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Structure-based targeting of orthologous pathogen proteins accelerates anti-parasitic drug discovery Vitul Jain, Arvind Sharma, Gajinder Pal Singh, Manickam Yogavel, and Amit Sharma ACS Infect. Dis., Just Accepted Manuscript • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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ACS Infectious Diseases

Structure-based targeting of orthologous pathogen proteins accelerates anti-parasitic drug discovery

Vitul Jain, Arvind Sharma, Gajinder Singh, Manickam Yogavel and Amit Sharma*

Molecular Medicine Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Road, New Delhi, 110067, India.

*Correspondence: [email protected]

Keywords: Drug discovery, Parasitic diseases, Structural biology

1 ACS Paragon Plus Environment


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Abstract Parasitic diseases caused by eukaryotic pathogens impose significant health and economic burden worldwide. The level of research funding available for many parasitic diseases is insufficient in relation to their adverse social and economic impact. In this perspective article, we discuss that extant 3D structural data on protein-inhibitor complexes can be harnessed to accelerate drug discovery against many related pathogens. Assessment of sequence conservation within drug/inhibitor-binding residues in enzyme-inhibitor complexes can be leveraged to predict and validate both new lead compounds and their molecular targets in multiple parasitic diseases. Hence, structure-based targeting of orthologous pathogen proteins accelerates the discovery of new anti-parasitic drugs. This approach offers significant benefits for jumpstarting discovery of new lead compounds and their molecular targets in diverse human, livestock and plant pathogens.

Main body Parasitic diseases (PDs) arise from infections caused by pathogens that are more common in poorer countries where they cause significant health and economic loss 1. The therapeutic options to treat several of these diseases are at times limited because of low efficacy, high toxicity and the emergence of resistance to available drugs 1. PDs have tremendous impact on quality of life, overall health and economic status of those infected; many parasites are ubiquitous and so can infect humans, livestock and/or plants. Besides the infection-associated morbidity and mortality in humans, parasites also cause misery to livestock and plants where they diminish farm and agricultural productivity. Some of the most prevalent and dreadful PDs that target humans, livestock and plants are summarized in table 1. Together, human parasites infect ~3 billion people per annum while livestock and plant infections cost >$100 billion each year. Hence, sustained investment into research and discovery of new drugs and 2 ACS Paragon Plus Environment

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their molecular targets remains a vital national and international necessity for controlling pathogens that target humans, livestock and plants.

Amongst the dominant human infections, Malaria remains a major public health problem because of resistance development against artemisinin and its partner drugs 2. However, unlike malaria, other PDs generally attract lower levels of research funding - an issue that is currently being addressed via increased public awareness of so-called neglected diseases (NDs). Of the ~3 billion humans infected in the world with one or the other parasite at any given time, most live in tropical and impoverished regions 1. Some parasitic infections by Babesia and Cryptosporidium affect not only livestock but also humans, and suffer from limited availability of therapeutics. Thus, there is an pressing need to fill the gap for rapid identification and validation of new druggable targets in various pathogens, preferably based on existing drugs or drug-like compounds that are active against related pathogens so as to quicken the discovery pace while remaining cost effective. Current methodologies focused on accelerating drug discovery mainly include repurposing of FDA approved drugs and/or cellbased high content phenotypic screenings of small molecule, drug-like libraries. The latter approaches, although beneficial, are time-consuming and can result in the identification of novel molecules that require extensive follow up mode-of-action studies. However, wherever conservation in sequence and structure of a protein drug-target is notable, identical/similar chemical scaffolds can be leveraged for attacking these ‘conserved’ active sites in orthologous pathogen proteins - and this can form the basis for testing across a whole spectrum of evolutionarily related or even unrelated parasites. With numerous examples, here we discuss and highlight the power of this methodology that in conjunction with phenotypic screening, modern genomics and drug resistance genomics can further propel drug discovery against infectious diseases.



