1. The molecule chosen for the analysis is Immunicillin -H. It is a transition state analogue for the enzyme purine nucleoside phosphorylase (PNP). Chemically Immunicillin -H is [(1S)-1-(9-deazahypozanthine-9yl)-1,4-dideoxy-1,4-imino-D-ribitol] (Kicska et al. 2001)
2. PNP is important for the degradation of nucleotides, especially deoxy guanosine. In the absence of this enzyme, deoxy guanosine accumulates and inhibits T cell function, causing T cell mediated immunosuppression. Inhibitors of PNP were found useful in treating T cell malignancies. Inhibiting PNP using transition state analogue, triggers apoptosis of T cells. Though Immunicillin-H inhibits stimulated malignant and nonmalignant T cells, it does not inhibit non stimulated normal T cells, and thus exhibits specific activity. This inhibitor will be very effective on faster dividing T cells like cancer cells than the normal T cells. (Kicska et al. 2001)
3. Immunicillin-H bind to human PNP, 739,00 times more tightly than the nature substrate inosine. Potent inhibition can be achieved even at the concentration of 56pM. Transition state is a transient state formed by the substrate with the enzyme, before it is converted to product. The arsenolysis of inosine by PNP was used to determine the transition state structure of PNP, and this was used to guide the designing of Immunicillin-H. Computational methods, along with kinetic isotope effect studies will provide information on bonds formed or broken in the transition state. These studies also help calculate fixed bond angle and bond length. Figure 1 is a schematic diagram showing the structure of inosine, transition sate of PNP and the structure of Immunicillin H. The inhibitory activity of the transition state analogue is a result of its strong structural resemblance to the transition state of the enzyme (PNP) and also the strong electrostatic interaction between the enzyme and the inhibitor. The transition state analogue can bind 105 more tightly than the natural substrate to PNP. Through molecular modelling, Immunicillin-H was provided with a ribo-oxocarbenium ion character and an elevated pKa at N7 as that observed in the transition state. (Schramm 2007)
4. Structural features: Immunicilin-H has two stereo chemical centers that were incorporated with the help of Mannich reaction. In this reaction there is a three-way condensation of 9 deazahypoxanthine, 3-hydroxy-4-methoxy-pyrrolidine and formaldehyde. The reaction is conducted in mild alkaline conditions. The resultant structure of this reaction is a compound that has structural similarity to the transition state of PNP. The structure of this compound (Immunicillin H) is shown in Figure 1. Unlike the physiological substrate inosine, the transition state analogue, does not have a group for the phosphate ester bond. In the natural substrate, the PNP cleaves the phosphate group to produce dephosphorylated product. (Evans et al. 2003; Schramm 2007)
5. The ability of Immunicillin -H to kill malignant T cells was studied in-vitro, using the apoptosis assay. For this assay, the MOLT-4 cell line was used. MOLT-4 is a Homo-sapiens, acute lymphoblastic leukemia cell line cultured from isolated patient’s cells. MOLT-4 is a rapidly proliferating T cell line. Inhibition of PNP in these cells will result in accumulation of deoxy guanosine, inhibition of proliferation and initiation of apoptosis. For this assay, MOLT-4 cell lines we cultured in RPMI1640 media and plated in petri-dishes at the rate of 1x 106 cells/ml. In one group, the cells were treated with varying concentration of Immunicillin-H and in another it was treated with varying concentration of deoxy guanosine or deoxy cytosine. The cells were incubated for 36 hours, after which the apoptosis was detected by Annexin staining. The apoptosis was quantified and the IC50 of the drug was also calculated. The cells tolerated a concentration of 5nM Immunicillin -H, above which the inhibitor was found to be toxic to T cells. A significant level of apoptosis was achieved with the inhibitor. (Kicska et al. 2001)
Figure 1: Structure of Inosine, PNP transition state and Immunicillin -H
Ref: Greg A. Kicska et al. PNAS 2001; 98:4593-4598
Imm-H (Immunillcin-H) has a structure similar to the PNP transition state, and thus binds with more affinity than its natural substrate inosine. Transition state analogues, act as competitive inhibitor to natural substrates inosine. The ionisation state of Immu-H helps it form a much stronger electrostatic bond with the enzyme. In the natural substrate, the PNP cleaves the phosphate group to produce dephosphorylated product.
References:
Evans, Gary B., Richard H. Furneaux, Peter C. Tyler, and Vern L. Schramm. 2003. "Synthesis of A Transition State Analogue Inhibitor Of Purine Nucleoside Phosphorylase Via The Mannich Reaction". Org. Lett. 5 (20): 3639-3640. doi:10.1021/ol035293q.
Kicska, G. A., L. Long, H. Horig, C. Fairchild, P. C. Tyler, R. H. Furneaux, V. L. Schramm, and H. L. Kaufman. 2001. "Immucillin H, A Powerful Transition-State Analog Inhibitor of Purine Nucleoside Phosphorylase, Selectively Inhibits Human T Lymphocytes". Proceedings of The National Academy Of Sciences 98 (8): 4593-4598. doi:10.1073/pnas.071050798.
Schramm, Vern L. 2007. "Enzymatic Transition State Theory and Transition State Analogue Design". Journal of Biological Chemistry 282 (39): 28297-28300. doi:10.1074/jbc.r700018200.
