Sniffing out New Ways to Combat Malaria
By: Carol A. Rouzer, VICB Communications
Published: May 19, 2011
The discovery of a molecule that stimulates mosquito olfactory receptors paves the way for new insect repellants to fight the spread of malaria.
Despite the major advances against infectious diseases during the 20th century, malaria remains a preeminent public health threat. In 2008, malaria was the cause of over 1 million deaths in sub-Saharan Africa, where one child dies of the disease every 45 seconds. Malaria is caused by a microscopic parasite Plasmodium falciparum, which infects red blood cells in humans (Figure 1). The disease is spread by the mosquito Anopheles gambiae (Figure 2). As the development of vaccines and drugs targeting the parasite continues to meet with limited success, researchers, including Vanderbilt Institute of Chemical Biology (VICB) member Larry Zwiebel, have turned to mosquito control to fight the disease. Now the Zwiebel lab reports on the discovery of VUAA1, a compound that provides a promising foundation for the development of new, highly effective, mosquito repellants [Jones et al. (2011) Proc. Natl. Acad. Sci. U.S.A., published online May 9, DOI: 10.1073/pnas.110245108].
Figure 1. Red blood cells infected with P. falciparum, seen as ring-shaped structures inside of the cells. Image courtesy of Wikimedia Commons under the GNU Free Documentation License.
An. gambiae mosquitoes take their blood meal primarily from humans, and they rely heavily on the sense of smell to locate and choose their hosts. Thus, the Zwiebel lab has focused on the mosquito’s olfactory system as a target for the discovery of new compounds that can serve as repellants or as lures to trap the insects for extermination. They have functionally characterized a large number of the 76 An. gambiae odorant receptor proteins (AgOrs), each of which binds a unique array of molecules that trigger a sensory response. It is thought that by targeting the odorant receptors involved in host seeking, one could disrupt this behavior, so AgOr10, a receptor that responds to important human odors, became one focus of study. In addition, every AgOr pairs in an as yet unknown manner with a single second protein, the odorant receptor coreceptor (AgOrco) in order to form a fully functioning receptor complex. Before these studies began, it was unknown whether the coreceptor could be chemically modulated, since no compound had yet been described that could bind to this protein and alter its function. If such a molecule could be found, then it would likely interfere with the mosquito’s entire olfactory system.
Figure 2. Anopheles gambiae. Image courtesy of Zwiebel Lab.
The complex of an AgOr with AgOrco forms an ion channel that alters intracellular calcium levels in response to an odorant molecule that is recognized by the AgOr. The Zwiebel lab reconstituted a functioning odorant receptor complex by expressing one AgOr (AgOr10) and AgOrco together in human embryonic kidney (HEK293) cells. With both proteins expressed, the cells exhibited changes in intracellular calcium in response to molecules, such as benzaldehyde, that stimulate AgOr10. This provided the foundation for a high-throughput screen of odorant receptor function, which was developed in collaboration with the VICB’s High-Throughput Screening Core. Testing of 118,720 compounds resulted in the identification of one molecule, VUAA1 (Figure 3), that activated calcium mobilization in the HEK293 cells. The finding that VUAA1 could increase the response of the cells to odorants such as benzaldehyde suggested that the molecule does not bind to the same protein site as the odorant, but acts at a distinct site. Thus, VUAA1 was classified as an allosteric agonist of odorant signaling.
Figure 3. Structure of VUAA1.
Since odorant receptors transmit their signals as ion channels, increasing the flow of positively charged ions across the cell membrane, further studies used whole cell patch clamp measurements to directly assess the effects of VUAA1 on ion flow in HEK293 cells expressing various odorant receptor protein combinations. The results showed that VUAA1 could stimulate ion flow in cells expressing AgOrco alone, or in cells expressing AgOrco plus a variety of AgOrs other than AgOr10. No response occurred in cells treated with VUAA1 in the absence of AgOrco. These results showed that AgOrco can form an ion channel in the absence of an AgOr, and that VUAA1 acts by stimulating AgOrco rather than the associated AgOr. Experiments conducted with the odorant receptor coreceptor proteins from Drosophila melanogaster (fruit fly, order diptera), Heliothis virescens (tobacco budworm, order lepidoptera), and Harpegnathos saltator (Jerdon’s jumping ant, order hymenoptera) indicated that VUAA1 could stimulate the ion channel function of all of these proteins. This result was consistent with the high level of conservation of AgOrco proteins across multiple orders of insects.
AgOr/AgOrco protein complexes are expressed in odorant receptor neurons, which are found in the antennae and maxillary palps of An. gambiae (Figure 4). Across the surface of the long maxillary palp sensory organs are almost 80 individual capitate peg (Cp) structures, each of which contains one sensory cell for carbon dioxide (CpA) and two odorant receptor neurons (CpB and CpC). Each odorant receptor neuron expresses a single AgOr protein in complex with AgOrco. To determine if VUAA1 could work in vivo, the Zwiebel lab measured extracellular electrophysiological signals from individual Cp’s in the maxillary palp of female An. gambiae mosquitoes. They discovered that VUAA1 markedly increased the signals from the CpB/CpC neurons, but not CpA, which does not express AgOrco. They confirmed CpA function by its unique signal pattern in response to a carbon dioxide pulse.
Figure 4. Close up of An. gambiae head showing maxillary palp. Image courtesy of Zwiebel Lab.
These results clearly demonstrate, for the first time, that insect odorant receptor coreceptors function as ion channels, even in the absence of associated odorant receptor proteins. They also characterize VUAA1 as the first directly acting modulator of odorant receptor coreceptor function, providing a prototype compound for the development of an insect repellant that acts by overwhelming the odorant receptor signaling system. One problem facing the further development of VUAA1 as an insect repellant is its current low volatility, making delivery to the mosquito’s odorant receptors difficult. Solving this problem is a current focus of VICB efforts. However, this compound serves as a useful tool for further study of odorant receptor coreceptor function and as an important lead for the future discovery of more potent and volatile modulators of insect olfactory sensation.