PKs in disease

PKB is also a principal upstream regulator of crucial intracellular processes responsible for cell survival, growth, proliferation, angiogenesis, metabolism, and probably cell migration and invasion [Manning B. D. and Cantley, 2007].

Interestingly, the PKB-catalyzed phosphorylation often serves for ‘switching off’ the functioning of its downstream targets; for example, the PKB-catalyzed phosphorylation of tuberous sclerosis 2 protein (TSC2), a component of the TSC2 complex, inhibits the GTPase-activating protein function of TSC1-TSC2 and thus stimulates the GTP-loading of RheB, which in turn potently activates mTOR [Toker, 2008]. In this way, PKB attenuates the inhibitory effects of the TSC1-TSC2 complex on the mTOR complexes mTORC1 and mTORC2, and thus creates two important feedback loops, as mTORC2 acts as PKB activator (positive feedback), while mTORC1 has been demonstrated to inhibit PKB (negative feedback) [Huang J. and Manning, 2009].

1.3. PKs in disease

While the normal functioning of PKs is essential for the sustainment of life of an organism, the errors at DNA-level (i.e., mutations of PK-encoding genes) or faults of PK expression, activation or feedback loops are connected to a variety of diseases [Chico et al., 2009; Knight et al., 2010; Manning B. D. and Cantley, 2007]. Probably the most explored group of diseases that are caused by dere-gulation of PKs is cancer. Within last decades, PKs and their direct activators

have evolved as the most frequently mutated oncogenes and tumor suppressors, thus representing the major path of signaling by which cancer cells evade normal physiological constraints on growth and survival [Zhang J. et al., 2009].

Moreover, some PKs expressed in tumor or in the surrounding tissues contri-bute to disease progression as a result of their normal functioning by enabling tumor to acquire possibilities for angiogenesis and metastases [Knight et al., 2010]. Whereas the identification of PKs contributing to tumor development and/or survival is a continuous process aided by the development of RNA-interference techniques and performance of large-scale forward and reverse genetic screens, the currently well-known examples of oncogenic PKs involve the following kinases [García-Echeverria et al., 2000; Keri et al., 2006; Knight et al., 2010; Zhang J. et al., 2009]:

 receptor tyrosine kinases – i.e., BCR-Abl in chronic myeloid leukaemia, EGFR in lung, head, neck, pancreatic and colourectal cancers, HER2 in breast cancer, VEGFR-2 in ovarian and kidney cancers, and PDGFR in a variety of tumors;

 STE kinases, MAPK pathway PKs – i.e., BRAF, MEK1 and MEK2 in ovarian and colourectal cancers;

 PI3K pathway PKs (including AGC-group) – i.e., PKB in a variety of tumors.

The consequences of PK malfunctioning are also tightly connected with another large group of diseases – central nervous system (CNS) disorders [Chico et al., 2009], although unveiling the role of PK in the CNS has been largely obstructed by the specific properties of the tissue itself (i.e., existence of blood-brain-barrier). Interestingly, in case of CNS disorders the major 'culprits' are not represented by Tyr-kinases (as in case of cancers) but by Ser/Thr kinases from different groups [Chico et al., 2009; Keri et al., 2006; Virdee et al., 2007]:

 CMGC-group – i.e., GSK3 involved in a vast spectrum of disorders involving Alzheimer's and Parkinson's diseases, depression, HIV-associated dementia, traumatic brain injury, etc.;

 STE-group – i.e., MAPK in Alzheimer's and Parkinson's diseases, cerebral ischaemia, spinal cord and traumatic brain injuries, etc.;

 AGC-group – i.e., PKC and ROCK, both in Alzheimer's disease, cerebral ischaemia and vasospasm, and additionally, ROCK in multiple sclerosis and epilepsy;

 CAMK-group – i.e., DAPK in acute brain injury and Alzheimer's disease.

The increased activity of a PK in the diseased tissue is in several cases accompanied by the increase of the concentration of the same PK in periferal tissues, i.e., body fluids. For instance, elevated levels of extracellular PKAc (ECPKA) and ECPKA autoantibodies have been detected in blood serum samples of patients with different malignant tumors (especially prostate, blad-der, breast, and colon cancers) [Nesterova et al., 2006]. Moreover, the valu-ability of ECPKA as a biomarker is not limited with diagnosis of cancer, but also allows monitoring and prognosis, as the good correlation between con-centration of ECPKA and stage of disease or success of anti-cancer therapy has

been demonstrated in recent tests [Wang H. et al., 2007]. Another PK suggested as a biomarker for cancers (i.e., melanoma) according to the studies of xeno-graft mouse models is PKC [Kang et al., 2009]; moreover, the latter may also serve as a biomarker for Alzheimers disease, in parallel with other PKC isozymes [Barry et al., 2010].

The examples mentioned above clearly illustrate that PKs belong to both, disease-associated and disease-modifying category of proteins, this fact rendering substantial interest in PKs as potential biological targets for pharma-ceutical industry. Importantly, in order to be classified as a therapeutic target, a protein should also be termed ‘druggable’, i.e. it should possess a well-defined binding site capable of development of multiple strong and specific interactions with a small drug molecule [Hopkins and Groom, 2002]. PKs fulfill the druggability requirement by virtue of incorporation of at least one suitable site represented by the ATP-binding site, and therefore belong to ca 3000 thera-peutic targets that comprise the ‘human druggable genome’ (which also includes GPCRs, nuclear hormone receptors, ion channels, metallopeptidases, proteases, PDEs, etc) [Hajduk et al., 2005]. On the other hand, it should be kept in mind that therapeutic targets might also be represented by bacterial, viral, fungal or parasitic enzymes (as in case of malarial PKs) [Doerig et al., 2010].

According to the currently reported state-of-the-art, there are only ca 330 targets that bind approved drugs, 270 encoded by the human genome and 60 belonging to pathogenic organisms; therefore, a vast majority of putative therapeutic targets remains to be explored [Landry and Gies, 2008]. In case of PKs, the difficulties for drug development rise not only due to the intrinsic complexity of PK signaling, but also due to the susceptibility of several thera-peutically important PKs to mutations that trigger resistance towards drug candidates designed to interfere with the non-mutated target [Krishnamurty and Maly, 2010]. It is therefore evident that the discovery of novel compounds able to interact with and suppress the activity of the disease-modifying PKs remains of utmost importance. Consequently, there is also a strong requirement for methods enabling assessment of PK inhibitors, exploration of PK structure, functions and regulation mechanisms, and determination of protein kinase activity (i.e., for PKs serving as biomarkers).