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QUESTION 1:
[39]
1.1) Explain why cancer cell lines which have mutant p53 phenotypes were
used as negative controls in this study. (4)

In this study, cell lines with mutant p53 gene (SW480; MDA-MB-435; and PC3)
were used as negative controls because they did not have the capacity to
activate MDM2 or any of the p53 pathway proteins such as p21. Therefore
incubation of MDM2 antagonists will not cause expression of MDM2 in any way.
This guaranteed the fact that in cell lines with wild-type p53 gene (HCT116, RKO
and SJSA-1), any detected levels of MDM2 or p21 were due to antagonistdependent stabilization of the p53 protein in cancer cells.

Because of absence of a functional p53 gene in cell lines such as SW480, MDAMB-435 or PC3, incubation with Nutlin-1 could not cause activation of the p53
pathway in these negative control cells. As such, the observed increase in the
mitotic index indicated lack of G1 and G2 cell arrest in these cells. However, G1
and G2 cell arrest was observed in cells that had a functional p53 gene,
confirming that Nutlin-1 indeed activates a major cellular function of the p53
pathway.
1.2) Western blots are routinely used to quantify the level of protein
expression in cells. Given the relationship between p53 and MDM2, interpret
the results seen in Figure 2A (also shown below). Make sure you consider
each cell line, the effect of drug treatment on each protein and what this
means for the cell on a molecular level. (15)

The high affinity of MDM2 for p53 and the ensuing complexation leads to
impairment of the p53 function in a three-thronged fashion: (i) MDM2 binding to
p53 thereby blocking p53’s ability to activate transcription; (ii) nuclear efflux of
p53 thereby reducing cytoplasmic p53 concentration and (iii) direct promotion of
cytosolic p53 degradation in a ubiquitin-dependent manner .

Figure 2A is a western blot that shows effects of varying concentrations of
Nutlin-1 (an antagonist of MDM2) on levels of p53, MDM2 and p21 in two distinct
cell lines: HCT116 and SW480 .Colon cancer cell line HCT116 has a wild-type
p53 gene but the other cell line (SW480) has a mutant p53 gene that cannot take
part in suppression of any cell proliferation.

In log-phase HCT116 cells, where wild-type p53 gene is active, dose-dependent
increase in the levels of all three proteins is evident on the western blot. That is
to say as concentration of Nutlin-1 is increased, from 0 M to 8 M, there is a
corresponding increase in levels of p53, MDM2 and p21. It is also notable from
the western blot that the increase in leveIs of p53 and p21 due to incremental
doses of Nutlin-1 are comparable. However, increase in MDM2 levels appears to
be higher than any of the other two proteins. These results can be explained by
the fact that the disruption of MDM2-p53 complexation or interaction in the
presence of the antagonist Nutlin-1 leads to accumulation of wild-type p53 and
this in turn leads to an elevation in the levels of MDM2 and p21. Increase in p21
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is due to activation of the p53 pathway. This deduction is consistent with
activation of the p53 pathway.

In contrast, log-phase SW480 cells exposed to the same conditions showed high
basal levels of mutant p53 protein but no detectable MDM2 or p21. During the
normal activation of p53, increase in concentration of p53 causes autoregulatory
feedback control whereby p53 activates MDM2 expression as a way of causing
repression of p53. Also expression of p21 is positively regulated during p53
activation. The reason why the two proteins p21 and MDM2 were absent in the
western blot wells for cell line SW480 is that the mutant p53 protein in this cell
line lacked the capacity to activate expression of p21 and MDM2.

In essence, this antagonist (Nutlin-1) mimics the interactions of the p53 peptide
and so it binds MDM2 in the p53-binding pocket and activates the p53 pathway
in cancer cells only, leading to cell cycle arrest in G1 and G2 phases, apoptosis,
and eventual inhibition of cell proliferation. Thus, the treatment of cells with an
inhibitor of MDM2-p53 binding should result in
˗
stabilization and accumulation of the p53 protein resulting from the
blockage of its nuclear export and degradation,
˗
activation of MDM2 expression, and
˗
activation of other p53-regulated genes and the p53 pathway such as p21.
1.3) Refer to Figure 3 B (also shown below). What is the purpose of β-actin on
the western blot? Explain. (4)

