Molecular modelling

5.4.1 Introduction

Pharmaceutical chemists today are facing numerous intricate challenges. The most demanding and rewarding one is the rational design of new therapeutic agents for treating human diseases (Cohen, 2007).

Recent scientific developments have changed the way pharmaceutical research produces new bio-active molecules. The traditional “trial and error” drug discovery processes are now slowly being replaced by structure based rational drug design. This includes the use of X-ray crystallography and NMR for the determination of the 3-dimensional structure of a target protein followed by various modelling techniques for the design of ligands that could interact with the target structure (Zsoldos et al., 2003).

Active conformation can be defined as the conformation, which a drug molecule adopts when bonding to either an enzyme or a receptor. This conformation is not necessarily the most energetically preferred one, but rather contains the correct spatial arrangement of all essential binding groups. Knowledge of the preferred conformation is nevertheless valuable as a means of explaining activity profiles and for the design of new analogues (Lucietto, 2004). The main challenge of structure based rational drug design is finding small molecular structures that bind strongly to a given protein due to steric and electrostatic complementarity and is readily available or easy to synthesise (Zsoldos et al., 2003).

The difficulty lies in keeping equal focus on both the above-mentioned criteria. Two main approaches are used to overcome this problem, each prioritising one of the criteria over the other:

 Flexible docking of potential ligands from a database of 3-D structures of known compounds. Here the availability criterion is a given, since the considered structures are all available (or synthesisable).

102

 Build up complimentary structures from scratch-de novo design focused on generating the best possible fit to the constraints (Zsoldos et al., 2003).

5.4.2 Methodology

The energetically favourable conformation distributions of cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) were elucidated by using Spartan‟10^® (Professional Version 1.10) computer molecular modelling software. Local minima conformations were obtained for each cyclic dipeptide by the use of molecular mechanics (AMBER force field) optimisation methods, due to the fact that it does not consider hydrogens explicitly (Wishart and Case, 2001).

The cyclic dipeptides cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) were initially built by using the amino acid database included in the Spartan‟10^® software. A step-wise approach to energy minimisation was then followed. Geometrical optimisation was achieved by an initial steepest-descent run, which was run for at least 200 cycles or until the root mean square (RMS) of 0.1 kcal.mol^-1Å^-1 was achieved. Geometrical optimisation of the cyclic dipeptides was then followed by a PolakRibiere (Conjugated gradient) minimisation, which was done in repeated runs of 32560 cycles, until suitable low energy conformations with RMS of less than 0.001 kcal.mol^-1Å^-1 was obtained. The conformational search was executed in order to identify the three lowest energy conformations, which could then be analysed to produce conclusions on the conformations of the cyclic dipeptides.

5.4.3 Results and discussion

The data for the three lowest energy conformers for each cyclic dipeptide are reported in Tables 5.4, 5.5, 5.6 and 5.7 respectively. The lower the heat of formation, or energy of the conformer, E (kJ/mol), the greater the stability of the conformer and thus the most likely to be found in nature, under standard conditions. The relative energy, Rel. E (kJ/mol), is calculated relative to the lowest energy conformer which is assigned a energy of 0 kJ/mol.

103 Table 5.4 Conform ational search results calculated for c yclo(Phe -4Cl-Pro) in the g as phase

Conformer Energy (kJ/mol) Rel. E (kJ/mol) Boltzmann Distribution

1 360.77 0 0.924

2 368.03 7.26 0.049

3 369.53 8.76 0.027

Figure 5.18 Conformation 1 of cyclo(Phe-4Cl-Pro) in the gas phase

Figure 5.19 Conformation 2 of cyclo(Phe-4Cl-Pro) in the gas phase

104 Figure 5.20 Conformation 3 of cyclo(Phe-4Cl-Pro) in the gas phase

Figure 5.21 Overlay of conformation 1 and 2 of cyclo(Phe-4Cl-Pro) in the gas phase

Figure 5.22 Overlay of conformation 1 and 3 of cyclo(Phe-4Cl-Pro) in the gas phase

105 Figure 5.23 Overlay of conformation 2 and 3 of cyclo(Phe-4Cl-Pro) in the gas phase

Figure 5.24 Overlay of conformation 1, 2 and 3 of cyclo(Phe-4Cl-Pro) in the gas phase

106 Table 5.5 Conform ational search results calculated for c yclo(D -Phe-4Cl-Pro) in the gas phase

Conformer Energy (kJ/mol) Rel. E (kJ/mol) Boltzmann Distribution

1 0 365.03 0.814

2 3.88 368.91 0.17

3 9.76 374.8 0.016

Figure 5.25 Conformation 1 of cyclo(D-Phe-4Cl-Pro) in the gas phase

Figure 5.26 Conformation 2 of cyclo(D-Phe-4Cl-Pro) in the gas phase

107 Figure 5.27 Conformation 3 of cyclo(D-Phe-4Cl-Pro) in the gas phase

Figure 5.28 Overlay of conformation 1 and 2 of cyclo(D-Phe-4Cl-Pro) in the gas phase

Figure 5.29 Overlay of conformation 1 and 3 of cyclo(D-Phe-4Cl-Pro) in the gas phase

108 Figure 5.30 Overlay of Conformation 2 and 3 of cyclo(D-Phe-4Cl-Pro) in the gas phase

Figure 5.31 Overlay of conformation 1, 2 and 3 of cyclo(D-Phe-4Cl-Pro) in the gas phase

