. Sulfamides class was first reported as ZAP-70 inhibitors in 2008 by research group of Cellzome Ltd. in patent published in 2009 (WO2009080638A3) [77]. In 2009, another novel class of ZAP-70 inhibitors i.e. sulphonamides has been reported by Cellzome research group (WO2009112490A1) [78]. Later in 2010, another patent was filed by them stating similar chemical classes of ZAP-70 inhibitors (WO2010146132A1) [79]. 2.6. Assay methods for ZAP-70 inhibitory activity Several numbers of assay procedures have been reported in literature for measuring inhibitory activity against ZAP-70. Basically tyrosine kinases convert ATP to ADP by transferring the phosphate to the tyrosine residue. The conversion of ATP to ADP via this reaction is measured quantitatively corresponding to some measurable signal i.e. luminescence, fluorescence, spectrophotometric signal etc. In 1999, Moffat et al. synthesized a series of pyrimidine derivatives and tyrosine kinase inhibitory activity against ZAP-70 was measured by capture assay [66]. In the assay method, the reaction mixture containing inhibitor, polyGlu-Tyr and enzyme was incubated at 30 °C for 15 min. The reaction was then terminated by the addition of stop reagent. A small aliquot was taken from the reaction mixture and washed to eliminate ATP. The bound phosphorylated 33PpolyGlu-Tyr was measured by scintillation counting of the filtermat in a Betaplate scintillation counter. The dpm obtained, being directly proportional to the amount of 33 P-polyGlu-Tyr produced by ZAP 70, was used to determine the IC50 for each compound. Hirabyashi and co-workers in 2008 developed a series of pyrimidine derivatives and evaluated them for ZAP-70 inhibitory activity by intracellular ZAP-70 kinase inhibition assay [67]. The authors express the ZAP-70 enzyme in the baculovirus expression system. A coupled

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