- Email: [email protected]
Repurposing of Potent Drug Candidates for Multiparasite Targeting
TREPAR 1595 No. of Pages 3
Forum
Repurposing of Potent Drug Candidates for Multiparasite Targeting Vitul Jain1 and Amit Sharma1,* Parasite-directed drug discovery efforts require sustained and substantial scientific resources. Many eukaryotic parasites share similarities in metabolic pathways and housekeeping genes, as evident from their underlying protein sequences. Their subsequent structural congruence within enzyme active sites can thus be leveraged for multiparasite targeting using similar or identical drug probes. This bodes well for delivering new anti-infectives. Targeting Protein Synthesis Many diverse and successful anti-infective drugs block the protein synthesis process, and this fact exemplifies the absolute cellular dependence on protein translation for its survival. It is well known that evolutionary adaptations of different organisms have provided significant molecular and atomic differences to allow selective chemical inhibition of bacterial over human ribosomes. In addition, cellular protein synthesis is dependent on enzymatic productivity of each of its aminoacyl-tRNA synthetases ([3_TD$IF]aaRSs), and lately [3_TD$IF]aaRSs druggability in the context of pathogens has been highlighted repeatedly [1]. Numerous groups have discovered the utility of targeting pathogenic aaRSs for discovering new drug-like molecules. Winzeler and colleagues performed phenotypic screening of 1.7 million compounds and identified cladosporin (CL) as a potent antimalarial agent that kills intraerythrocytic parasites [2]. They
found CL to be equipotent against both liver and blood-stage malaria parasites, and CL displayed high selectivity against mammalian cells [2]. In their follow up study, they discovered malaria parasite lysyl-tRNA synthetase (PfKRS) as the molecular target of cladosporin [3]. Shortly thereafter, our group resolved the structural basis for binding of CL to the active site of PfKRS [4]. It was discovered that CL mimics the adenosine moiety of the natural substrate ATP, and hence acted via competitive inhibition of the enzyme substrate [3,4]. We had also noted the remarkable sequence and structural congruence in the active sites of not only other malaria parasite KRSs but also in KRSs of diverse eukaryotic pathogens such as Loa loa and Schistosoma mansoni [4]. In line with this, we have recently validated the proposed Loa loa and Schistosoma mansoni KRSs as valuable new targets for development of antihelminthic drugs using CL as the probe molecule [5]. Hence, CL can be repurposed and utilized against a number of additional pathogens where the KRS active site residues are invariant [4_TD$IF](Figure 1[5_TD$IF]A). Similarly, Cusack and colleagues had shown the interactions and efficacy of the Anacor pharmaceutical compound AN2690 (benzoxaborole – a boron-containing pharmacophore) against leucyltRNA synthetase (LRS), where the drug bound to the enzyme's editing subdomain called edt-LRS [6]. Their structural studies revealed an oxaborole tRNA trapping (OBORT) mechanism that was dependent on the unique boron atom in AN2690based series [6]. Boron formed covalent bonds with the 20 and 30 oxygen of the ribose ring of A76-tRNA to yield a stable tRNA-AN2690 adduct in the LRS editing domain, which thence blocked the LRS activity and arrested protein synthesis [6]. This led to the successful clinical trials of AN2690 (Tavaborole) against onychomycosis, a fungal disease caused by Candida albicans, and eventually resulted in the release of the now marketed drug KerydinTM [6]. More recently, compounds
from the AN2690-based series have been shown to target editing domains of LRSs from many parasites, including Plasmodium falciparum, Cryptosporidium muris[1_TD$IF] and Toxoplasma gondii[6_TD$IF] (Figure 1B) [7,8]. Hence the above two examples [7_TD$IF]exemplify how valuable repurposing of potent lead molecules can be for targeting orthologous proteins in multiple parasites where active site conservation is evident.
