Introduction

1.1 Antifungal drug research and development

Approximately 1.2 billion individuals worldwide suffer from fungal infections, and the occurrence of these infections has significantly increased in recent years due to a rise in the number of immunocompromised patients, such as patients with AIDS or those with cancer, organ transplant, or autoimmune disease who require immunosuppressive therapy ¹. Unlike superficial infections that cause local, benign, or self-limiting diseases, invasive fungal infections (IFIs) are deep-seated and include bloodstream and systemic infections as well as infection of specific organs. IFIs are frequently caused by yeast pathogens such as Candida and Cryptococcus, filamentous fungi such as Aspergillus, Fusarium, or Mucor, or, less frequently, dimorphic fungi, including Coccidioides, Blastomyces, or Histoplasma.

Currently, only three main classes of antifungals are approved for treatment of patients with IFIs: polyenes, triazoles, and echinocandins. These agents target ergosterol, lanosterol 14-α-demethylase, and β-1,3 glucan synthase, respectively ². Because our current antifungal therapies have problems like great efficacy but with significant toxicities, emergence of drug resistance and low potency against specific fungal pathogen, new drugs are needed to extend the limited antifungal arsenal ³.

1.2 Cryptococcus neoformans

The pathogenic yeast Cryptococcus species belong to Basidiomycota and are divergent from other human fungal pathogens Candida and Aspergillus, which belong to ascomycetous fungi. Two major Cryptococcus species including C. neoformans and C.

gattii attribute to cryptococcosis in human. The virulence factors of the pathogen include the capability of survival in host temperature (37 ºC), the production of capsule and

melanin which are specific structures for escaping from immune system and restricted environment ⁴. Current studies also reported quorum sensing between fungal populations and unique changing in cell morphology as important virulence factors in host ⁵. Cryptococcus spp. are ubiquitous in the environment and usually abundant in avian excreta and various trees with waxier cuticles ⁶. Therefore, the epidemiology of cryptococcosis demonstrated the worldwide distribution of clinical cases. It is estimated that one million cases of cryptococcosis were diagnosed each year and over half of the patients were died from cryptococcal meningoencephalitis ^{7, 8}. Meanwhile, high mortality in different countries is noticed. Two main reasons contributed to the high mortality possibly include drug shortage in low-resources countries and strong association with human immunodeficiency virus infection ⁹. Others people at risk are the immunocompromised patients who take immunosuppressive medications ⁹. Hence, the adequate treatment and new antifungal for prophylaxis and management of cryptococcosis is important.

1.3 Currently used antifungal agents for Cryptococcus

C. neoformans is an opportunistic pathogen that colonizes in different parts of human body and is life-threatening if no appropriate therapeutic treatments performed. The pathogen infects individuals by inhalation into lungs and thus cause pulmonary cryptococcosis.

Afterward, C. neoformans can spread quickly to other organs and disseminate to central nerve system and lead to overwhelming cryptococcal meningoencephalitis. Cryptococcosis is a challenging disease due to limited utilizable antifungal drugs. Currently, the practical guideline for management of cryptococcosis only articulate two regimens: fluconazole monotherapy and amphotericin B with flucytosine as combination therapy ¹⁰. Besides,

amphotericin B can cause severe nephrotoxicity, thus newer formulation liposomal amphotericin B is used when it comes to renal impairment patients. Fluconazole is a fungistatic agents widely used as an initial therapy. It is a safer and cheaper antifungal drugs compared with others. However, the cases of fluconazole resistance in patients with cryptococcal meningitis have more frequently been reported in Africa ¹¹. Due to the risk of terrible side effects and emergence of drug resistant isolates ¹², more novel antifungal agents are urgent to be discovered and applied to the clinic.

Multiple approaches have been launched to discover lead compounds for antifungal investigation. We could follow the target-based or phenotype-based methods to find out the potential drug targets or the candidate compounds. Nowadays, several antifungal agents are in pre-clinical stages. T-2307, an arylamidines derivatives, has been reported its broad-spectrum antifungal activity ^{13, 14}. Structure of arylamidine is similar to pentamidine which is utilized in anti-parasitic and anti-pneumocystis. Though the modes of action are still unclear, the in vitro and in vivo results both demonstrated that T-2307 was more effective than the conventional echinocandins or polyenes against human fungal pathogens ¹⁴. Besides, a series of 1,2-benzisothiazol-3(2H)-one derivatives (BZT) were also reported to exhibit antifungal activity. The mode of action of BZT was proposed by interfering fungal mitochondrial respiration system ^{13, 15}. In addition, a group of phosphoinositide dependent kinase inhibitors KP-372-1, OSU-03012 and UCN-01 have been discovered as potent antifungal compounds against Candida and Cryptococcus species ¹⁶. BHBM and D0 have been demonstrated as new class of antifungal agents with the ability to target the fungal sphingolipids, GlcCer ¹⁷. It has been demonstrated that these compounds exhibit antifungal activity against wide ranges of human fungal pathogens and are highly effective in in vivo

test.

