LIMITATIONS OF THE CURRENT TREATMENT REGIMEN FOR SURRA

There is no vaccine currently available for surra. Hence, the control and prevention is supported by drug treatment and eradication of the vector. Most of the drugs used for treatment of surra have limited therapeutic windows, with hypersensitivity and other toxicities as side effects. Further, reports of resistance and use of drugs having unexplained modes of action pose challenges in administration of the required dosage. The information related to the pharmacokinetic (PK) profiles of these drugs is unclear. Currently, there are only five scaffolds available in the market, namely phenanthridine, diminazine, suramin, quinapyramine and melarsomine (Fig. 2). There are nearly 20 generic brands from these five scaffolds, as listed in Table 1, which are used currently for treatment. All these generics have either intramuscular or subcutaneous routes of administration. Phenanthridine compounds are toxic because of their DNA intercalating action (MacGregor and Johnson, Reference MacGregor and Johnson1977). Melarsomine is ineffective against CNS infection in horses and dogs (Desquesnes et al. Reference Desquesnes, Holzmuller, Lai, Dargantes, Lun and Jittaplapong2013) and the toxic effects of this compound include diarrhoea, fever, panting, restlessness, salivation, vomiting, nephrotoxicity and CNS toxicity. Suramin treatment shows a high relapse rate and has reports of resistance in Sudan (El Rayah et al. Reference El Rayah, Kaminsky, Schmid and El Malik1999). Diminazine aceturate is known to have poor efficacy and less tolerance in horses and mules and is toxic to camels (Tuntasuvan et al. Reference Tuntasuvan, Jarabrum, Viseshakul, Mohkaew, Borisutsuwan, Theeraphan and Kongkanjana2003). Also, there are reports of meningoencephalitis after treatment with diminazene aceturate and quinapyramine sulphate (Ranjithkumar et al. Reference Ranjithkumar, Saravanan, Yadav, Kumar, Singh and Dey2013). The emergence of resistance to quinapyramine is also reported (Liao and Shen, Reference Liao and Shen2010). Hence, the identification of a new class of drugs is essential for the control of surra.

Fig. 1. Treatment of T. evansi-infected mice by 17-AAG. Group 1 (n = 5) received vehicle alone, Group 2 (n = 5) received 17-AAG (30 mg kg⁻¹ body weight, for 7 days) after 4 h of injection with parasites. Whereas, Group 3 (n = 3) received 17-AAG (50 mg kg⁻¹ body weight, for 6 days) upon confirmation of infection. Mice in group 2 and 3 survived with no relapse of infection.

Table 1. List of combination drugs available for the treatment of surra

HEAT SHOCK PROTEIN 90 AS A DRUG TARGET AGAINST SURRA

Hsp90 is a molecular chaperone which helps in supporting cellular dynamics under stress conditions. It belongs to the GHKL family of ATPases, with three distinct functional domains; N-terminal domain (NTD) with ATP binding pocket, which connects to catalytic middle domain via variable linker region and C-terminal domain. It is a dimeric protein and it functions as part of a multi-chaperone complex with accessory co-chaperones. The co-chaperones either regulate ATPase activity of Hsp90 or provide client specificity (Pearl and Prodromou, Reference Pearl and Prodromou2006; Taipale et al. Reference Taipale, Jarosz and Lindquist2010).

In biological systems, the unfolded client protein interacts with the ADP-bound complex of Hsp90. To fold an unfolded client protein, Hsp90 works in association with Hsp70 and Hsp40 as a part of a multi-chaperone complex. The dimeric protein forms a closed conformation when ATP is bound to the N-terminal pocket. Further, in the presence of co-chaperones, the multi-chaperone complex performs folding of unfolded client proteins (Siligardi et al. Reference Siligardi, Hu, Panaretou, Piper, Pearl and Prodromou2004). Inhibition of Hsp90 function causes activation of proteosomal degradation of unfolded client proteins (Taipale et al. Reference Taipale, Jarosz and Lindquist2010). Hsp90 serves as a regulatory hub of many cellular processes including signal transduction, accumulation of drug resistance and immunity. These properties make Hsp90 an important drug target in both cancer and infectious diseases (Taipale et al. Reference Taipale, Jarosz and Lindquist2010; Rochani et al. Reference Rochani, Singh and Tatu2013). Hsp90 has been shown to play an essential role in the life cycle of various parasitic protozoa such as Plasmodium falciparum, Giardia lamblia, Leishmania donovani, Entamoeba histolytica, T. cruzi and T. evansi (Wiesgigl and Clos, Reference Wiesgigl and Clos2001; Graefe et al. Reference Graefe, Wiesgigl, Gaworski, Macdonald and Clos2002; Banumathy et al. Reference Banumathy, Singh, Pavithra and Tatu2003; Pallavi et al. Reference Pallavi, Roy, Nageshan, Talukdar, Pavithra, Reddy, Venketesh, Kumar, Gupta, Singh, Yadav and Tatu2010; Nageshan et al. Reference Nageshan, Roy, Hehl and Tatu2011; Singh et al. Reference Singh, Shah and Tatu2014).