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Targeting of orthologous pathogen proteins The publicly available 3D structural data on protein-drug inhibitor complexes from a given parasitic lineage can be utilized to assess conserved drug-binding residues amongst orthologous proteins, and to predict drug activity against different pathogens and/or their proteins (Figure 1). To start this process, the PDB structure coordinates are retrieved from the public database (RCSB). The PDB file is visualized in freely available softwares like Chimera

3-4

or PyMol, and drug interacting residues in the target protein are identified using

free online servers like PLIP

5

or Ligplot+

6-7

. In cases where structural information is

missing due to lack of crystal structure (or its resolution is limited), homology modeling (in cases where the sequence identity is significant) can be employed to predict probable binding sites and interacting residues. However, caution is required in cases of poor target conservation as it might be harder to predict the exact modes of binding interactions for potential drugs. Nonetheless, the drug can be tested directly against orthologous enzyme targets. Then, using an annotation of protein residues that recognize the drug pharmacophore, a sequence alignment is generated of multiple orthologous proteins from diverse parasites (using free online services from EBI – Clustal-Omega) 8-11. Clustal Omega alignment helps to understand variability at the residue level, and changes in the interacting residues may allow prediction of possible steric hindrances. Orthologous pathogen proteins can additionally be identified using the free online database orthoMCL 12-14. The level of sequence conservation amongst drug-contacting residues then forms the basis to test the drug either via phenotypic screening or target-based approaches. Sequence conservation assessment must go together with experimental testing of the drug against the enzyme target. For the former, EC50 values can quickly provide an estimate of the drug inhibition potential – however, despite failure in this it may be worthwhile to proceed with drug testing against orthologous pathogen proteins. This may be achieved via either enzyme activity inhibition or biophysical assays for direct


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protein-drug binding. Some commonly used techniques include Thermal Shift Assays (TSA), Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC), all of which can provide estimates of inhibitory potency and/or binding affinity

15-21

. If reasonable

inhibition and/or shift in thermal melting temperature are observed at this step (say with IC50 values between 0.1 nM and 10 µM, and/or either negative or positive alterations of ~2 °C to 10 °C in the melting profiles, and/or KD values better than 10 µM) then host cell toxicity via various cell viability tests (such as MTT or ATP assays) can be performed so that short lists of most active and selective scaffolds can be generated

22-23

. We have proposed to test

enzymes first, before cell-based screening, because at times some hits molecules can be missed or underscored when only tested in cell-based assays because of poor drug solubility profiles and/or low membrane permeability. This situation of poor availability can in some cases be circumvented by chemical modifications of the candidate drug. Given the necessity of knowledge about the molecular target of a drug in current times, enzyme-based screening first (which has its own caveat of possibly missing high potency molecules) can form an alternative approach24. The above steps can thus build a biological toolbox of drugs/inhibitors/derivatives that can provide the basis for synthesis and modifications of useful compounds and their development into potential lead compounds. Others and we have shown the benefits of above approaches that are now implemented in many parasite biology laboratories worldwide 19-21, 25-27 28 .

Here, we illustrate benefits of the above-described modules using six examples based on 3D structural information retrieved from PDBs. The target enzymes we discuss are diverse and include aminoacyl-tRNA synthetases, proteases and nucleotide biosynthesis enzymes. It is particularly noteworthy that many of these parasitic enzymes have counterparts in the human or animal host too, and yet specific lead compounds have been discovered or synthesized that



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are able to discern subtle differences in the active sites between parasite and host enzymes. Contrary to the commonly held idea that it is best to target unique enzymes in pathogens (so as to avoid host toxicity), these six examples disregard enzyme uniqueness as a prerequisite for parasite-specific drug discovery. It naturally follows that it may be reasonable to target a whole gamut of housekeeping pathogen enzymes as long as they are essential, unique and indispensable for cellular metabolic processes. Such enzymes may have been excluded from traditional focus due to their (partially) conserved nature with host proteins. Small and subtle differences in active sites or drug interaction binding residues between host and pathogen proteins (evolutionary similar within parasites but divergent from the host) may indeed be able to provide a window for specific inhibitor development, as illustrated by the examples below.

Aminoacyl-tRNA synthetases To initiate and complete the process of protein translation, ribosomes require correctly paired amino acid-transfer-RNA complexes to be available. In this process, fidelity is determined by the correct pairing of amino acids with their tRNAs – and this crucial coupling is conducted by a remarkable family of enzymes called aminoacyl-tRNA synthetases (aaRSs)

19-21, 25-27 28-

29

. Most cells synthesize proteins using a minimum of 20 different aaRSs, one for each of the

20 amino acids – and in many cases additional aaRSs are present in organelles

30

. The

selectivity for amino acids is determined by many factors including amino acid size and shape, along with the presence of editing domains that can be appended in some aaRS

31

.

