The cell signalling pathways are often regulated by kinases1. The perturbation to these pathways because of kinase dysfunction caused due to mutations, translocations, and upregulation events often results in unanticipated detrimental consequences such as diabetes, inflammation, and mainly cancer2. Cancer is a primary health concern worldwide3. According to the Cancer Facts & Figures of American Cancer Society, the number of new cancer cases was expected to be approximately 1.9 million in the United States (US) in 2021. However, intriguingly, the Cancer Statistics 2021 reports the decline of death rate from cancer in the US consistently by 31% over the past two decades. This drop in the cancer mortality rate is predominantly due to a reduction in smoking and advancement in the disease diagnosis and treatment4. With the success of the discovery of the imatinib, the first kinase inhibitor, a multitude of the current drug development efforts considerably focuses on targeting aberrant kinases with the selective inhibitors, thus, uncovering their tremendous treatment potential for cancer and other diseases3, 5. Nevertheless, many factors, such as tumour microenvironment, specific tumour genetics, pharmacogenomics, and drug resistance puzzle the clinical efficacy of these molecules and present remarkable challenges towards kinase drug discovery6. Until now, the overall focus of kinase drug development and clinical efforts has been exclusive to oncology, with only one JAK inhibitor tofacitinib for the non-oncology applications of arthritis3, 7. Owing to the challenges, there are only a few FDA approved kinase inhibitors available for the treatment of breast and lung cancer, after the first protein kinase inhibitor in 20018, 9. Hence, there is a compelling need to devise improved methods to modulate the kinase function. The kinases were authenticated as important molecular targets by the development of leading edge chemical biology techniques. In 1991, the three dimensional crystal stricture of a Protein Kinase-A domain provided a fundamental model for the inhibitor design. In the following years, the number of human catalytic domain model structures increased expeditiously to over more than 200, thus, emphasizing the role of structural biology in understanding the translational aspects of this strikingly dynamic protein family10. Notably, the high-resolution crystal structure analysis of the selective inhibitor-protein complexes offer explanations for the inhibitor selectivity and identify their unique molecular mechanisms, hence, providing an excellent opportunity in developing a kinase target area. It also provides the basis for optimism that we might overcome the problems with the existing antikinase therapies10, 11. Precisely, the catalytic domains are the major sites involved in the kinetic reactions and are highly targeted for kinase inhibitor therapy. It is pertinent to overproduce soluble kinase domains in comparison to their full-length counterparts. Since the kinase domain can easily be engineered and expressed in high yield, the development of selective inhibitors would also be fostered, thereby offering an advantage over currently available high throughput platforms12–14. All the 518 protein kinases encoded by a human genome share a highly conserved catalytic domain in regards to their sequence and structure. However, they mainly differ in their regulation. The major catalytic site is the ATP-binding pocket, called as the hinge region, that is located between the N-terminal and C-terminal lobes10, 11. This catalytic site is highly explored especially for targeted drug design to achieve selectivity and to understand the molecular structure of the kinase10. The determination of kinase domain structures by employing high-resolution X-ray crystallography requires a protein to be produced in high yields15. The human kinase expression achieved in insect cells is substantial16, 17. Although successful, this method suffers from high experimental costs and is time demanding. Alternatively, the recombinant bacterial expression system offers comparable performance advantages over insect cell culture, including shorter generation times, low costs and increased protein yields. While the removal of the regulatory and other auxiliary regions can adversely affect the expression of the catalytic domains, coexpression along with a protein phosphatase from bacteriophage λ is shown to enhance their expression in a soluble and homogeneously dephosphorylated form greatly18, 19. In our previously published report, we could successfully demonstrate the soluble and homogeneously dephosphorylated expression and purification of wild-type, full-length Human Tousled Like Kinase, (hTLK1B) using such coexpression strategy20. Since TLKs are involved in DNA repair mechanism and often upregulated in case of breast or prostate cancer, they are considered to be clinically relevant molecular targets for anticancer therapy21. However, surprisingly, the automated screen panel on kinase domain constructs generated by Parton et al. did not report the expression profile of Human TLK1B kinase domain22. Hence, we envisaged if this simple, robust and efficient protocol can be extensively applied to the hTLK1B kinase domain (hTLK1B-KD) construct as well.
As a first step towards our research goal, we cloned hTLK1B-KD into a pETDUET-1 vector harbouring a coexpression capacity of two target genes. Impressively, we could obtain ample amounts (~ 10 mg per litre of the bacterial culture) of the homogeneously dephosphorylated and soluble, form of biologically active hTLK1B-KD with exceptional purity in a single purification step via 6x-polyhistidine tags, which can then be used for structural, functional and in vitro drug screening studies. Our protocol provides a significant improvement upon the three-step approach described for purification of hTLK2-KD23, thus, increasing the protein yields together with managing time, costs and resources. We also evaluated the therapeutic viability of the purified hTLK1B through inhibition studies and identified two potent in-house synthesised compounds. These second generation inhibitors were synthesised to complete the structure-activity relationship studies of phenothiazine analogue with respect to TLKs, as our previously reported inhibitor J54 had shown interesting results during the in vivo screening24. We speculate that our sincere efforts in understanding the kinase domain structure, hTLK1B-KD in this case might help the new design of combination and single-agent therapies to enhance the prospects of mitigating challenges involved in kinase drug discovery.