During transmission and manifestation of infection, these parasites experience high levels of stress including heat, osmotic and nutritional stress. Owing to the levels of stress experienced by these parasites, they require a robust stress response machinery. Hsp90 is a key member of this response machinery, which is highlighted by the growth arrest of these protozoa upon Hsp90 inhibition.

Studies on different Trypanosoma species show their dependence on Hsp90 function for their growth and survival. For example, it was observed that transcripts of Hsp70 and Hsp90 were abundant in the blood stage of T. brucei compared with the insect stages, suggesting that the stress response causes up-regulation of Hsp90 (Van der Ploeg et al. Reference Van der Ploeg, Giannini and Cantor1985). In T. brucei, Hsp90 (TbHsp83) inhibition leads to growth arrest of both the procyclic and the bloodstream forms. Compared with humans, T. brucei (bloodstream form) shows 50- to 60-fold higher sensitivity to inhibition by GA derivative 17-allylamino 17-demethoxy geldanamycin (17-AAG) (Jones et al. Reference Jones, Anderson, Singha and Chaudhuri2008). 17-AAG treatment resulted in increased sensitization of T. brucei to heat shock, leading to severe morphological abnormalities (Meyer and Shapiro, Reference Meyer and Shapiro2013). Mouse studies using a soluble GA derivative, 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), cured mice of T. brucei infection (Meyer and Shapiro, Reference Meyer and Shapiro2013).

Studies on T. cruzi have shown that Hsp90 plays an important role in stage transition. Inhibition of Hsp90 by GA not only inhibits proliferation but also arrests epimastigotes in the G₁ phase of the life cycle. Although in the presence of GA, trypomastigotes (bloodstream form) converted to spheromastigote-like forms, differentiation into epimastigotes was permanently blocked (Graefe et al. Reference Graefe, Wiesgigl, Gaworski, Macdonald and Clos2002).

In the case of T. evansi, stress can be attributed to two levels of heat shock experienced by the parasite. First, when T. evansi gets transmitted to the host where the host body temperature is higher than the vector; second, during fever experienced by the host on infection with T. evansi. Studies on T. brucei and T. cruzi can be extrapolated to T. evansi, as T. evansi is thought to be evolved from T. brucei by losing maxi-circle DNA from kinetoplastids (Wells, Reference Wells1984) and thus these species are very closely related. Further, Pallavi et al. (Reference Pallavi, Roy, Nageshan, Talukdar, Pavithra, Reddy, Venketesh, Kumar, Gupta, Singh, Yadav and Tatu2010) demonstrated the in vivo efficacy of Hsp90 inhibition for the treatment of surra (Pallavi et al. Reference Pallavi, Roy, Nageshan, Talukdar, Pavithra, Reddy, Venketesh, Kumar, Gupta, Singh, Yadav and Tatu2010). This study showed that 17-AAG specifically binds to T. evansi Hsp90 (TeHsp90) and was effective against surra in the pre-clinical rodent model. Intra-peritoneal administration of 17-AAG at 30 mg kg⁻¹ body weight for 4 continuous days was able to cure T. evansi infection in mice with a 60% survival rate with no relapse of infection for the 90 days that the mice were monitored (Pallavi et al. Reference Pallavi, Roy, Nageshan, Talukdar, Pavithra, Reddy, Venketesh, Kumar, Gupta, Singh, Yadav and Tatu2010). Additionally, treatment with an increased dose of 50 mg kg⁻¹ body weight showed 100% survival, as shown in Fig. 1 (unpublished data). This pre-clinical study for surra treatment, along with studies on different Trypanosoma spp., demonstrates the potential of Hsp90 as a chemotherapeutic target for T. evansi infection.