Since protein synthesis is an indispensable activity for any live cell, aaRSs are an absolute necessity for cellular viability. Hence, by deduction, they seem as excellent drug targets – an idea that is quickly poisoned with the realization that the host cells have orthologs for these enzymes too. In this scenario, series of very elegant experimental studies from numerous


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laboratories have shown that despite the obvious sequence and structure overlap between host and pathogen aaRSs, it is indeed feasible to selectively target pathogen aaRSs

20-21, 32-36 37

.

The culmination of decades of cutting-edge work in phenotypic screening, cell biology, genetics, genomics, structural parasitology and medicinal chemistry have resulted in two outstanding examples of approved drugs against aaRSs. There are Tavaborole (Kerydin®, for the treatment of onychomycosis) and mupirocin (Bactroban®, antibacterial) - these drugs inhibit leucyl- and isoleucyl-tRNA synthetases respectively 38-41. Driven by such discoveries, numerous research groups have now shown that indeed many other parasitic aaRSs can be selectively targeted – and below we detail four aminoacyl-tRNA synthetases that exemplify this thrust: prolyl-, lysyl-, threonyl- and leucyl-tRNA synthetases.

The malarial prolyl-tRNA synthetase and its inhibitor halofuginone (HF) have been extensively investigated, and we now have substantial structural insights into HF’s unique mode of inhibition (Figure 2A)

19-20, 42-47

. Interestingly, from P. falciparum prolyl-tRNA

synthetase–HF complexes (Figure 2B) it became evident that the key drug binding contacts were invariant in many other pathogen prolyl-tRNA synthetases including in Toxoplasma gondii

19-20

. Via cell-based screening, it was shown that HF was highly potent against

Toxoplasma gondii (EC50 of 1 nM) infections in poultry

20

, just as it was for Eimeria and Cryptosporidium

48-51

. Further, given the extensive structural and sequence conservation

in HF-active site binding residues within pathogen prolyl-tRNA synthetases, it may be predicted that HF-like drugs will also disable PRSs from L. major, T. cruzi, C. parvum, S. mansoni and C. albicans. Although host toxicity remains an issue for HF-like drugs, it may be feasible to exploit the differences in active site surrounding residues to develop specificity against PRSs. Based on sequence conservation, it seems that HF-like molecules may be effective against parasites like C. parvum and B. bovis, and for infectious plant fungi like P.



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infestans and P. graminis (Figure 2B, C). So, although PRSs are druggable, it so far seems difficult to build in selectivity and thus safety for compounds targeting the highly conserved PRS binding site. Going forward, HF-based drug scaffolds may need to be individually tailored to exploit differences in host and pathogen cellular characteristics e.g. in membrane permeability, metabolic processes and expression of various drug efflux pumps.

We also highlight examples from three other aaRSs that by virtue of either sequence or structural conservations have been exploited for similar cross-parasitic drug discovery. Examples include: (a) the fungal metabolite cladosporin (Figure 2A &B) that inhibits the P. falciparum lysyl-tRNA synthetase (PfKRS)

21, 27, 35, 52

. The selectivity of cladosporin for the

parasite enzyme over the human counterpart most likely arises from residues situated outside the active site. Cladosporin has been shown to be of utility against unrelated human pathogens such as those from the nematode and trematode worms Loa loa and Schistosoma mansoni due to notable sequence conservation in active site regions

53

. Other pathogens

where KRS active sites are similarly constructed include plant fungi like P. infestans and P. graminis (Figure 2C), (b) the natural polyketide called borrelidin (BN) (Figure 3A & B) or its derivative inhibit bacterial and eukaryotic threonyl-tRNA synthetases (TRS)

54-56

been targeted by BN for bacterial infections caused by Brucella abortus

57

. TRS has

, and BN is

effective against plant pathogen fungus Phytophthora sojae that causes soybean root rots 58. In addition, given the evident sequence conservation across pathogen TRSs, these enzymes can be investigated for human parasites including Plasmodium spp., Toxoplasma spp., Leishmania spp., Trypanosoma spp., Cryptosporidium spp. Schistosoma spp. and Loa spp.; livestock parasites Cryptosporidium spp., Eimeria spp., Babesia spp., Theileria spp., Trichomonas spp.; and for several fungi that cause human and/or plant infections like Candida spp., Aspergillus spp., Phytophthora spp., Ustilago spp. and Puccinia spp. amongst


