University of Nebraska Medical Center University of Nebraska Medical Center

DigitalCommons@UNMC DigitalCommons@UNMC

Posters: 2021 Summer Undergraduate Research Program Summer Undergraduate Research Program

2021

Characterization of MAS1-86 Activity in Malaria Parasites Characterization of MAS1-86 Activity in Malaria Parasites

Leah A. Keim University of Nebraska Medical Center

Tessa M. Firestone University of Nebraska Medical Center

Emily K. Bremers University of Nebraska Medical Center

Sukhithasri Vijayrajratnam University of Nebraska Medical Center

Mohamed A. Seleem University of Nebraska Medical Center

See next page for additional authors

Follow this and additional works at: https://digitalcommons.unmc.edu/surp2021

Recommended Citation Recommended Citation Keim, Leah A.; Firestone, Tessa M.; Bremers, Emily K.; Vijayrajratnam, Sukhithasri; Seleem, Mohamed A.; Conda-Sheridan, Martin; and Ng, Caroline L., "Characterization of MAS1-86 Activity in Malaria Parasites" (2021). Posters: 2021 Summer Undergraduate Research Program. 50. https://digitalcommons.unmc.edu/surp2021/50

This Poster is brought to you for free and open access by the Summer Undergraduate Research Program at DigitalCommons@UNMC. It has been accepted for inclusion in Posters: 2021 Summer Undergraduate Research Program by an authorized administrator of DigitalCommons@UNMC. For more information, please contact digitalcommons@unmc.edu.

Author Author Leah A. Keim, Tessa M. Firestone, Emily K. Bremers, Sukhithasri Vijayrajratnam, Mohamed A. Seleem, Martin Conda-Sheridan, and Caroline L. Ng

This poster is available at DigitalCommons@UNMC: https://digitalcommons.unmc.edu/surp2021/50

Conclusions

Investigate MAS1-86 impact on the apicoplast’s metabolic pathways and apicoplast biogenesis in greater detail.

Test cross resistance in MAS1-86 selected synchronized ring stage cultures to ART since ring stages exhibit ART resistance.

Test cross resistance in WLL-resistant mutants to MAS1-86 to investigate MAS1-86 inhibition of the proteasome.

Investigate UPR after treatment of MAS1-86.

Future Directions

Days

Characterization of MAS1-86 activity in malaria parasitesLeah Keim1, Tessa M. Firestone1, Emily K. Bremers1, Sukhithasri Vijayrajratnam1, Mohamed A. Seleem2, Martin Conda-Sheridan2, Caroline L. Ng1

1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA2 Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USASummer Undergraduate

Research Program

MV

Analogs of MAS1-86 were then tested against P. falciparum. MAS1-86 was identified as most potent inhibitor.

Background

Figure 5. MAS1-86 selected parasites display 6-23-fold increase in resistance to MAS1-86. (A) MAS1-86 growth inhibition assays tested against various asynchronous parasites as indicated, and the (B) corresponding IC50 +/- SEM values. Parasitemia was assessed 72 hours after compound addition. Parasite DNA was stained with SYBR Green I and respirating mitochondria stained by MitoTracker Deep Red, then assessed by flow cytometry. Statistical significance was assessed using Student’s t-test with Welch’s correction. *p < 0.05; **p <0.01. n=3.

0 1 2 3 40

50

100

Log[MAS1-86], (nM)

% In

hibi

tion

Cam3.II K13 WT

Cam3.II K13 R539T

Cam3.II K13 C580Y

N=3

Cam3.II K13C580Y Flask 1

0 2 4 60

50

100

Log[MAS1-86], (nM)

% In

hibi

tion

Cam3.II K13 C580Y

65X (3200 nM) Flask 1

10X (500nM) Flask 1

10X (500nM) Flask 2

65X (3200 nM) Flask 2

0 1 2 30

50

100

Log[WLL], (nM)

% In

hibi

tion Cam3.II K13 C580Y

50X (2500nM) Flask 1

50X (2500nM) Flask 2

160X (8000nM) Flask 2

Figure 4. MAS1-86 stepwise resistance selections. Two replicates, Flask 1 and Flask 2, of Cam3.II K13C580Y parasites were exposed to MAS1-86 in increasing concentrations, as indicated on the right axis. Parasitemia over time is tracked, as indicated on the left axis. Green dots correspond to profiled parasites in Figure 2.

24 Hours 48 Hours 54 Hours0 Hours 36 Hours 48 Hours 24 Hours

250 nM MAS1-86

DMSO

500 nM MAS1-86

24 Hours 48 Hours 54 Hours24 Hours0 Hours 48 Hours 58 Hours24 Hours 48 Hours 54 Hours0 Hours 18 Hours 24 Hours 30 Hours

A CB

1WHO. World Malaria Report, (2021).2Rosenthal, Melissa R. et al. “Plasmodium falciparum Artemisinin Resistance: The effect of heme, protein damage, and parasite cell stress response.” ACS Infectious Diseases, (2020).3Dondorp et al. “Artemisinin resistance in Plasmodium falciparum malaria.” N England J Med, (2009).4Ng, C. L. et al. “Protein degradation systems as antimalarial therapeutic targets.” Trends in Parasitology,(2017).5Guggisberg, A. M. et al. “Isoprenoid biosynthesis in Plasmodium falciparum.” Eukaryotic Cell, (2014).6Yeh, E., & DeRisi, J. L. “Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum.” PLoS Biology, (2011).7Florentin, A., Cobb. “PfClpC is an essential Clp Chaperone required for plastid integrity and Clp Protease stability in Plasmodium falciparum”. Cell Reports, (2017).8Fetzer C, “A chemical disruptor of the ClpX chaperone complex attenuates the virulence of multidrug-resistant Staphylococcus aureus.” Angew Chem Int Ed Engl, (2017).