 -actin is one of the highly conserved and ubiquitously expressed proteins in
mammalian cells. Its expression is therefore not controlled by presence or
absence of any of enantiomers of Nutlin-3 molecule. As such, its purpose on this
particular western blot was primarily to serve as a loading control where it made it
possible to
˗ check the integrity of cells and hence any possible protein degradation in the
samples.
˗ confirm that protein synthesis in cells was successful
˗ normalize amounts of protein pipetted in wells by confirming that protein
loading was uniform or consistent across the gel so as to enable proper and
accurate interpretation of the western blot
1.4) Protein expression levels were detected using western blots. Describe, in
detail, how gene or transcript expression levels were detected. (10)


Three cancer cell lines with wild-type p53 gene (HCT116, RKO, and H460a) were
treated with Nutlin-1 for 8 hours, and the change in the level of transcription was
measured by quantitative PCR (or real-time PCR) where fold induction of gene
expression was monitored and measured and compared with the untreated
control.
The transcription of p21 increased in a dose-dependent manner in all the three
cancer cell lines. However, expression of the p53 gene itself was not affected by
presence of the drug Nutlin-1 at all concentrations tested.
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
These observations indicate that the Nutlin-1 does not affect expression of p53
gene at transcriptional level but it up-regulates p53 protein by means of a posttranslational mechanism.

Since, expression of p21 gene occurs as a direct response to p53 pathway
activation, then Nutlin-1 first positively influences p53 protein levels posttranslationally and the p53 then directly upregulates p21 expression at
transcriptional level.
1.5) The authors of the article evaluated the viability of various cell lines in
response to nutlin-1 treatment using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT) assay. What is the underlying principle of
MTT assays? (6)

This colorimetric assay measures cell viability as a function of reductive
activity and capacity cells to enzymatically convert the yellow tetrazolium dye
(MTT) to water-insoluble formazan purple crystals using microsomal and
mitochondrial NADPH-dependent dehydrogenases (or oxidoreductases).(Lu
et al., 2012; Stockert et al., 2012).

After a 24-hr incubation at 37˚C, the water insoluble purple formazan crystals
are then dissolved in dimethyl sulfoxide (DMSO), acidified ethanol solution, or
acidified SDS. Then, the absorbance at 570nm of this solution is measured by
a multiwell scanning spectrophotometer (or ELISA Reader)

Optical density values of the treated cells are compared with the optical
density values of the control cells and results are presented as a percentage
of cell survival. Cell number is then calculated based on the calibration curve.

When cells die they lose their ability to convert MTT into formazan, thus,
colour formation presumably serves as a marker of the viable cells.
QUESTION 2:
[16]
2.1) In contrast to the article by Vassilev et al (2004) where a library of MDM2
inhibitors were screened experimentally, how are the library of SARS-CoV-2
main protease (Mpro) inhibitors screened in this article? (4)


Screening was done using molecular docking studies. Here, the 3D structures
of more than 75 FDA-approved antiviral drugs were downloaded from a
PubChem database
Computational docking studies using AutoDock Vina software was done. This
software uses a Lamarckian genetic algorithm in combination with grid-based
energy estimation to check the docking accuracy.
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2.2) What is drug re-purposing? Elaborate on how this may assist in drug
discovery research activities. (4)
 Drug repurposing re-assignment of already approved and existing drugs to treat
new diseases which they would have shown to have efficacy against. Drug repurposing starts with an informed idea when then translates into clinical trial to
confirm the efficacy (effect) of the drug in the new patient and disease
population.

Developing novel drugs is a long process that may take years yet a disease like
the current COVID19 pandemic will be ravaging people. Thus drug repurposing is
a good strategy of redeploying readily available and certified safe drugs for use in
humans to treat new diseases. So from a drug discovery point of view, time
required to develop disease-specific drugs is shortened and lives arte saved in
due time.
2.3) Name one pre-requisite (relating to the target protein) in order to conduct
docking studies with potential inhibitors. (2)
The target protein’s X-ray crystallographic structure, showing a potential binding site
should be elucidate first.
2.4) Lopinavir-Ritonavir binds to the main protease (Mpro) with a value of
-10.6kcal/mol. What does this value tell you? Differentiate between negative
and positive values. (6)