Targeting Protein Degradation Proteostasis – the cellular balance of protein synthesis and degradation – is vital for cell survival, and even subtle alterations in it can lead to cell death [9]. As mentioned earlier, biomolecular cohorts that drive protein synthesis, including ribosomes, translational factors[1_TD$IF] and aminoacyl-tRNA synthetases, are increasingly being exploited for pathogenfocused drug discovery. Befittingly, a recent study discovered that the protein degradation pathway, via its 20S proteasome, equally offers itself as highly druggable in the context of human parasites that cause leishmaniasis, Chagas disease[1_TD$IF] and sleeping sickness [10]. These three infections affect predominantly poor communities in Latin America, Asia[1_TD$IF] and Africa, where they affect >20 million people and cause >50 000 deaths annually. Khare et al. performed phenotypic screening with 3 million compounds in proliferation assays of Leishmania donovani, Trypanosoma cruzi[1_TD$IF] and Trypanosoma brucei [10]. They discovered an azabenzoxazole compound called GNF6702 that was capable of stopping growth of all three parasites [10]. Their subsequent mechanistic studies to identify the pan-kinetoplastid target of GNF6702 (using resistant strain generation) identified 26S proteasome b4 subunit as the molecular target [10]. They revealed that GNF6702 blocked the proteolytic (chymotrypsin-like) activity possessed by the b5 subunit (which is bound to the b4 subunit) in a noncompetitive manner [10]. Further validation of the parasite proteasomes as the selected
Trends in Parasitology, Month Year, Vol. xx, No. yy
1
TREPAR 1595 No. of Pages 3
Drugs
Targets
Effecve against
(A)
P. falciparum L. loa S. mansoni
OH O
O OH
O
Cladosporin Lysyl-tRNA synthetase
(B)
P. falciparum C. muris T. gondii C. albicans
OH B O
F
AN2690
Leucyl-tRNA synthetase
(C)
L. donovani T. cruzi T. brucei
F N
N N
N N
O N
GNF6702
O
N
20S proteasome
Figure 1. Drugs That Target Multiple Parasites. (A) Cladosporin is shown here with its molecular target lysyl-tRNA synthetase (PDB code: 4PG3). Cladosporin is active against Plasmodium falciparum, Loa loa[1_TD$IF] and Schistosoma mansoni lysyl-tRNA synthetases. (B) Tavaborole or AN2690 is shown here along with its molecular target leucyl-tRNA synthetase-editing domain (colored in green, PDB code: 2V0G). Tavaborole is being marketed for the treatment of onychomycosis, and its derivatives have been recently shown to inhibit the growth of Plasmodium falciparum, Cryptosporidium muris[1_TD$IF] and Toxoplasma gondii. (C) Drug molecule GNF6702 and its molecular target the 20S proteasome (PDB code: 1IRU). GNF6702 has been shown to kill three kinetoplastid parasites (Leishmania donovani, Trypanosoma cruzi[1_TD$IF] and Trypanosoma brucei) from diverse niches in mouse models of disease.
Repurposing Going Forward It is evident that drug discovery pipelines worldwide already have a growing volume of new data from both extensive phenotypic screening as well as target-based approaches for several prevalent human and livestock pathogens. However, given the paucity of, and access to, new druglike libraries, discovery of potent leads against many pathogens will additionally need to rely on drug repurposing. Although seemingly hackneyed, repurposing can be very powerful in delivering new hope [10_TD$IF]for rekindling drug discovery efforts, as evidenced by the currently pursued druggable targets of [1_TD$IF]parasite aaRSs and proteasomes. It is noteworthy that these two classes of molecular targets were perhaps partly ignored earlier, given their housekeeping and conserved roles and assumed cross-reactivity with host machinery. Other repurposed drug targets, such as kinases and phosphodiesterases, have also been pursued for trypanosomatid diseases and malaria [11,12]. Repurposing has given [12_TD$IF]several protein targets a rebirth, and so far this renewal looks very promising against a host of human pathogenic diseases. Ribosomes have already been extensively exploited for drug development, and now aaRSs and proteasomes represent the new incarnates worthy of attention for drugging. These molecular targets participate jointly to maintain balance in the cellular proteostasis machinery, and they together promise to deliver more antiparasitic drugs. [13_TD$IF]Acknowledgment This research was supported by DBT grant PR6303 to
target of GNF6702 was also supported via point mutations in the gene encoding the b4 subunit that were able to confer resistance to GNF6702 [10]. Finally, the lead compound GNF6702, when tested, seemed highly selective for the parasite proteasome, and did not inhibit the human counterpart, thus lending hope for lower or no toxicity to the host. Hence, either GNF6702, which is under evaluation in preclinical toxicity studies
2
Trends in Parasitology, Month Year, Vol. xx, No. yy
[10], or its analogs, have the potential to yield either a unified single new treatment for multiple kinetoplastid infections, or [8_TD$IF]multiple tailored drugs based on the potent GNF6702. So, once again it is evident that the conservation in housekeeping proteostasis machinery (proteasome) across diverse parasites has allowed repurposing of one potent lead molecule against related parasites[9_TD$IF] (Figure 1C).
