Department of Life Sciences

Molecular Biology: Theory & Practice - BSS020N234Y

maira.freschi@roehampton.ac.uk

Bioinformatics Practical
Tutor: Dr. Maira Freschi

Introduction:
Recently, a breakthrough was made in our understanding of how the Ebola virus infects
people. Many outbreaks of the Ebola virus have occurred in Africa, and infection is often deadly. In
August 2011, two different groups of researchers reported that in order to enter our cells and infect our
bodies, the Ebola virus must bind to a protein called Niemann-Pick C1 ("NPC1" for short). To figure
this out, one of the research groups (Carette et al.) first took a large number of cells and randomly
disrupted different genes in the different cells. Eventually, the number of genes disrupted, or mutated,
in all of the different cells combined was in the millions.The researchers exposed these mutated cells
to the Ebola virus and then checked to see if any cells were resistant to infection. They analyzed the
resistant cells to see what genes were mutated in these specific cells. They realized that the genes,
especially NPC1, that were mutated in the resistant cells probably play key roles in infection. This is
how the researchers found that NPC1 is essential for the Ebola virus to infect cells.
NPC1 is found along the membrane of endosomes, which are small compartments in our
cells that transport molecules from the outside of the cell to the inside. Inside the cell, molecules in
endosomes are carried to lysosomes, which are compartments that break down molecules and cell
debris. Normally, NPC1 is important for transporting cholesterol in cells, but the Ebola virus uses
NPC1 to gain entry into the endosomes and causes the endosomes to burst, releasing the virus into
the cell.
One of the research groups (Ct et al) already developed two anti-viral drugs that can block
infection by binding the NPC1 protein. To find these successful drugs, the
researchers first tested a large number of small molecules on cells exposed to the Ebola virus to see
if any of the small molecules could prevent infection. Sure enough, one small molecule, labeled 3.0,
stopped infection. The researchers made 50 small molecules similar to this one and found that one of
these 50, labeled 3.47, worked even better at preventing Ebola infection. (3.0 is technically a
benzylpiperazine adamantine diamide molecule, and 3.47 is just like this molecule but has a
methoxycarbonyl benzyl group added to it.)
While the researchers (Ct et al) found that their drugs bind NPC1, and that this can block
Ebola infection, extensive pharmaceutical testing still needs to be done before doctors can use these
drugs to fight Ebola infection in people. For example, it needs to be determined whether the drugs
bind other proteins that are similar to NPC1. Additionally, because researchers had the goal to study
how the drugs prevent infection, using only cells grown in a lab, they did not find out how the drugs
affect the overall health and function of the cells. For example, the drugs might interfere with
important signalling pathways (biochemical pathways). Or the drugs may even affect the body of an
animal as a whole.
In summary, currently researchers do not know whether these will be good clinical drugs.
In this practical you will use bioinformatics tools (computer tools used to explore
biological processes), to explore how these Ebola virus drugs could bind non-target
proteins, that is, proteins other than NPC1.
Bibliography
To do this practical you will need to use these databases:
NCBI. (n.d.). BLAST: Basic Local Alignment Search Tool. Retrieved September
9, 2011, from http://blast.ncbi.nlm.nih.gov/Blast.cgi
NCBI. (n.d.). Gene. Retrieved September 9, 2011, from
http://www.ncbi.nlm.nih.gov/gene
These resources are a good place to start gathering information about drugs, the Ebola
virus, and NPC1:
Centers for Disease Control and Prevention. (n.d.). Ebola Hemorrhagic Fever.
Retrieved September 9, 2011, from
http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/ebola.htm
Genetics Home Reference (n.d.). NPC1. Retrieved September 9, 2011, from

Department of Life Sciences

Molecular Biology: Theory & Practice - BSS020N234Y
maira.freschi@roehampton.ac.uk

http://ghr.nlm.nih.gov/gene/NPC1
The Naked Scientists. (2011, August 24). Ebola virus blocked. Retrieved
September 7, 2011, from
http://www.thenakedscientists.com/HTML/content/news-archive/news/2354/
Sullivan, N., Yang, Z., and Nabel, G. (2003, September). Ebola Virus
Pathogenesis: Implications for Vaccines and Therapies. Journal of Virology. Vol
77, No 18, 9733-9737. Retrieved September 9, 2011, from
http://jvi.asm.org/cgi/reprint/77/18/9733.pdf
These are the two recent studies that discovered that the Ebola virus enters cells using the
NPC1 protein and developed small molecule drugs to bind NPC1 and block infection:
Carette, J. et al. (2011, August 24). Ebola virus entry requires the cholesterol
transporter Niemann-Pick C1. Nature. Published online. Retrieved September 7,
2011, from
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10348.html
Ct, M. et al. (2011, August 24). Small molecule inhibitors reveal Niemann-Pick
C1 is essential for Ebola virus infection. Nature. Published online. Retrieved
September 7, 2011, from
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10380.html