Fig. 2. Drugs currently used for treatment of animal trypanosomiasis.

HSP90 TARGETED ANTI-TRYPANOSOMAL COMPOUNDS

Ansamycin scaffolds were the first to be explored for inhibitory activity against Hsp90 and are specific towards the GHKL family of ATPases. They are a class of antibiotics which are extensively used against microbial infections. They are polyketide compounds obtained from natural sources. Ansamycins are categorized as benzoquinone and naphthoquinone derivatives. The benzoquinone ansamycins family includes geldanamycin, the parent molecule of 17-AAG. Geldanamycin was first isolated from the soil bacteria Streptomyces hygroscopicus var. geldanus. Although being a potent Hsp90 inhibitor, the molecule remained out of clinical reach due to its hepatoxicity (Samuni et al. Reference Samuni, Ishii, Hyodo, Samuni, Krishna, Goldstein and Mitchell2010). The relatively safe medicinal profile of 17-AAG enabled it to enter clinical trials for cancer in humans (Rochani et al. Reference Rochani, Singh and Tatu2013). It is noted that the binding affinity of the geldanamycin nucleus to Hsp90 from parasites such as Plasmodium and Trypanosoma is stronger compared with ATP (Pallavi et al. Reference Pallavi, Roy, Nageshan, Talukdar, Pavithra, Reddy, Venketesh, Kumar, Gupta, Singh, Yadav and Tatu2010). As discussed in the previous section, Trypanosoma shows higher sensitivity to Hsp90 inhibitors compared with host cells. Apart from 17-AAG, there are three other molecules, namely IPI-504, IPI-493 and 17-DMAG, from the ansamycin class which are being explored for their Hsp90 inhibitory activities in cancer and can be repositioned to see their effect on Trypanosoma infection.

Another important scaffold of Hsp90 inhibitors belongs to the purine family. The purine analogues are known to target the ATP binding site at NTD to inhibit ATPase activity. Some of the purine analogues, like allopurinol, have shown inhibitory activity against T. cruzi amastigotes in vitro with a 50% growth inhibitory concentration of 3 μ m (Nakajima-Shimada et al. Reference Nakajima-Shimada, Hirota and Aoki1996). 3′-Deoxyinosine and 3′-deoxyadenosine also suppressed T. cruzi growth in host cells, although with less sensitivity (Nakajima-Shimada et al. Reference Nakajima-Shimada, Hirota and Aoki1996). The adenosine analogues PU-H71, Debio 0932, MPC-3100 and CNF 2024 (BIIB021) have entered clinical trials for cancer and are known to have safe profiles. These compounds can be tested against TeHsp90 as a repositioning strategy. The structures of these adenosine analogues are shown in Fig. 3.

Fig. 3. Structures of Hsp90 inhibitors.

Pizarro et al. (Reference Pizarro, Hills, Senisterra, Wernimont, Mackenzie, Norcross, Ferguson, Wyatt, Gilbert and Hui2013) have shown the binding affinities of purine and benzamide analogues towards NTD of TbHsp83 protein. It is interesting to note that one of the benzamide derivatives (NVP-AUY922) is reported to have a K _d value of 3 nm. This study also reported that GA and its derivatives such as 17-AAG and 17-DMAG, along with the ansamycin compound macbecin, show higher binding affinities to TbHsp83 than human Hsp90. GA derivatives and compounds synthesized by the group showed sub micro-molar EC₅₀ for T. brucei (Pizarro et al. Reference Pizarro, Hills, Senisterra, Wernimont, Mackenzie, Norcross, Ferguson, Wyatt, Gilbert and Hui2013). This study can be extrapolated to T. evansi for exploring their effect as anti-surra drugs since TeHsp90 and TbHsp83 are 100% identical (Pallavi et al. Reference Pallavi, Roy, Nageshan, Talukdar, Pavithra, Reddy, Venketesh, Kumar, Gupta, Singh, Yadav and Tatu2010).