A.S and DBT grant PR3084 to A.S. A.S is additionally supported by the JC Bose fellowship. A Senior Research Fellowship from DBT to V.J. is also acknowledged. [2_TD$IF]We thank I. Aves, B.C. Indus and I. Pantig for constant encouragement. 1 Molecular Medicine Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Road, New Delhi[1_TD$IF] 110067, India
*Correspondence: [email protected] (A. Sharma). http://dx.doi.org/10.1016/j.pt.2016.12.007
TREPAR 1595 No. of Pages 3
References 1. Jain, V. et al. (2015) Structure of prolyl-tRNA synthetaseHalofuginone complex provides basis for development of drugs against malaria and toxoplasmosis. Structure 23, 819–829 2. Plouffe, D. et al. (2008) In silico activity profiling reveals the mechanism of action of antimalarials discovered in a highthroughput screen. Proc. Natl. Acad. Sci. U. S. A. 105, 9059–9064 3. Hoepfner, D. et al. (2012) Selective and specific inhibition of the Plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin. Cell Host Microbe 11, 654–663
4. Khan, S. et al. (2014) Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin. J. Struct. Funct. Genom. 15, 63–71 5. Sharma, A. et al. (2016) Protein translation enzyme lysyl-tRNA synthetase presents a new target for drugdevelopment against causative agents of loiasis and schistosomiasis. PLoS Negl. Trop. Dis. 10, e0005084
8. Palencia, A. et al. (2016) Cryptosporidium and toxoplasma parasites are inhibited by a benzoxaborole targeting leucyltRNA synthetase. Antimicrob. Agents Chemother. 60, 5817–5827 9. Hetz, C. et al. (2015) Proteostasis control by the unfolded protein response. Nat. Cell Biol. 17, 829–838 10. Khare, S. et al. (2016) Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness. Nature 537, 229–233
6. Rock, F.L. et al. (2007) An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site. Science 316, 1759–1761
11. Muller, J. and Hemphill, A. (2016) Drug target identification in protozoan parasites. Expert Opin. Drug Discov. 11, 815–824
7. Sonoiki, E. et al. (2016) Antimalarial benzoxaboroles target Plasmodium falciparum leucyl-tRNA synthetase. Antimicrob. Agents Chemother. 60, 4886–4895
12. Klug, D.M. et al. (2016) Repurposing strategies for tropical disease drug discovery. Bioorg. Med. Chem. Lett. 26, 2569–2576
Trends in Parasitology, Month Year, Vol. xx, No. yy
3
Forum
Repurposing of Potent Drug Candidates for Multiparasite Targeting Vitul Jain1 and Amit Sharma1,* Parasite-directed drug discovery efforts require sustained and substantial scientific resources. Many eukaryotic parasites share similarities in metabolic pathways and housekeeping genes, as evident from their underlying protein sequences. Their subsequent structural congruence within enzyme active sites can thus be leveraged for multiparasite targeting using similar or identical drug probes. This bodes well for delivering new anti-infectives. Targeting Protein Synthesis Many diverse and successful anti-infective drugs block the protein synthesis process, and this fact exemplifies the absolute cellular dependence on protein translation for its survival. It is well known that evolutionary adaptations of different organisms have provided significant molecular and atomic differences to allow selective chemical inhibition of bacterial over human ribosomes. In addition, cellular protein synthesis is dependent on enzymatic productivity of each of its aminoacyl-tRNA synthetases ([3_TD$IF]aaRSs), and lately [3_TD$IF]aaRSs druggability in the context of pathogens has been highlighted repeatedly [1]. Numerous groups have discovered the utility of targeting pathogenic aaRSs for discovering new drug-like molecules. Winzeler and colleagues performed phenotypic screening of 1.7 million compounds and identified cladosporin (CL) as a potent antimalarial agent that kills intraerythrocytic parasites [2]. They
found CL to be equipotent against both liver and blood-stage malaria parasites, and CL displayed high selectivity against mammalian cells [2]. In their follow up study, they discovered malaria parasite lysyl-tRNA synthetase (PfKRS) as the molecular target of cladosporin [3]. Shortly thereafter, our group resolved the structural basis for binding of CL to the active site of PfKRS [4]. It was discovered that CL mimics the adenosine moiety of the natural substrate ATP, and hence acted via competitive inhibition of the enzyme substrate [3,4]. We had also noted the remarkable sequence and structural congruence in the active sites of not only other malaria parasite KRSs but also in KRSs of diverse eukaryotic pathogens