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others (Figure 3C), (c) the 3D structure of Thermus thermophilus leucyl-tRNA synthetase editing domain complexed with the drug AN2690 (Tavaborole, Figure 4A) contributed to the use of benzoxaboroles to target LRS editing domains (LRS-edt)

40

. Analysis of C. albicans

LRS-edt bound to AN3018 proved the utility of benzoxaborole scaffold to target additional eukaryotic parasites

59

. Recently, two compounds called ZCL039 and AN6426 (Figure 4A) 60-61

were found to be effective against Streptococcus pneumoniae and Plasmodium

. Note

from figure 4B that structural architectures of the LRS editing domains amongst deposited structures from humans, C. albicans 59, P. falciparum 61, E. coli 62, S. pneumoniae tuberculosis

63

60

and M.

are essentially identical. Hence, it is likely that the benzoxaborole scaffold-

based compounds may be useful in targeting LRS-edt in other parasites including P. vivax, T. gondii, C. muris, L. major, T. cruzi, T. brucei and S. mansoni (Figure 4C). Indeed, the AN6426 bound complex structure of LRS-edt from Cryptosporidium muris has recently been deposited in PDB 64.

Cysteine proteases Cysteine proteases, also known as thiol proteases, are enzymes that degrade proteins by a catalytic mechanism that involves nucleophilic cysteine thiol in a catalytic dyad/triad These enzymes play multi-faceted roles in physiology and development currently pursued protein targets in T. cruzi

67

and P. falciparum

68

36, 65

.

65-66

. Amongst

that cause Chagas and

malaria respectively, cysteine proteases called cruzain and falcipain are considered highly valuable65. Of these, cruzain has been exploited for long, and currently a plethora of inhibitors with varying degree of activity target it

66

. Irreversible inhibitors of cruzain like

peptidyl diazomethylketones and vinyl sulphones are able to block the differentiation steps in the parasite life cycle, thus effectively killing it (Figure 5A & B)

69

. For example, a vinyl

sulphone derivative disables T. cruzi by inducing an accumulation of unprocessed cruzain in



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the Golgi cisternae and thus interfering with its secretory pathway (Figure 5A & B) 70. Based on cruzain, inhibitors for falcipain are also being studied as both these proteases are structurally orthologous and hence possibly are druggable with related hit compounds

66

.

Cysteine proteases from H. sapiens, T. gondii and T. rhodesiense have similar active site architectures, but few critical differences between the active sites of human and parasite cysteine proteases highlights the observed selectivity for the irreversible inhibitors

71-73

.

Figure 5A and 5B show notable invariance in drug-binding regions and residues within cysteine proteases from Plasmodium spp., Toxoplasma spp., Leishmania spp., Trypanosoma spp., Cryptosporidium spp.; worms like Schistosoma spp. and Loa spp. (Figure 5C). Furthermore, livestock parasites Cryptosporidium spp., Eimeria spp., Babesia spp., Theileria spp., Trichomonas spp. and several fungi that cause human and /or plant infections like Candida spp., Aspergillus spp., also display notable conservation in their cysteine protease enzymes (Figure 5C). This suggests that cruzain-inhibiting compounds may be useful across a wider spectrum of pathogens.

Dihydrofolate reductase The enzyme dihydrofolate reductase (DHFR) converts dihydrofolate to the active tetrahydrofolate that is required for nucleotide precursor biosynthesis. DHFR has been a widely exploited drug target

74-75

. DHFR was the first enzyme to be utilized for cancer

chemotherapy, and the drug used then was aminopterin which binds to the enzyme a thousand times more tightly than folate, thereby inactivating DHFR 76-77. Many of the studied organisms encode slightly sequence tinkered versions of DHFR, and several groups have developed selective drugs (pyrimethamine, methotrexate and trimetrexate) that take advantage of these subtle active site differences in DHFRs