109 Table 5.6 Conform ational search results calculated for c yclo( Phe-4Cl-Pro) in the

solvated (water) phase

Conformer Energy (kJ/mol) Rel. E (kJ/mol) Boltzmann Distribution

1 287.86 0 0.942

2 296.19 8.33 0.033

3 296.84 8.98 0.025

Figure 5.32 Conformation 1 of cyclo(Phe-4Cl-Pro) in the solvated (water) phase

Figure 5.33 Conformation 2 of cyclo(Phe-4Cl-Pro) in the solvated (water) phase

110 Figure 5.34 Conformation 3 of cyclo(Phe-4Cl-Pro) in the solvated (water) phase

Figure 5.35 Overlay of conformation 1 and 2 of cyclo(Phe-4Cl-Pro) in the solvated (water) phase

111 Figure 5.36 Overlay of conformation 1 and 3 of cyclo(Phe-4Cl-Pro) in the solvated (water) phase

Figure 5.37 Overlay of Conformation 2 and 3 of cyclo(Phe-4Cl-Pro) in the solvated (water) phase

112 Figure 5.38 Overlay of Conformation 1, 2 and 3 of cyclo(Phe-4Cl-Pro) in the solvated (water) phase

Table 5.7 Conform ational search results calculated for c yclo(D -Phe-4Cl-Pro) in the solvated (water) phase

Conformer Energy (kJ/mol) Rel. E (kJ/mol) Boltzmann Distribution

1 291.37 0 0.791

2 294.83 3.46 0.916

3 301.54 10.16 0.013

113 Figure 5.39 Conformation 1 of cyclo(D-Phe-4Cl-Pro) in the solvated (water) phase

Figure 5.40 Conformation 2 of cyclo(D-Phe-4Cl-Pro) in the solvated (water) phase

114 Figure 5.41 Conformation 3 of cyclo(D-Phe-4Cl-Pro) in the solvated (water) phase

Figure 5.42 Overlay of conformation 1 and 2 of cyclo(D-Phe-4Cl-Pro) in the solvated (water) phase

Figure 5.43 Overlay of Conformation 1 and 3 of cyclo(D-Phe-4Cl-Pro) in the solvated (water) phase

115 Figure 5.44 Overlay of Conformation 2 and 3 of cyclo(D-Phe-4Cl-Pro) in the solvated (water) phase

Figure 5.45 Overlay of Conformation 1, 2 and 3 of cyclo(D-Phe-4Cl-Pro) in the solvated (water) phase

116 Table 5.8 Conform ational search results calculated for c yclo(Phe-4Cl-Pro) in dim eth yl sulphoxide (DMSO)

Conformer Energy (kJ/mol) Rel. E (kJ/mol) Boltzmann Distribution

1 -3315302.5 0 0.648

2 -3315300.02 2.49 0.238

3 -3315298.2 4.3 0.114

Figure 5.46 Conformation 1 of cyclo(Phe-4Cl-Pro) in DMSO

117 Figure 5.47 Conformation 2 of cyclo(Phe-4Cl-Pro) in DMSO

Figure 5.48 Conformation 3 of cyclo(Phe-4Cl-Pro) in DMSO

118 Figure 5.49 Overlay of conformation 1 and 2 of cyclo(Phe-4Cl-Pro) in DMSO

Figure 5.50 Overlay of conformation 1 and 3 of cyclo(Phe-4Cl-Pro) in DMSO

119 Figure 5.51 Overlay of conformation 2 and 3 of cyclo(Phe-4Cl-Pro) in DMSO

Figure 5.52 Overlay of conformation1, 2 and 3 of cyclo(Phe-4Cl-Pro) in DMSO

120 Table 5.9 Conform ational search results calculated for c yclo(D -Phe-4Cl-Pro) in DMSO

Conformer Energy (kJ/mol) Rel. E (kJ/mol) Boltzmann Distribution

1 -3315307 0 0.673

2 -3315304 2.05 0.295

3 -3315299 7.54 0.032

121 Figure 5.53 Conformation 1 of cyclo(D-Phe-4Cl-Pro) in DMSO

Figure 5.54 Conformation 2 of cyclo(D-Phe-4Cl-Pro) in DMSO

122 Figure 5.55 Conformation 3 of cyclo(D-Phe-4Cl-Pro) in DMSO

Figure 5.56 Overlay of conformation 1 and 2 of cyclo(D-Phe-4Cl-Pro) in DMSO

123 Figure 5.57 Overlay of conformation 1 and 3 of cyclo(D-Phe-4Cl-Pro) in DMSO

Figure 5.58 Overlay of conformation 2 and 3 of cyclo(D-Phe-4Cl-Pro) in DMSO

124 Figure 5.59 Overlay of conformation1, 2 and 3 of cyclo(D-Phe-4Cl-Pro) in DMSO

The aromatic cyclic side chain is extended and not folded onto the cyclic dipeptide ring due to steric hindrance caused by the halogen atom. This is a possible indication that the keto-groups are free to bind to a complimentary receptor.

125 autonomous growth of tissue not subject to the rules and regulations of normal growing cells. Tumour is a general term indicating any abnormal mass or growth of tissue. The main characteristics of benign tumours can be summarised as encapsulation, slow growth, and non-invasive growth into surrounding tissue, in other words lack of the ability to metastasise. Malignant tumours grow rapidly, are not encapsulated and incomplete if present, invade surrounding tissues and metastasise. Benign growths generally have a normal complement of chromosomes, exhibit good differentiation, and have limited cell division whereas the opposite is characteristic of malignant neoplasms (Warshawsky and Landolph, 2006). Table 6.1 illustrates the main type of cancers.

Table 6.1 Main t ypes of cancers (W arshawsk y and Landolph, 2006).

Tissue Benign Malignant

Epithelial

Squamous cell papilloma Squamous cell or epidermoid carcinoma

Basal cells (skin) - Basal cell carcinoma