such as Loa loa and Schistosoma mansoni [4]. In line with this, we have recently validated the proposed Loa loa and Schistosoma mansoni KRSs as valuable new targets for development of antihelminthic drugs using CL as the probe molecule [5]. Hence, CL can be repurposed and utilized against a number of additional pathogens where the KRS active site residues are invariant [4_TD$IF](Figure 1[5_TD$IF]A). Similarly, Cusack and colleagues had shown the interactions and efficacy of the Anacor pharmaceutical compound AN2690 (benzoxaborole – a boron-containing pharmacophore) against leucyltRNA synthetase (LRS), where the drug bound to the enzyme's editing subdomain called edt-LRS [6]. Their structural studies revealed an oxaborole tRNA trapping (OBORT) mechanism that was dependent on the unique boron atom in AN2690based series [6]. Boron formed covalent bonds with the 20 and 30 oxygen of the ribose ring of A76-tRNA to yield a stable tRNA-AN2690 adduct in the LRS editing domain, which thence blocked the LRS activity and arrested protein synthesis [6]. This led to the successful clinical trials of AN2690 (Tavaborole) against onychomycosis, a fungal disease caused by Candida albicans, and eventually resulted in the release of the now marketed drug KerydinTM [6]. More recently, compounds
from the AN2690-based series have been shown to target editing domains of LRSs from many parasites, including Plasmodium falciparum, Cryptosporidium muris[1_TD$IF] and Toxoplasma gondii[6_TD$IF] (Figure 1B) [7,8]. Hence the above two examples [7_TD$IF]exemplify how valuable repurposing of potent lead molecules can be for targeting orthologous proteins in multiple parasites where active site conservation is evident.
Targeting Protein Degradation Proteostasis – the cellular balance of protein synthesis and degradation – is vital for cell survival, and even subtle alterations in it can lead to cell death [9]. As mentioned earlier, biomolecular cohorts that drive protein synthesis, including ribosomes, translational factors[1_TD$IF] and aminoacyl-tRNA synthetases, are increasingly being exploited for pathogenfocused drug discovery. Befittingly, a recent study discovered that the protein degradation pathway, via its 20S proteasome, equally offers itself as highly druggable in the context of human parasites that cause leishmaniasis, Chagas disease[1_TD$IF] and sleeping sickness [10]. These three infections affect predominantly poor communities in Latin America, Asia[1_TD$IF] and Africa, where they affect >20 million people and cause >50 000 deaths annually. Khare et al. performed phenotypic screening with 3 million compounds in proliferation assays of Leishmania donovani, Trypanosoma cruzi[1_TD$IF] and Trypanosoma brucei [10]. They discovered an azabenzoxazole compound called GNF6702 that was capable of stopping growth of all three parasites [10]. Their subsequent mechanistic studies to identify the pan-kinetoplastid target of GNF6702 (using resistant strain generation) identified 26S proteasome b4 subunit as the molecular target [10]. They revealed that GNF6702 blocked the proteolytic (chymotrypsin-like) activity possessed by the b5 subunit (which is bound to the b4 subunit) in a noncompetitive manner [10]. Further validation of the parasite proteasomes as the selected
Trends in Parasitology, Month Year, Vol. xx, No. yy
1
TREPAR 1595 No. of Pages 3
Drugs
Targets
Effecve against
(A)
P. falciparum L. loa S. mansoni
OH O
O OH
O
Cladosporin Lysyl-tRNA synthetase
(B)
P. falciparum C. muris T. gondii C. albicans
OH B O
F
AN2690
Leucyl-tRNA synthetase
(C)
L. donovani T. cruzi T. brucei
F N
N N
N N
O N
GNF6702
O
N
20S proteasome
Figure 1. Drugs That Target Multiple Parasites. (A) Cladosporin is shown here with its molecular target lysyl-tRNA synthetase (PDB code: 4PG3). Cladosporin is active against Plasmodium falciparum, Loa loa[1_TD$IF] and Schistosoma mansoni lysyl-tRNA synthetases. (B) Tavaborole or AN2690 is shown here along with its molecular target leucyl-tRNA synthetase-editing domain (colored in green, PDB code: 2V0G). Tavaborole is being marketed for the treatment of onychomycosis, and its derivatives have been recently shown to inhibit the growth of Plasmodium falciparum, Cryptosporidium muris[1_TD$IF] and Toxoplasma gondii. (C) Drug molecule GNF6702 and its molecular target the 20S proteasome (PDB code: 1IRU). GNF6702 has been shown to kill three kinetoplastid parasites (Leishmania donovani, Trypanosoma cruzi[1_TD$IF] and Trypanosoma brucei) from diverse niches in mouse models of disease.