Repurposing of off-patent drugs is another strategy for antifungal discovery. Zhai et al.

screened the Johns Hopkins Clinical Compound Library and found an early stage antibiotics, polymyxin B, for Gram-negative bacterial infection and later for, human fungal infections ^{18, 19}. Furthermore, polymyxin B is synergistic with fluconazole and exhibits fungicidal effect at lower concentration. Other repurposing cases such as tamoxifen and toremifene, acted as estrogen receptor antagonists in breast cancer therapy, have been found to exhibit anti-cryptococcal activity ²⁰. Miltefosine which is a medication for treating leishmaniasis was reported its in vitro activity against Cryptococcus species ²¹. The miltefosine analogs also exhibits broad-spectrum activity to human fungal pathogen ²². While the in vivo test of miltefosine and its analogs revealed unsuccessful efficacy. In spite of the fact that more and more antifungal agents have been discovered and identified, none of them represents as a new class of antifungal agent approved in pharmacy market yet.

1.4 Natural product tryptanthrin

Natural alkaloid tryptanthrin is a yellow needle-like crystal which could be purified from several natural sources or be synthesized through organic procedure. Tryptanthrin sublimated from a plant Indigo is first identified in 1879 by Sommargua ^{23, 24}. Afterward, it is denominated tryptanthrin due to the high production of this compound by culturing Candida lipolytica in tryptophan-contained media ^{25, 26}. The empirical formula of tryptanthrin is C15H8N2O2 (M.W. = 248.24) and its chemical structure is planar.

Tryptanthrin could be isolated from many organisms, from microorganism Schizophyllum commune, Leucopaxillus cerealis to higher plants Isatis indigotica, Polygonum tinctorium

and Wrightia tinctoria. In this study, we identify tryptanthrin from the secondary metabolites of Nocardiopsis alba, an actinobacterium existed symbiotically in the intestine of honeybee. Tryptanthrin has been mentioned to its various medical activities, including anti-microbial, anti-trypanosomal, anti-inflammatory and anti-cancer properties.25, 27, 28

Tryptanthrin possesses anti-bacterial activity against both Gram positive and negative bacteria including Bacillus subtilis, Mycobacterium tuberculosis, Porphyromonas gingivalis and methicillin-resistant Staphylococcus aureus. It could inhibit the growth of different pathogenic protozoa such as Trypanosoma brucei, Leishmania donovani and Plasmodium falciparum. Otherwise, tryptanthrin inhibits cellular expression of inflammation-related enzyme COX-2 and 5-LOX. Tryptanthrin exhibits various anti-cancer activity via decrease the secretion of growth factor, inhibition of multidrug resistance gene, cytotoxicity to specific cancer cells. All of the above demonstrated that tryptanthrin is a multifunction bioactive compound potential for pharmacological use. But little of studies reported that tryptanthrin also exhibits anti-fungal activity ^{29, 30}. Few studies described that tryptanthrin exhibits antifungal activity to topical infection fungus such as Trichophyton mentagrophytes and Malassezia furfur ^{31, 32}. As to systemic infection fungus, none of the report has mentioned tryptanthrin exhibits antifungal activity.

Studies about the mechanism of tryptanthrin bioactivity are still finite and incomplete.

Bandekar et al. indicated that tryptanthrin inhibits the growth of Escherichia coli by DNA intercalation ³³. On the other hand, few studies reported that tryptanthrin exhibits anti-cancer activity by arresting cell cycle at G0/G1 phase and inhibiting the proliferation on myeloid leukemia and neuroblastoma cells ^34-36. Conversely, tryptanthrin exhibits anti-angiogenesis by arresting cell cycle at G2/M phase ³⁷. However, the definite drug targets or

associated pathways that tryptanthrin interacts with remain elusive ³⁸.