Radicicol is a naturally occurring Hsp90 inhibitor isolated from Diheterospora chlamydosporia which has an in vitro IC₅₀ value of 20–23 nm for cancer cell lines (Sharma et al. Reference Sharma, Agatsuma and Nakano1998; Wang et al. Reference Wang, Shen and Blagg2006). Against the bloodstream form of T. brucei, radicicol shows potency at the nano-molar scale (Meyer and Shapiro, Reference Meyer and Shapiro2013). However, radicicol is not a very good choice of inhibitor owing to its reduced stability in vivo but the resorcinol scaffold of radicicol can be used for developing new derivatives which can be explored for their Hsp90 inhibitory activity. Combining resorcinol with triazoles, isoxazoles and benzisoxazoles resulted in increased potency of Hsp90 inhibitors (Janin, Reference Janin2005; Du et al. Reference Du, Moulick, Rodina, Aguirre, Felts, Dingledine, Fu and Chiosis2007; Gopalsamy et al. Reference Gopalsamy, Shi, Golas, Vogan, Jacob, Johnson, Lee, Nilakantan, Petersen, Svenson, Chopra, Tam, Wen, Ellingboe, Arndt and Boschelli2008; Taldone et al. Reference Taldone, Sun and Chiosis2009). Further, the lead optimization of a resorcinol heterocyclic hybrid gave rise to STA9090 or Ganetispib and NVP-AUY922. Both the molecules are under clinical trials for use as anti-cancer drugs (Dutton et al. Reference Dutton, Kitson, Parry-Morris, Roe, Prodromou and Moody2014). They have shown promising results for human applications. Their use for the treatment of surra remains unexplored.

The Plant Kingdom has also been explored extensively for newer scaffolds which can be optimized for designing novel Hsp90 inhibitors. A triterpenoid called gedunin, isolated from Azadirachta indica, is shown to have affinity towards PfHsp90 (Brandt et al. Reference Brandt, Schmidt, Prisinzano and Blagg2008) and the methanolic extract of A. indica in combination with diminazene aceturate provided anti-trypanosomal activity against mice infection with T. evansi (Omoja et al. Reference Omoja, Anaga, Obidike, Ihedioha, Umeakuana, Mhomga, Asuzu and Anika2011). Hence, it will be of interest to find out if the anti-trypanosomal activity of this extract is due to inhibition of Hsp90 from the parasite. Derivatives of coumarin scaffold have emerged as novel options of exploring new Hsp90 inhibitors. Novobiocin was the first coumarin analogue shown to have affinity towards the C-terminal domain of Hsp90 (Marcu et al. Reference Marcu, Schulte and Neckers2000; Donnelly and Blagg, Reference Donnelly and Blagg2008). Novobiocin shows growth inhibition of Trypanosoma at micro-molar levels (Meyer and Shapiro, Reference Meyer and Shapiro2013). Epigallocatechin-3-gallate (EGCG) is a polyphenolic coumarin molecule which binds to the C-terminal domain of Hsp90. It is shown to bind to a stretch of 538–728 residues of C-terminal Hsp90 (Yin et al. Reference Yin, Henry and Gasiewicz2009). The exact mechanism of inhibition is still unknown. It has been observed that EGCG is prone to epimerization and auto-oxidation (Khandelwal et al. Reference Khandelwal, Hall and Blagg2013). Hence, attempts were made to develop stable derivatives for increasing the stability of molecule by modifying substitutions on EGCG rings. The structure activity relationship (SAR) study of this molecule showed that the addition of a phenolic group on Ring A and Ring D (Fig. 3) has positive and negative impacts respectively on Hsp90 inhibition. Also, the inclusion of novobiocin-derivative phenyl benzoate in place of gallic acid of EGCG was found to be suitable for Hsp90 inhibition, as shown in Fig. 3. It is also noted that EGCG exhibits inhibition of T. evansi in a mouse model of infection at ∼30 μ m concentration (Vigueira et al. Reference Vigueira, Ray, Martin, Ligon and Paul2012). This increased inhibitory concentration can also be attributed towards the instability of the molecule, as stated previously.

These studies highlight the potential of repositioning currently available Hsp90 inhibitors for treatment of surra. In addition to repositioning current Hsp90 inhibitors, there is a need to develop new inhibitors with even higher specificity towards TeHsp90.