Repurposing Going Forward It is evident that drug discovery pipelines worldwide already have a growing volume of new data from both extensive phenotypic screening as well as target-based approaches for several prevalent human and livestock pathogens. However, given the paucity of, and access to, new druglike libraries, discovery of potent leads against many pathogens will additionally need to rely on drug repurposing. Although seemingly hackneyed, repurposing can be very powerful in delivering new hope [10_TD$IF]for rekindling drug discovery efforts, as evidenced by the currently pursued druggable targets of [1_TD$IF]parasite aaRSs and proteasomes. It is noteworthy that these two classes of molecular targets were perhaps partly ignored earlier, given their housekeeping and conserved roles and assumed cross-reactivity with host machinery. Other repurposed drug targets, such as kinases and phosphodiesterases, have also been pursued for trypanosomatid diseases and malaria [11,12]. Repurposing has given [12_TD$IF]several protein targets a rebirth, and so far this renewal looks very promising against a host of human pathogenic diseases. Ribosomes have already been extensively exploited for drug development, and now aaRSs and proteasomes represent the new incarnates worthy of attention for drugging. These molecular targets participate jointly to maintain balance in the cellular proteostasis machinery, and they together promise to deliver more antiparasitic drugs. [13_TD$IF]Acknowledgment This research was supported by DBT grant PR6303 to
target of GNF6702 was also supported via point mutations in the gene encoding the b4 subunit that were able to confer resistance to GNF6702 [10]. Finally, the lead compound GNF6702, when tested, seemed highly selective for the parasite proteasome, and did not inhibit the human counterpart, thus lending hope for lower or no toxicity to the host. Hence, either GNF6702, which is under evaluation in preclinical toxicity studies
2
Trends in Parasitology, Month Year, Vol. xx, No. yy
[10], or its analogs, have the potential to yield either a unified single new treatment for multiple kinetoplastid infections, or [8_TD$IF]multiple tailored drugs based on the potent GNF6702. So, once again it is evident that the conservation in housekeeping proteostasis machinery (proteasome) across diverse parasites has allowed repurposing of one potent lead molecule against related parasites[9_TD$IF] (Figure 1C).
A.S and DBT grant PR3084 to A.S. A.S is additionally supported by the JC Bose fellowship. A Senior Research Fellowship from DBT to V.J. is also acknowledged. [2_TD$IF]We thank I. Aves, B.C. Indus and I. Pantig for constant encouragement. 1 Molecular Medicine Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Road, New Delhi[1_TD$IF] 110067, India
*Correspondence: [email protected] (A. Sharma). http://dx.doi.org/10.1016/j.pt.2016.12.007
TREPAR 1595 No. of Pages 3
References 1. Jain, V. et al. (2015) Structure of prolyl-tRNA synthetaseHalofuginone complex provides basis for development of drugs against malaria and toxoplasmosis. Structure 23, 819–829 2. Plouffe, D. et al. (2008) In silico activity profiling reveals the mechanism of action of antimalarials discovered in a highthroughput screen. Proc. Natl. Acad. Sci. U. S. A. 105, 9059–9064 3. Hoepfner, D. et al. (2012) Selective and specific inhibition of the Plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin. Cell Host Microbe 11, 654–663
4. Khan, S. et al. (2014) Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin. J. Struct. Funct. Genom. 15, 63–71 5. Sharma, A. et al. (2016) Protein translation enzyme lysyl-tRNA synthetase presents a new target for drugdevelopment against causative agents of loiasis and schistosomiasis. PLoS Negl. Trop. Dis. 10, e0005084
8. Palencia, A. et al. (2016) Cryptosporidium and toxoplasma parasites are inhibited by a benzoxaborole targeting leucyltRNA synthetase. Antimicrob. Agents Chemother. 60, 5817–5827 9. Hetz, C. et al. (2015) Proteostasis control by the unfolded protein response. Nat. Cell Biol. 17, 829–838 10. Khare, S. et al. (2016) Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness. Nature 537, 229–233
6. Rock, F.L. et al. (2007) An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site. Science 316, 1759–1761
11. Muller, J. and Hemphill, A. (2016) Drug target identification in protozoan parasites. Expert Opin. Drug Discov. 11, 815–824
7. Sonoiki, E. et al. (2016) Antimalarial benzoxaboroles target Plasmodium falciparum leucyl-tRNA synthetase. Antimicrob. Agents Chemother. 60, 4886–4895
12. Klug, D.M. et al. (2016) Repurposing strategies for tropical disease drug discovery. Bioorg. Med. Chem. Lett. 26, 2569–2576
Trends in Parasitology, Month Year, Vol. xx, No. yy
3