1. B
Abstract
MMV006169 inhibits growth of T. gondii and C. parvum
Conclusions/Future Directions
Acknowledgements/Current
Status References
The Life Cycle of Toxoplasma gondii
Our yeast 3-hybrid system contains the Gal4 DNA binding domain
(DBD) and activation domain (AD) both expressed as fusion proteins
to DHFR and CDC48. Interaction between our two fusion proteins
and our chemical inducer of dimerization (CID) ensures binding of
the DBD to a transcription reporter gene upstream activating
sequence (UAS). Inducible transcription of the reporter gene
ensures growth on selective media.
MMV006169 (pink) was attached via a triazole linker (green) to
methotrexate (blue). Methotrexate is an anti-cancer agent known to
interact with dihydrofolate reductase (DHFR), attaching these two
chemicals to MMV006169 made a compound suitable for a yeast 3-
hybrid assay.
The life cycle of Toxoplasma gondii is
quite intricate. Its ability to take on a
number of different cellular forms
provides it with the ability to infect
such a wide range of organisms. Its
definitive host is the feline, this is the
only organism that features the
sexually reproducing form of
Toxoplasma, the sporozoite. These
parasites form oocysts which can be
excreted in the feces of the feline
and contaminate water, soil, etc. If
the oocyst is ingested by an
intermediate host, the parasite takes
on the replicative form, the
tachyzoite, and eventually converts
into bradyzoites resulting in the
formation of tissue cysts [1].
The Lytic Cycle
Once the parasite enters the body it invades the intestinal epithelial cells. Upon
entrance into the parasite forms a parasitophorous vacuole in which it
replicates. As a result of replication the vacuole becomes filled with parasites. At
this point the parasites secrete proteases that poke holes in the host cells
membrane. The parasite vacuole then pushes itself out of the host cell through
a process called egress. One replicative cycle can be assayed or multiple rounds
of the cycle can be assayed to analyze growth within the host cell [1].
The drug MMV006169 shows potent inhibitory effects on both T. gondii and C. parvum growth [2]. The drug DBeQ is a
compound which is known to inhibit p97 in yeast [3]. Due to the structural similarities between MMV006169 and DBeQ, we
hypothesize that MMV006169 may be interacting with p97 (CDC48) in T. gondii. Toxoplasma has two forms of CDC48, a
cytoplasmic and an apicoplast form, Cryptosporidium only the cytoplasmic form because Cryptosporidium lacks an apicoplast.
CDC48 is a protein involved in proper trafficking of proteins from the endoplasmic reticulum to the proteasome during
endoplasmic reticulum associated degradation [4]. In order to perform yeast 3-hybrid we needed to identify analogs of 6169
that would allow for linker attachment. FT14.026.2 showed promise for chemical inducer of dimerization (CID) development.
Due to the fact that the FT14.026.2 analog retains potency against Cryptosporidium and does not against Toxoplasma we believe
that this analog has been made specific to inhibit only the cytoplasmic version of CDC48.
Toxoplasma gondii is an obligate, intracellular, parasitic protozoan that causes the
disease toxoplasmosis. It can be transmitted in a number of different ways including
the consumption of undercooked meat, contact with contaminated water or soil,
and congenital transmission. It has the ability to infect virtually any tissue within any
warm-blooded animal; a major contribution to why this is possible is the numerous
different cellular forms the parasite can take on within its host. The most concerning
of all cellular forms would be the tachyzoite, this is the replicative form of the
parasite which is critical for its ability to invade and proliferate within the host cell.
To inhibit invasion of Toxoplasma one must focus on the tachyzoite form of the
parasite. The drug MMV006169 (6169) has been shown to inhibit growth in both
Toxoplasma and Cryptosporidium, a related apicomplexan parasite. Although the
details of the protein-drug interaction are unknown, an analog of 6169 called DBeQ
is know to interact with the p97 protein in yeast. We hypothesized that 6169,
because of its structural similarities to DBeQ, may be interacting with p97 (CDC48)
in Toxoplasma and therefore inhibiting growth of the parasite. By expressing the
two forms of Toxoplasma CDC48 in AH109 yeast cells we can determine whether or
not there is a protein-drug interaction through the use of a yeast 3-hybrid assay
which utilizes a gene reporter system to identify such interactions.