75 78 79

(Figure 6A). Amongst the


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battery of developed drugs are methotrexate (MTX), aminopterin, trimethoprim (TMP) and pyrimethamine (PYR) (Figure 6A). Notably, TMP and PYR are selective parasite DHFR inhibitors, and often their co-administration with sulfonamides is required to provide synergistic effects for clinical utility 75 80. Trimetrexate (TMQ) and piritrexim (PTX) are two potent non-classical inhibitors, neither of which exhibit selectivity for pathogen DHFRs and therefore must be used with host rescue 75. Structures of DHFR from T. cruzi 81, C. hominis 82

, B. bovis

83

, T. gondii

84

, C. albicans and of T. brucei and P. falciparum are expectedly

related and conserved 85 (Figure 6B). Their analyses indicates that either pyrimethamine or its derivatives may be useful against parasites such as Cryptosporidium spp., Eimeria spp., Babesia spp., Theileria spp., Trichomonas spp; against several fungi that cause human and/or plant infections like Candida spp., Aspergillus spp., and against Phytophthora spp., Ustilago spp., Puccinia spp. (Figure 6C). However, in case of Cryptosporidium a thymidine kinase (TK) gene has been integrated, most likely through horizontal transfer from bacteria – and this offers an alternate route for dTMP during DHFR inhibition. Hence, Cryptosporidium is resistant to folate inhibitors 86. This example suggests the utility of both cell-based screening and extensive genomic analyses as vital tools in the drug targeting process as it may be that validated enzyme targets (such as DHFR) are not always druggable in the context of a pathogen. Ideally, each enzyme target should be assessed given its metabolic network, essentiality and cell-stage specificity.

Orthologous pathogen protein targeting jumpstarts drug discovery The above case studies indicate that drug discovery against parasitic diseases can be further hastened by exploiting 3D structural conservation amongst phylogenetically diverse sets of pathogen enzymes. Structural underpinnings implicit in these studies allow a definition of discrete atomic contacts that define the spatial interactions within protein-drug complexes.



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This information, once collated and aligned with orthologous pathogen proteins using multiple sequence alignments, provides a useful resource for testing drugs across a spectrum of conserved pathogen protein active sites. Although these efforts will not necessarily reveal lead compounds (there might be unresolved issues of drug solubility, bioavailability, drug permeability etcetera), drug testing via both enzyme-based and cell-based assays is still warranted so as to initiate structure-activity relationships that guide medicinal chemistry efforts in drug development (Figure 1).

This Structure-based orthologous targeting of pathogen proteins (succinctly abbreviated as STOPP) does not rely on FDA-approved drugs as a starting point (i.e. it is not a drug repurposing routine). The currently limited yet available libraries of drug-like molecules (like the ‘Malaria box’ or the ‘Pathogen box’) in context of their target protein structures are together sufficient to initiate STOPP in many cases. To improve drug-like properties, specific libraries of derivatives for a particular molecule will need to be synthesized in many cases, but this is a more reasonable approach than blind screening of large libraries. For example, once a breakthrough like the discovery of cladosporin (that targets malaria parasite lysyltRNA synthetase) has been achieved35, 3D structural information from protein-drug complexes can be immediately analyzed and pathogens shortlisted where active site conservation suggests cladosporin potency in unrelated parasites

53

. We believe that such

efforts, when combined with phenotypic screening and host toxicity studies, can together greatly accelerate drug discovery against pathogens as they rely on shared 3D structural architectures, conservation in drug action mechanisms, and common end points of targeting pathogens over the host.


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Concluding remarks Discovery of new drugs against human, animal and plant parasites requires emphasis on both target-based and phenotypic screening methods, and the road ahead necessarily involves a balance between these two methodologies. We believe that categorizing and providing an acronym such as STOPP for ‘structure-based targeting of orthologous pathogen proteins’ is timely as it will alert and encourage researchers working in unrelated streams of pathogen biology to converge for exploitation of drugs and their molecular targets (Figure 7). As illustrated by examples above, it is evident that potentially active, drug-like compounds can be identified via STOPP – especially when additional information via de novo phenotypic screening is available. It is thus evident that parasite genomics and experimental validations together with STOPP are indispensable for identification of druggable targets and useful scaffolds. Some caveats for STOPP remain such as when salvage pathways are active for the target enzyme product, or when the target is either not required at a particular cell cycle stage, and/or it is chemically inaccessible in the organism. The structural parasitology literature is replete with examples of 3D structures of unique pathogen enzymes, which although initially appear to be attractive as drug targets, do not necessarily succeed as druggable entities largely due to paucity of potent small molecules that bind to them. This is largely because identification of small molecule chemical ‘hits’ that target novel protein architectures can be cumbersome, time consuming and fund intensive. Indeed, the examples we have elaborated here reveal that conserved enzymes (between hosts and pathogens and within parasites) can be very attractive drug targets as long as chemical entities that are able to differentiate subtle sequence/structure variations between host and pathogen targets can be discovered, either using natural (re) sources or via human ingenuity in synthesizing unique chemical equities 87, 88.



