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.