Combinatorial in-silico modeling and bioinformatics analysis of immune proteins and small-molecular weight inhibitors: a potential for cancer chemotherapy.

Abstract

The immune system carries out pivotal functions in the protection of the body from damaging substances, microbes, and cellular alteration that could affect the health of an individual. The component of the immune system comprises of proteins, cells, and diverse organs. Individuals remain in a good state of health and wholeness if the immune system is working optimally, but if it becomes incapacitated toward fighting off germs or other harmful foreign substances, a diseased state set in. The innate and adaptive systems are the two sub-categories of the immune system. They work synergistically in the defence of the body and fighting off germs that triggered an immune response. Several proteins have been discovered to play pivotal roles in immune evasion and have therefore become attractive targets. Three of these proteins form the core of this thesis. Programmed death-ligand 1 (PD-L1), is an immune checkpoint protein which upon binding with another inhibitory checkpoint protein programmed cell death protein 1 (PD-1), elicit a cascade of reaction that leads to the reduction of proliferating antigen-specific T cells. The upregulation of PD-L1 can therefore, lead to evasion of the immune system by cancer cells. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is made up of three parts (a transmembrane part, extracellular part, and a cytoplasmic part). CTLA-4 has been implicated in the downregulation of immune response and blocking CTLA-4 activity results in a surge in immune functions. It has also been found that CTLA-4 negatively controls the T-cells. Natural killer group 2, member D (NKG2D) is located on the surface of immune cells where it acts as an activating receptor and regulator of the adaptive and innate immune system upon binding to its constitutive ligands such as UL16-binding protein (ULBP6). ULBP6 is a highly polymorphic protein, hence, the interaction between NKGD2 and ULBP6 is often altered. This effect has a great impact on the function of NKGD2 as a regulator of the immune system. Various humanized antibodies have been developed to specifically target PD-L1 and CTLA4. Some have been approved while others are at different phases of clinical trial. Ipilimumab and tremelimumab are designed as CTLA-4 inhibitors. Durvalumab and atezolimab are designed as anti-PD-L1 inhibitor. However, due to the limitations that have characterized the use of humanized antibodies inhibitors such as production cost, instability, and low tumour penetration, etc, small molecule inhibitors have been considered as a better alternative to humanized antibodies inhibitors. This thesis explored the mechanism of inhibition of newly synthesised PD-L1 inhibitors (BMS- 1166 and BMS-1001). Also, per-residue based virtual screening was employed to predict potential CTLA-4 inhibitors. Computational methods such as molecular docking, molecular dynamic simulation, virtual screening, and SNPinformatics were employed. These computational techniques revealed that BMS-1166 and BMS-1001 caused a motional movement in the monomers of PD-L1 to form a dimer, thereby preventing PD-L1-PD-1 interaction. Although the PD-L1 monomers have the same residues, their affinity for the BMS compounds differ. Two compounds ZINC04515726 and ZINC08985213 were identified as possible targets of CTLA-4. These two compounds elicited favourable interaction with CTLA- 4 facilitated by some crucial residues. Furthermore, the non-synonymous Single Nucleotide Polymorphism (nsSNPs) associated with ULBP6 were identified, and the effect of these nsSNPs on the interaction between NKGD2 and ULBP6 was also investigated. The first study (Chapter 4) investigates the structural dynamics and also provides insights into the mechanism of inhibition of BMS-1166 and BMS-1001 on PD-L1.

The second study (Chapter 5) determines the binding site landscape of CTLA-4 and also employs binding site similarities between unrelated proteins to repurpose an inhibitor to target CTLA-4. The third study (Chapter 6) identifies deleterious polymorphisms associated with ULBP6. The effect of these polymorphisms on NKGD2-ULBP6 binding as a consequent on immune response is also explored in this chapter. Chapter 7 gives a detailed report on how the use of bioinformatics tools and strategies have aided and advanced the field of cancer immunotherapy. This study provides a thorough insight into the in-silico design, development and mechanism of action of small molecule inhibitors of PD-L1 and CTLA-4. Furthermore, this study gives insight into the polymorphic nature of ULBP6. Thence, the work presented in this study would serve a s a platform towards the design of small molecule inhibitors of CTLA-4 and PD-L1 with high therapeutic and less toxicity.