4. DISCUSSION
A novel compound named, Phormidin was isolated from the marine cyanobacteriumPhormidium ambiguum , from the shores of Ezhara beach, Kannur district, Kerala, India (coordinates 11°49’09.9”N 75°25’02.9”E). Structure of the compound was elucidated by a combination of UV, FTIR, NMR and LC MS/MS methods. The isolated compound was found to have a molecular weight of 349.475 Daltons and has a structure similar to C75 and Cerulenin, known fatty acid synthase inhibitor drugs. Phormidin has a 20 carbon skeleton composed of a long aliphatic chain with an amide group attached to 13thcarbon and a 6 carbon open ring at one end. The two non polar methyl groups at one end make Phormidin lipophilic in nature. The compound is readily soluble in chloroform and DMSO.
How good a lead molecule can act as a drug candidate is termed ‘drug likeness’, a measure of the compound’s bioavailability and potency which is calculated based on properties like solubility in aqueous and organic phases, molecular weight etc. Lipinski’s rule of five is a set of four rules that can be correlated to check how much the compound is ‘druglike’ (Lipinski et al ., 2001). Phormidin fits to all the four of the ‘Lipinski’s rule of five’ which also predicts whether a compound is orally active or not. The Lipinski’s rule wants the number of hydrogen bond donors to be less than 5 and number of hydrogen bond acceptors to be less than 10. The number of hydrogen bond acceptors is 5 and donors are 4 for Phormidin, extending towards the bacterial FAS II. Likewise, the molecular weight of Phormidin is 349.4 Daltons and it is within the molecular weight limit of 500 Dalton for any compound to be an oral drug. And, finally the logp value which is the ratio of solubility of a compound in aqueous phase to that of an organic phase and has a maximum value of 5, for Phormidin it was found to be 2.75. Since it obeys all 4 of Lipinski’s rules, Phormidin can be considered as a good drug lead molecule. Along with Lipinski’s rule of five there are some more discussed criteria which are now used to establish the ‘druglikeness’ of any compound like molar refractivity, total number of atoms present and the total topological surface area of a compound etc. The molar refractivity of Phormidin comes to around 104.76 which indicate the polarizability of the compound. The total number of atoms in the title compound is 25 and for a good drug lead the total number of atoms must be from 20 to 70, including H-bond donors. Similarly, the maximum topological surface area (TPSA) of a good drug lead is considered to be around 140 Å2, exceeding which usually results in poor cell membrane permeation. Phormidin is having a TPSA value of 103.78 Å2 indicating a moderate cell membrane penetrating capacity. All the above discussed properties and specifications of Phormidin place it better than the known FAS inhibitors, Cerulenin and C75.
Enzyme inhibition assay with both bovine FAS I and bacterial FAS II showed that Phormidin is a better inhibitor compared to the standard FAS inhibitor Cerulenin. Also Phormidin showed better inhibition against bacterial FAS II compared to bovine FAS-I. This could be either due to the difference in activity towards FAS I and FAS II or might be related to the extent of purity of the enzymes used in the assay. The bovine enzyme extract used for the assay was partially purified which may consist of both cytoplasmic FAS I and mitochondrial FAS II enzymes. This aspect has to be clarified further by assaying with pure FAS I and FAS II enzymes.
The new compound did not show marked cytotoxicity (IC50>30 µg/mL) on the tested normal cell lines, and was simultaneously FAS I and FAS II inhibitors. Likewise, effect of Phormidin on cultured human alveolar basal epithelial cells; A549 cells showed an IC50 value 76.034 µg/mL, compared to 86.419 µg/mL for Cerulenin and 99.034 µg/mL for C75. This shows that Phormidin acts on normal cell line in a limited way only, but is more active against cancer cell lines. This could be because Phormidin acts on FAS expression and its activity is limited (Deepa et al ., 2013). Since there is added free fatty acids present in cultured media, inhibition of FAS may limit the cell growth but would not affect much on its growth rate. But in cancer cell line, since there is an increased need for fatty acids beyond what is available in culture media due to increased expression of FAS, inhibiting FAS will prevent the formation of new fatty acids and thus may directly affect the growth rate of cancer cell. Interestingly, FAS assay using crude Bacillus subtilis FAS II and bovine FAS I showed similar pattern of activity with Phormidin where it showed higher activity than Cerulenin and C75. This was well in accordance with the docking simulation results obtained comparing the three different inhibitors against FAS. As seen from the Figures 6a. and b. and Tables 4, 5 & 8. For both FAS II and FAS I docking glide scores as well as binding energies are in the order Phormidin<Cerulenin<C75 demonstrating that Phormidin would be better than Cerulenin and C75.
In designing good FAS inhibitor, as a lead compound Phormidin stands out as a promising candidate. The two other inhibitors, both Cerunenin and C75 are used as FAS inhibitors in different scenarios but have limitations. Cerulenin contains the reactive epoxy group that may interact with other proteins and may act on other off targets other than FAS like interfering with palmitoylation (a posttranslational modification, there by fatty acids are attached to amino acids allowing key signaling proteins to attach to the plasma membrane), cholesterol synthesis and proteolysis (Lupu and Menendez, 2006). Also, this epoxy group contributes to lower stability of Cerulenin. C75 was designed to avoid this epoxy group to increase stability and availability, but this redesign reduced potency of C75 (Kuhajda et al ., 2000). Phormidin does not contain this epoxy group and thus might have less side effects compared to Cerulenin and C75 due to non binding to off targets and also improved stability.
Results of docking studies carried out to investigate the mode of interaction of Phormidin with FAS I and FAS II was in accordance with the assay data (Figures 9 and 10). In docking simulations with FAS I and FAS-II, Phormidin showed better score compared to Cerulenin and C75. Even though the active site residues are different for both FAS IKS MAT domain and FAS II FasF domain interaction of Phormidin showed remarkably similar results.
From the enzyme assay and docking studies it is clear that Phormidin is more active than Cerulenin, even without the reactive epoxy group and hence, it must be also stable than Cerulenin. It is also better active than C75 as found in this study but Phormidin’s interaction with CPT has to be investigated and can be further re-engineered as a good lead molecule for suitable uses.