MMV006169
IC50 MMV006169
-1 0 1 2
-50
0
50
100
150
log[MMV006169]µM
%Inhibition
T.g. IC50=1.15μM
C.p. IC50=1.50μM
pGADCDC48Ap pGADCDC48Cy
Both forms of Toxoplasma CDC48 were cloned into pGADT7 via in vivo cloning. This was made possible by amplifying our genes of interest
from cDNA with flanks homologous to the vector. When co-transformed into yeast double homologous recombination takes place resulting in
the vectors shown below [5]. (A) Finalized constructs for both the cytoplasmic and apicoplast forms cloned into the pGADT7 vector in such a
way that they are expressed as a Gal4AD/CDC48 fusion protein. (B) Diagnostic digest confirming proper cloning using enzyme indicated.
• Both forms of Toxoplasma CDC48 were cloned into pGADT7
confirmed by diagnostic sequencing.
• Perform targeted yeast 3-hybrid using both constructs.
• Clone cryptosporidium CDC48 into our pGADT7 vector and test
whether the same interaction is present in the parasite.
• Clone the Cryptosporidium version of CDC48 into Toxoplasma
and determine whether or not it has a lethal effect on the
parasite.
I’d like to acknowledge Gary Ward and Jenna Foderaro for helping
me through this whole process. Throughout the course of this
semester I’ve spent my time attempting to clone the cytoplasmic
version of the CDC48 gene into the pGADT7 vector to fix a mutation
in our initial construct. Complications with my initial cloning strategy
led me to develop an alternative strategy where I will use the
already constructed pGADCDC48Ap plasmid to form a gapped
pGADT7 plasmid that can be used to construct the pGADCDC48Cy
plasmid via in vivo homologous recombination.
[1] Weiss, Louis M., and Kami Kim. Toxoplasma Gondii: The Model Apicomplexan: Perspectives and
Methods. London: Academic, 2007. Print.
[2] Bessoff, K., T. Spangenberg, J. E. Foderaro, R. S. Jumani, G. E. Ward and C. D. Huston (2014).
"Identification of Cryptosporidium parvum active chemical series by Repurposing the open access malaria
box." Antimicrob Agents Chemother 58(5): 2731-2739.
[3] Chou, T. F., S. J. Brown, D. Minond, B. E. Nordin, K. Li, A. C. Jones, P. Chase, P. R. Porubsky, B. M. Stoltz, F.
J. Schoenen, M. P. Patricelli, P. Hodder, H. Rosen and R. J. Deshaies (2011). "Reversible inhibitor of p97,
DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways." Proc Natl Acad Sci
U S A 108(12): 4834-4839.
[4] Agrawal, S., G. G. van Dooren, W. L. Beatty and B. Striepen (2009). "Genetic evidence that an
endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in
import of apicoplast proteins." J Biol Chem 284(48): 33683-33691.
[5] Hua, S. B., M. Qiu, E. Chan, L. Zhu and Y. Luo (1997). "Minimum length of sequence homology required
for in vivo cloning by homologous recombination in yeast." Plasmid 38(2): 91-96.
Yeast Three-Hybrid
DBeQ FT14.026.2
C.p. IC50=2.331μM
T.g. IC50=6.574μM
2kb
2kb
A)
B)
CDC48 Cloning
Cloning Two Forms of CDC48 for Targeted Yeast Three-Hybrid
Interaction With MMV006169-Based Chemical Inducer of
Dimerization
McKenzie L. Stannard1, Jenna E. Foderaro1, and Gary E. Ward1.
1Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405