If you like exploring the forest, one thing to be careful of is “Malaria” or jungle fever because Thailand is not yet free from this disease. Symptoms of this disease are fever, shivering, headache, and muscle pain. The scary thing about this disease is that if the patient does not receive treatment, it can lead to complications such as liver failure, kidney failure, or cerebral malaria and the patient can pass away easily. A report from the World Health Organization states that in 2022, 247 million people were infected with malaria and more than 600,000 people passed away. Malaria is found mostly in countries with hot and humid climates.

               Global warming also causes the number of Anopheles mosquitoes (Anopheles spp.) which are disease vectors to increase. In addition, the medicines used to treat them are starting to become ineffective because the germs are becoming more resistant to the drugs. Researchers are therefore trying to develop new drugs to deal with this tropical disease. Synchrotron is a powerful tool that can help prove the effects of drugs. However, researchers must know the weak points of malaria first, before developing the drugs to cure malaria disease.

               The real cause of malaria is a protozoan organism in the Plasmodium group that lives in Anopheles mosquitoes. The Plasmodium species found in Thailand and causing the most severe symptoms in humans is the Falciparum species. We get this infection from the bite of a female Anopheles mosquito. The germs hidden in mosquito saliva are passed into the bloodstream and become embedded in liver cells where they multiply and increase in number. It takes about 2 weeks until the liver cells break, then it releases germs into the bloodstream and enters the red blood cells to digest hemoglobin which is an important protein as food for growth and increment in numbers. It can cause the red blood cells to change their original shape depending on the growth period (Trophozoite) which is the stage where the infection increases in number. And when the number increases to the dividing stage (Schizont), the nucleus of the germ will divide rapidly causing red blood cells to break down in the bloodstream. It causes a cycle of fever and shivering for the patient and enters the red blood cells again.

                The malaria bacteria's digestion of hemoglobin releases Heam that remains from the decomposition. This substance is toxic to cells. This causes a mechanism to change Heam into alpha-hematin (α-hematin), which can be paired together and crystallized into Hemozoin within the cell. Therefore, the formation of hemozoin crystals allows the survival of infectious cells in the red blood cells. A drug that has been used to treat malaria for decades, such as Chloroquine will interfere with the conversion of hematin to hemozoin. This drug is more effective at disinfecting Plasmodium in the ring stage, which is the first stage in which the infection enters red blood cells, than in the growth stage.

                 Scientists can detect malaria infection or test the effectiveness of malaria drugs by examining hemozoin crystals with a high-quality microscope. This requires polarized light and highly skilled examiners. Or it can be done by mixing pyridine into the sample solution, which will result in a clear yellow solution made from a heme-pyridine complex that can absorb light at wavelengths of 370 nanometers. However, this technique also complicates sample preparation. Therefore, a method for detecting hemozoin crystals was developed using Fourier transform infrared spectroscopy (FTIR Spectroscopy), a technique that relies on the principle of absorption of infrared radiation. Plasmodium can be detected in the patient's blood at all three stages, and if the infrared absorption measurement technique from synchrotron light is used in conjunction with an infrared microscope, this will allow us to detect malaria infection in each red blood cell by measuring the absorption of infrared light in the wave number range 1,210 - 1,220.

                  From the above knowledge, Dr. Buaban Kuaprasert, beamline scientist at Synchrotron Light Research Institute (Public Organization), or SLRI, together with researchers from many agencies including the National Center for Genetic Engineering and Biotechnology (Biotech), Phramongkutklao College of Medicine, Vidyasirimedhi Institute of Science and Technology and Kasetsart University has discovered a new type of antimalarial drug from the anticancer drug database with codes NSC45545, NSC45570 and NSC45507 that can resist the growth of Plasmodium in the ring stage at a concentration similar to Chloroquine. The chemical structures of all 3 substances are compared to Chloroquine as shown in Figure 1.

Figure 1 Chemical structure of NSC45545, NSC45570, NSC45607 compared with Chloroquine.

                  When samples of red blood cells infected with Plasmodium ring stage treated with NSC45545 were compared with samples treated with Chloroquine and analyzed using the Synchrotron FTIR microspectroscopy technique from the Siam Photon Laboratory at SLRI (Figure 2), there was no absorption of infrared in the wave number range 1,210 - 1,220. Unlike infected red blood cells that were not treated with the drug, they clearly absorbed infrared in this wavelength range (Figure 3), thus confirming that NSC45545 can inhibit the growth of malaria bacteria.

Figure 2. Image taken under an IR microscope of malaria-infected red blood cells treated with NSC45545 in an IR window.

The green square is an optical field measuring 10 x 10 square micrometers that uses infrared light to measure samples.

Figure 3 shows a comparison of secondary FTIR spectra obtained from measuring red blood cell samples infected with malaria treated with NSC45545 (green) and Chloroquine (red) and a control which were untreated Plasmodium-infected red blood cells (blue).

The wave number 1,220 is due to the absorption of infrared by the carbon-oxygen (C-O) bond in the propionate functional group (red dotted circle) in the hemozoin structure (insert picture bottom right).

                   This research can be used to develop a new type of antimalarial drug that can inhibit the production of hemozoin by malaria bacteria. All three prototype drugs must be tested for toxicity to normal human cells before further testing in animals.

 

Reference:  Kuaprasert, B. et al. Dual role of azo compounds in inhibiting Plasmodium falciparum adenosine deaminase and hemozoin biocrystallization. Exp. Parasitol. 243, 108384 (2022).

Article by Dr. Buaban Kuaprasert, Beamline scientist at Synchrotron Light Research Institute (Public Organization)

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