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Table 1: Examples of parasitic diseases in humans, livestock and plants.

Disease name

Major causative pathogen

Human parasites Malaria Plasmodium falciparum Toxoplasmosis Toxoplasma gondii Chagas disease Trypanosoma cruzi African sleeping sickness Trypanosoma brucei Leishmaniasis Leishmania major Cryptosporidiosis Cryptosporidium hominis Trichomoniasis Trichomonas vaginalis Aspergillosis Aspergillus fumigatus Candidiasis Candida albicans Cryptococcal meningitis Cryptococcus neoformans Ascariasis Ascaris lumbricoides Onchocerciasis Onchocerca volvulus Nematodiasis Dracunculiasis Dracunculus medinensis Filariasis Wuchereria bancrofti Cestodiasis

Echinococcosis Echinococcus multilocularis cysticercosis Taenia solium Schistosomiasis Schistosoma mansoni Fascioliasis Fasciola hepatica Trematodiases Clonorchiasis Clonorchis sinensis Paragonimiasis Paragonimus westermani Livestock parasites Coccidiosis Eimeria tenella Cryptosporidiosis Cryptosporidium parvum Babesiosis

Babesia bovis

Theileriosis Histomoniasis Aspergillosis Ascaridiasis Candidiasis Sugarcane Smut Corn Smut Cereal Rusts Potato Blight Rice blast

Theileria annulata Histomonas meleagridis Aspergillus fumigatus. Ascaridia galli Candida albicans Plant fungal parasites Sporisorium scitamineum Ustilago maydis Puccinia graminis Phytophthora infestans Magnaporthe grisea


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ACKNOWLEDGMENT We thank I. Aves and I. Pantig for constant encouragement.

Funding information This research was supported by department of Biotechnology (DBT), govt. of India grant PR6303 to A. S and DBT grant PR3084 to A. S. and M. Y. A Senior Research Fellowship from DBT to V. J. is also acknowledged. A.S is additionally supported by the JC Bose fellowship, India.



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Kamchonwongpaisan, S.; Charman, S. A.; McLennan, D. N.; White, K. L.; Vivas, L.; Bongard, E.; Thongphanchang, C.; Taweechai, S.; Vanichtanankul, J.; Rattanajak, R.; Arwon, U.; Fantauzzi, P.; Yuvaniyama, J.; Charman, W. N.; Matthews, D., Malarial dihydrofolate reductase as a paradigm for drug development against a resistance26 ACS Paragon Plus Environment

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O'Neil, R. H.; Lilien, R. H.; Donald, B. R.; Stroud, R. M.; Anderson, A. C.,

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87.

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Kato, N.; Comer, E.; Sakata-Kato, T.; Sharma, A.; Sharma, M.; Maetani, M.; Bastien,

J.; Brancucci, N. M.; Bittker, J. A.; Corey, V.; Clarke, D.; Derbyshire, E. R.; Dornan, G. L.; Duffy, S.; Eckley, S.; Itoe, M. A.; Koolen, K. M.; Lewis, T. A.; Lui, P. S.; Lukens, A. K.; Lund, E.; March, S.; Meibalan, E.; Meier, B. C.; McPhail, J. A.; Mitasev, B.; Moss, E. L.; Sayes, M.; Van Gessel, Y.; Wawer, M. J.; Yoshinaga, T.; Zeeman, A. M.; Avery, V. M.; Bhatia, S. N.; Burke, J. E.; Catteruccia, F.; Clardy, J. C.; Clemons, P. A.; Dechering, K. J.; Duvall, J. R.; Foley, M. A.; Gusovsky, F.; Kocken, C. H.; Marti, M.; Morningstar, M. L.; Munoz, B.; Neafsey, D. E.; Sharma, A.; Winzeler, E. A.; Wirth, D. F.; Scherer, C. A.; Schreiber, S. L., Diversity-oriented synthesis yields novel multistage antimalarial inhibitors. Nature 2016, 538 (7625), 344-349. DOI: 10.1038/nature19804. 88. Jain, V.; Sharma, A., Repurposing of potent drug candidates for multi-parasite targeting. Trends in Parasitology, accepted.