The value -10.6kcal/mol is called the binding free energy which tells me the
enthalpy of the protein-ligand complex.
In a binding process between a protein and a ligand molecule, the enthalpy is
given by the difference between the final enthalpy (bound complex) and the initial
enthalpy (before binding to the ligand).
A negative enthalpy variation indicates a favorable number of "connections" and
that the bound state is energetically more favourable than an unbound state
(protein alone). A positive value indicates that the protein-ligand interaction is not
energetically favourable and in drug discovery the drug or ligand is not going to
work in as far as treating the disease in question concerned.
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Question 3
[45]
In the previous article by Kumar et al (2020), three potential drug candidates
targeting the SARS-CoV-2 main protease were identified using in silico
methods. However, the authors state that the efficacy of these drugs must be
corroborated
experimentally
through
biochemical
and
structural
investigations. You have been tasked with validating the protease-inhibitor
interactions experimentally.
3.1) Assume the main protease gene (918 bp) was amplified using PCR and
cloned into the pET28a plasmid vector between the NcoI and NotI restriction
sites as shown on the next page. Describe how you would insert this plasmid
into a host such as Escherichia coli? (10)

During the insertion and ligation of the protease gene (918bp) between
restriction sites NcoI and NotI , various possible ligation products are formed:
˗ Successfully ligated protease gene
˗ Re-circularised PE28a plasmid
˗ Uncut pET28a plasmid
Making competent cells
 Inserting the recombinant plasmid into E. coli cells is called transformation
and there are a number of procedures one can use to introduce the plasmid
into a host bacterial cell. But before inserting the plasmid into the host cell, the
host cell needs to be made competent so that it is ready to take in foreign
naked DNA.
 The protocols for making competent cells vary depending on whether
transformation is to via heat shock or electroporation. In either case, a single
colony of the desired strain is taken from an agar plate and inoculated into
liquid medium to make a starter culture that will be monitored for optical
density at 600 nm (OD600).
 It is desirable that in order to obtain high transformation efficiency, when
OD600 is between 0.4 and 0.9, the bacteria are at the log-phase and are
ready for harvesting and transformation.
 Care must be taken to use sterile tools and always work aseptically.
 Harvested cells are then processed according to the method of
transformation, whether by heat shock or electroporation
 Incubating host cells in Ca2+ (aq) can make the bacterial cells competent. This
is followed by incubation on ice (0–4°C ) of the E. coli and plasmid DNA
mixture.


Determination of transformation efficiency
Once prepared, competent cells should be evaluated for transformation
efficiency, aliquoted to small volumes to minimize freeze/thaw cycles, and
stored at an appropriate temperature to maintain viability.
The transformation efficiency of competent cells is usually measured by the
uptake of subsaturating amounts of a supercoiled intact plasmid (e.g., 10–500
pg of pUC DNA). The results are expressed as the number of colonies formed
(transformants), or colony forming units (CFU), per microgram of plasmid
DNA used (CFU/μg).
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
Transformation of competent bacterial cells
When ready for the transformation step, competent cells should be thawed on
ice and handled gently to retain viability. Cells can be mixed by gentle
shaking, tapping, or pipetting, but vortexing should be avoided.

According to specific method or protocol, the mixture of plasmid and
competent cells is incubated to allow transformation. A quick dip of the tube
into a warm water bath (heat-shock method) or a quick pulse of electricity
(electroporation) can be employed instantly open pores in the bacteria cells
that allow the plasmids to enter the cells.

In this solution of bacteria, there are various possible cells with different
genotypes:
˗ Bacteria that are successfully transformed with the recombinant plasmid
that carries the 918 bp protease gene (true transformants)
˗ Bacteria that have been transformed by a re-circularised plasmid or
uncut plasmid
˗ Bacteria that have failed to be transformed.