Procedure
Identifying Non-Target Proteins
How accurately does a drug bind to its target protein (the protein it is supposed to bind to)? Are there
other proteins the drug might bind to that it should not? In this practical you will be looking at what
non-target proteins the Ebola drugs might interact with, based on how similar the target protein is to
other proteins.
1. Learn as much as you can about the Niemann-Pick CI gene and protein. You can do
this using the NCBI Gene Database.
a) You will use the Niemann-Pick C1 gene name, NPC1, for your searches.
Make sure to choose the top result that says "NPC1" and is in humans (Homo
sapiens).
b) What does the database tell you about the NPC1 gene and protein? What
disease do defects in this gene cause? What part(s) of the cell is the NPC1
protein located in?
2. Retrieve the amino acid sequence of the NPC1 protein.
a) From within the NCBI Gene page about NPC1, navigate to the "Related Information"
sidebar on the right-hand side of the page. Click on the "Protein" link.
b) Click on the result for the NPC1 protein, as shown in Figure 1, scroll down until find
Niemann-Pick C1 protein precursor [Homo sapiens].
c) On this page, scroll down to the bottom, where the amino acid sequence for the protein is
given, or click FAST. Copy the entire amino acid sequence (including the numbers at the
start of each line), as shown in Figure 2. It is a good idea to save this sequence in a word
or text file in case you need it later

Department of Life Sciences

Molecular Biology: Theory & Practice - BSS020N234Y
maira.freschi@roehampton.ac.uk

Figure 1. Searching for the amino acid sequence for the Niemann-Pick C1 protein will generate
many results. Many of these are variants of the NPC1 protein. Pick the result that is in humans
([Homo sapiens]).

Figure 2. The amino acid sequence of the protein will be located at the bottom of the protein page
for the NPC1 protein.

3. Next, use the NCBI Basic Local Alignment Search Tool (BLAST) to identify any other human
proteins that have a similar amino acid sequence. If there are any, they are candidates for
proteins that the Ebola drugs might also bind to and interfere with. a. Under "BLAST
Assembled Genomes" click on "Human," as shown in Figure 3.

Department of Life Sciences

Molecular Biology: Theory & Practice - BSS020N234Y
maira.freschi@roehampton.ac.uk

Figure 3. The NCBI BLAST website allows you to submit an amino acid sequence of a
protein or a nucleotide (DNA or RNA) sequence of a gene and see what proteins, or genes,
share similar sequences. To search the human genome, click on "Human," In this practical
we are interested in looking for proteins with similar amino acid sequences to NPC1.
4. On the top of the page, click the "blastp" tab, as shown in Figure 5, circled in red.
5. Paste the amino acid sequence you copied before in the box under "Enter Query Sequence",
as shown in Figure 4. Click the blue "BLAST" button on the bottom of the page.

Figure 4. Using the NCBI BLAST tool, you can search for proteins with similar amino acids by
clicking on the "blastp" tab. In this tab, enter the amino acid sequence you want to search for in the
box circled in green. Hit the "BLAST" button.
a) The next page may take several seconds to load. After a short wait, a page that looks like
Figure 5 should appear. This page gives you a lot of information on how the amino acid
sequence you submitted matches other sequences in the database.

Department of Life Sciences

Molecular Biology: Theory & Practice - BSS020N234Y
maira.freschi@roehampton.ac.uk

b) At the top of this results page, in the blue section, click on the "Distance tree of results" link,
as shown in Figure 5,

Figure 5. Searching for similar amino acid sequences on the NCBI BLAST website will probably
generate many results that match your sequence to varying degrees. Clicking on the "Distance
tree of results" link, circled in green, will take you to a helpful visual representation of your
results. For a visual representation of different protein domains, or regions, that your protein
shares with other proteins.
c) Just how similar are these two proteins to the NPC1 protein? To find out, scroll down to the
"Descriptions" section, as shown in Figure 6. Locate the names of your two proteins under
"Description," in Figure 6, and see what their "Query coverage" is.
d) To see exactly how the amino acid sequences match each other, click on the "Max score" for
any result, which is circled in yellow in Figure 6. In this section, the NPC1 amino acid
sequence you searched for is shown as the "Query" and the protein it is being compared to is
the "Sbjct." Amino acids that the two sequences have in common are listed by their letter
between where these two sequences are aligned, while a "+" between the aligned sequences
indicate that the two amino acids are similar. Dashes in either sequence indicate a gap in the
match.

Department of Life Sciences

Molecular Biology: Theory & Practice - BSS020N234Y
maira.freschi@roehampton.ac.uk

Figure 6. To see how similar proteins are to the amino acid sequence you submitted, scroll down
to the "Descriptions" section of your NCBI BLAST results page. The "Description" column, circled in
green, lists the names of the different matching proteins. For each matching protein you can see
what percentage of amino acids it shares with your submitted sequence under the "Query
coverage" column.

e) At the top of the NCBI BLAST results page, in the "Graphic Summary" click on the
image immediately below where it says "Putative conserved domains have been
detected
f) This page shows the amino acid sequence you submitted at the top and below it, the
other amino acid sequences that matched it (in grey rectangles). Hover your mouse over the
sequence matches to learn more about them.
a. Where do the "Patched" proteins match your sequence? (The numbers given
correspond to the amino acid sequence of the protein.) Researchers do not know where the drugs
bind the NPC1 protein, but where could the drugs bind and probably not also bind the Patched
proteins?
b. Overall, how likely do you think it is that a drug that binds the NPC1 protein would
also bind the Patched proteins?
c. You have read about the NPC1 protein, but what do the two similar proteins you
picked in step 3g do? Go back to the NCBI Gene website, which you explored in step 1, and instead
of searching for NPC1, search for each of these two similar proteins (enter their names instead of
their abbreviations, for example "patched homolog 1"). Click on the top search result that is a human
gene.
d. Read about the genes in the "Summary" section. What do they have in common with
NPC1, and how are they different? Based on their function, how damaging do you
think it might be if a drug blocked the proteins encoded by these genes?

Using this information we could further investigate:

Investigating Complicated Interactions - How even if a drug only binds its target protein, it may still
disrupt delicate biological processes.