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Figures

Figure 1

3D structure of target-inhibitor complex

P. fal MAD-EWTHHIKLPM-NVC-AEDKER T. gon. MAEQEWTHIIKLPM-NVC-AEDGTRF T. bru. MAKSEWTHIIKLPM-NVC-AEDGTED

Multiple sequence alignment (via ClustalOmega)

Conserved binding residues (via PDBePISA)

MAD-EWTHHIKLPM-NVC-AEDKER MAEQEWTHIIKLPM-NVC-AEDGTRF MAKSEWTHIIKLPM-NVC-AEDGTED

Test enzyme inhibition & binding

Test for parasite growth inhibiton

Derivatization and optimisation; hit-based libraries

Share library resources

Figure 1. Steps involved in structure-based targeting of orthologous parasitic proteins.



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Figure 2. Study cases of pathogen prolyl- and lysyl-tRNA synthetases. (A) The chemical structures of halofuginone (HF) and cladosporin (CL). (B) Key active site residues of Plasmodium falciparum prolyl-tRNA synthetase and lysyl-tRNA synthetases that interact with HF and CL respectively. (C) The multiple sequence alignment of PRS and KRS from various eukaryotic parasites along with residues that recognize HF and CL respectively. Eukaryotic pathogens shown here are: Plasmodium falciparum, Toxoplasma gondii, Trypanosoma cruzi, Leishmania major, Schistosoma mansoni, Cryptosporidium parvum, Babesia bovis, Candida albicans, Puccinia graminis and Phytophthora infestans. PDB codes are in brackets.


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Figure 3

B D564 Q562

A

Y540 F539

H590 Zn

T560

C413

S386

BN Borrelidin (BN) M411 Q460

H391 H388 Y392

R442

H. sapiens

Homo sapiens TRS-BN (4P3N)

C Threonyl-tRNA synthetase (TRS) 5 9 5

P .fa lc ip a r u m P .v iv a x T .g o n d ii C .p a r v u m L .m a jo r T .c r u z i T .b r u c e i S .m a n s o n i H .s a p ie n s L .lo a

6 0 1 6 1 8

S G H YQ N Y S G H YQ N Y S G H YQ N Y S G H YFAY S G H W EKY S G H W D KY S G H W D KY S G H W Q H Y S G H W Q H Y S G H W EH Y

6 2 3 6 5 0 6 5 3 6 6 7 6 7 07 4 6

KPM N C P KPM N C P KPM N C P KPM N C P KPM N C P KPM N C P KPM N C P - - - - - KPM N C P KPM N C P

H R N E H R N E H R N E H R N E H R N E H R N E H R N E - - - H R N E H R KK

7 5 0 7 6 7

7 7 7 8 7 18 7 3 I HR I HR I HR I HR I HR I HR I HR I HR VHR I HR

FQ Q DG AFYG C G TI Q LDFQ LP FQ Q DG AFYG C G TI Q LD FQ LP FQ Q DG AFYG C G TI Q LD FQ LP FQ Q DG AFYG C G TI Q LD FQ LP FQ Q DG AFYG C ATI Q LD FNLP FQ Q DG AFYG C ATVQ LD FNLP FQ Q DG AFYG C ATI Q LD FNLP - - - - G AFYG C ATI Q LD FQ LP FQ Q DG AFYG C ATI Q LD FQ LP FQ Q DG AFYG C ATI Q LD FQ LP

Figure 3. Case of threonyl-tRNA synthetases. (A) Structure of borrelidin (BN). (B) Active site residues of homo sapien threonyl-tRNA synthetase (TRS) that binds with BN. (C) Multiple sequence alignment of TRSs from different parasites along with interacting residues. Eukaryotic pathogens shown here are P. vivax, T. gondii, C. parvum, L. major, S. mansoni, L. loa and T. brucei.