The plasmid/E-coli solution is incubated at 37˚C overnight. But to avoid
propagation of bacteria that were not transformed or those that took up a nonrecombinant plasmid, the growth media should be laced with antibiotic
kanamycin.
3.2) Making use of a diagram depicting an agarose gel, demonstrate how you
can use restriction digests to confirm that the plasmid containing your target
gene was successfully inserted into the E. coli cells. (10)

Digestion of the purified plasmid with restriction enzymes NcoI and NotI will
generate restriction fragments of the following sizes:
˗ 918 bp (insert)
˗ 5369 bp (plasmid vector)

On agarose gel, the 918 bp will migrate faster than the 5369 bp and the visuals
will be as shown below.
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3.3) The recombinant SARS-CoV-2 main protease must now be over-expressed and
purified from the E.coli cells in order to use in subsequent experiments. The main
protease has a molecular weight of 34 kDa and a theoretical pI of 5.95. Using this
information, along with the vector map provided on the next page, suggest a series
of chromatographic steps that can be used to purify this protein. (15)
 Given molecular weight and isoelectric point (pI), the purification method I can
suggest 2-D Gel electrophoresis whereby I will employ Isoelectric Focusing
(IEF) in one dimension and SDS-PAGE in the perpendicular dimension.

In IEF, the gel shall be made to have a pH gradient whereby linear migration
of proteins is based on differences in their isoelectric points (the pH) at which
they are zwitterionic and have a zero charge and hence they stop migrating in
the electric field.

In the second dimension, the molecules are then separated at 90 degrees
from the first electropherogram according to molecular mass.






Isoelectric Focusing
A specially prepared gel with a pH will be subjected to an electric potential
difference across it whereby one end is more positive (anode) than the other
which will be more negative relative to the other (cathode). At all pH values
other than their isoelectric point, proteins will be charged. If they are acidic
and therefore negatively charged, they will be pulled towards the more
positive end of the gel but if they are (basic) and therefore positively charged,
they will be pulled to the more negative end of the gel. The proteins applied in
the first dimension will move along the gel and will stop migrating at their
isoelectric point
The protein in this example has a pI of 5.95 and so when it reached a point in
the gel where the pH is 5.95, it stops there. Other proteins will continue
migrating.
It may so happen that several proteins in the cell lysate have the same pI of
5.95, so another dimension of separation which uses difference in molecular
weight should be employed.
SDS-PAGE
Before separating the proteins by mass, they are treated with sodium dodecyl
sulfate (SDS), a detergent that denatures the proteins so that they unfold
along
Also, the SDS molecules will impart a uniform negatively charge to all the
proteins on the gel so that separation of these proteins is solely due to size
not charge
In the second dimension, an electric potential difference is applied again, but
perpendicular to the first electric field. The proteins will be attracted to the
more positive side of the gel (because SDS is negatively charged), however,
proportionally to their mass-to-charge ratio which is technically the same for
all proteins. The migration of proteins will be slowed by friction in the gel
which acts like as molecular sieve when the current is applied. Larger proteins
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will migrate slowly while smaller proteins pass through the molecular sieve
quite easily.
Visualisation and identification of protein of interest.
 Protein of interest and other resolved proteins can then be detected by stains
such as silver or and Coomassie Brilliant Blue staining.
 In the case of silver staining, a silver colloid is applied to the gel and silver
binds to cysteine groups within the protein. Then the silver is darkened by
exposure to ultra-violet light. The amount of bound silver is proportional to the
darkness, and therefore the amount of protein at a given location in the gel.
 From the gel, a protein of molecular weight of 34 kDa will be located by use of
a protein larder or marker
 It is the cut out of the gel and concentrated by various salting methods.
3.4) Presume you have successfully purified the main protease. Recommend
and describe one technique that can be used to validate the binding between
the main protease and the identified inhibitors. (10)

So many methods for validation of protease-inhibitor binding are available such
as
˗ High Performance Liquid Chromatography (HPLC),
˗ Fluorescence resonance energy transfer (FRET) assays,
˗ Fluorescence intensity assays monitoring change from dual-label quenched
pairs,
˗ Fluorescence polarization (FP) assay and
˗ Fluorescence intensity assays

I would recommend Fluorescence Intensity Assays for validating proteaseinhibitor binding activity. In this approach, a fluorescent dye containing a
reactive amine group is covalently attached to the -COOH end the substrate
through the amide bond.

The peptide sequence gives protease specificity, while the dye acts as a
reporter for enzyme activity.

The target protease cleaves the amide bond when the peptide binds and
forms a complex.

The fluorescence of the attached dye is quenched when covalently attached
to the peptide and it increases sharply when released by a protease cleavage
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