We would like to thank Dr. Conda-Sheridan and Mohamed for synthesizing the compounds used in these experiments. Thanks to Dr. Reid for allowing us access to the flow cytometer. Lastly, thanks to SURP for this research opportunity.

[MAS1-86], uM

Days

% P

aras

item

ia

Cam3.II K13C580Y Flask 2

[MAS1-86], uM

% P

aras

item

ia

Days

Results

0 1 2 3 40

50

100

Log[MAS1-86]

% In

hibi

tion

Cam3.II K13 C580Y - no IPP

Cam3.II K13 C580Y - w/IPP

Figure 7. Morphology of parasites exposed to MAS1-86 across the asexual blood stages. (A) Ring stage, (B) trophozoitestage, and (C) schizont stage Cam3.II K13 C580Y parasites were treated with DMSO, 250 nM, or 500 nM MAS1-86 for the hours indicated above. Thin blood smears were made, stained with Giemsa, and imaged by light microscopy using a 100X objective.

Figure 8. K13 haplotype does not influence parasite susceptibility to MAS1-86. Asynchronous K13 mutants were exposed to MAS1-86 for 72 hours, and the dose responses are plotted. n=3.

Figure 9. MAS1-86-resistant parasites do not show cross-resistance to WLL. Dose response curves of indicated parasites exposed to WLL for 72 hours. n=3

Figure 6. IPP does not rescue MAS1-86 parasite inhibition. (A) 72-hour growth inhibition assays of MAS1-86 in the absence or presence of IPP tested against Cam3.II K13 C580Y. (B) MAS1-86 IC50 values assayed at 72 hours and 120 hours. A 10-fold reduction in IC50 values at 120 hours compared to72 hours indicates a delayed death phenotype.

A B

A B

In 2019, ~ 229 million malaria cases were reported globally, causing 409,000 deaths 1.

Malaria is caused by the Plasmodium parasite with cyclical infection in human and Anopheles mosquito host. P. falciparum is the most prevalent species 1 .

Blood stage parasites cause malaria symptoms. The lifecycle begins with merozoites that invade red blood cells. They develop into ring stages (0-23 hours post invasion, hpi), trophozoite stages (24-39 hpi), and mature into schizont stages (40-48 hpi) 2.

Artemisinin-based combination therapy (ACT) is the first-line treatment for uncomplicated falciparum malaria 3.

Resistance to all artemisinin (ART) is a widespread problem 3.

Point mutations in Kelch 13 confer ART resistance. The C580Y mutation is the most abundant in SE Asia. The R539T mutation displays high levels of resistance in vitro.

P. falciparum’s apicoplast, an essential organelle that generates fatty acids, heme, and isoprenoid precursors, is a promising drug target since humans lack this organelle 4.

The apicoplast’s primary function in asexual life stages is to produce isoprenoid precursor isopentenyl pyrophosphate (IPP) via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. IPP supplementation has shown to chemically rescue MEP inhibited cultures 5,6.

Delayed death phenotype is when growth of treated parasite is unaffected, but growth arrest is observed in the progeny. This is seen when apicoplast biosynthesis and metabolic pathways are inhibited 6.

The apicoplast-located PfClpC/P complex degrades proteins and has chymotrypsin-like proteolytic activity 4. PfClpC is a chaperone to the PfClpP protease 4.

Aberrant schizont morphology with fewer nuclei in auto-inhibited PfClpC has been reported 7.

P. falciparum 26S proteasome is a cytoplasmic protease. The β1, β2, and β5 subunits have caspase-like, trypsin-like and chymotrypsin-like activity, respectively 4. WLL inhibits the β2 and β5 subunits 4.

The unfolded protein response (UPR) upregulates proteasome activity 4.

PfClpC has 27% identity to the Staphylococcus aureus homolog ClpX. MAS1-86 inhibited multi-drug resistant S. aureus 8.

Conclusions MAS1-86 selected parasites display 6-23-fold increased resistance to MAS1-86. MAS1-86 does not target the MEP pathway, since IPP fails to rescue MAS1-86 inhibition. MAS1-86 inhibition caused a delayed progression in late trophozoite through schizont stages,

with fewer nuclei observed in schizonts. MAS1-86 kills artemisinin-sensitive and artemisinin-resistant parasites. MAS1-86 resistant parasites do not show cross resistance to proteasome β2 and β5 subunit

inhibitor, WLL, which has the same chymotrypsin-like activity as ClpP. References

Acknowledgements

Figure 1. P. falciparum asexual lifecycle. Credit to 2 for figure

Figure 2. Synthesis of MEP pathway and isoprenoid products in P. falciparum. Adapted from 5

Figure 3. MAS1-86 and analogs tested against P. falciparum. MAS1-86 circled in red.

Cam3.I

I K13

C58

0Y

10X (5

00 nM

) Flas

k 1

10X (5

00 nM

) Flas

k 2

65X (2

500n

M) Flas

k 1

65X (3

250 n

M) Flas

k 20

2000

4000

6000

8000

MAS

1-86

IC50

, (nM

)

✱

✱✱

✱

✱✱