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Figure 4

A

AN2690

AN3018

AN6426

ZCL039

B

E. coli (4ASI)

C

C. albicans (2WFG)

P. falciparum (5FOC)

H. sapiens (2WFD)

M. tuberculosis (5AGT)

S. pneumoniae (4K47)

Leucyl-tRNA synthetase editing domain 274 P. falciparum P. vivax T. gondii C. muris L. major T. cruzi T. brucei H. sapiens S. mansoni

Q Q Q Q Q Q Q Q Q

P. falciparum P. vivax T. gondii C. muris L. major T. cruzi T. brucei H. sapiens S. mansoni

S A G G G G G G G

E E E E E E E E E

281

394

Y F Y Y Y Y Y Y Y

T T T T T T T T T

L L V L L L V L L

I I V I V V V L I

K K K K K K K K K

I I L L L L L L L

S S A A A A A A A

T T T T T T T T T

L L L L L L L L L

K K R R R R R R R

P P P P P V P P P

400

579

638

G A G G G G G G G

I I V I V V V V V

V V V V V V V V V

P P M T M M M T T

C C S S S S S S S

V V V V V V V V V

D D D D D D D D D

S S A S S S S S S

T A P P P P P P P

D D D D D D D D D

D D D D D D D D D

L L L L L L L L L

564 T T T T T T T T T

S S P P P P P P P

S S S S S S A S S

E E E E E E E E E

Y Y Y Y Y Y Y I W

551 L L I I I I I I V

T T T T T T T T T

Q Q Q K E E E A Q

559

Y Y Y F Y Y Y Y F

I I A I T T T V V

L L L L L L L L L

P P P P P P P P P

M M M M M M M M M

T T L F A Q Q L M

K K E Q E E E E E

I V I A A A A A A

K K K K K K K K K

E E E E K K K E E

V S E E V L V K K

L L V I C V I I V

T T T S T T S T S

I I I I I I I I I

646 Y Y Y Y Y Y Y Y Y

Figure 4. Case of pathogen leucyl-tRNA synthetases. (A) Structures of benzoxaborolebased inhibitors (AN2690, AN3018, AN6426 and ZCL039) that target the editing site (LRSedt) of Thermus thermophilus, Candida albicans, Cryptosporidium parvum and Streptococcus pneumoniae leucyl-tRNA synthetase. (B) Conserved domain architectures and inhibitor binding sites in the LRS-edt of H. sapiens (PDB: 2WFD), P. falciparum (PDB: 5FOC), C. albicans (PDB: 2WFG), E. coli (PDB: 4ASI), S. pneumoniae (PDB: 4K47) and M. tuberculosis (PDB: 5AGT) are evident. (C) Multiple sequence alignment and conserved drug binding residues amongst P. falciparum, P. vivax, T. gondii, C. muris, S. mansoni, L. major, T. cruzi T. brucei and H. sapiens are shown. 32 ACS Paragon Plus Environment

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Figure 5. Case of cysteine proteases. (A) Structures of known inhibitors for Homo sapien, Trypanosoma rhodesiense and Trypanosome cruzi cysteine proteases. (B) Note the conserved structural architectures and inhibitor binding sites in cysteine proteases from H. sapiens (PDB: 1M6D), T. rhodesiensis (PDB: 2P7U), T. cruzi (PDB: 3IUT), T. gondii (PDB: 3F75) and P. falciparum (PDB: 1YVB). (C) Multiple sequence alignment and conserved drug binding residues amongst P. falciparum, P. vivax, T. gondii, L. major, T. cruzi and T. brucei.



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Figure 6. Case of dihydrofolate reductases. (A) Structures for dihydrofolate reductase (DHFR) inhibitors - pyrimethamine, methotrexate and trimetrexate that are active against P. falciparum, T. brucei and T. cruzi respectively (B) DHFR domain architectures and active site residues are similar in many pathogens: Trypanosoma cruzi (PDB: 3HBB), Cryptosporidium hominis (PDB: 1QZF), Trypanosoma brucei (PDB: 3QFX), Babesia bovis (PDB: 3I3R), Toxoplasma gondii (PDB: 3ECK), C. albicans (PDB: 4HOF) and P. falciparum (PDB: 3QGT). (C) Multiple sequence alignment and conserved drug binding residues amongst P. falciparum, P. vivax, T. gondii, C. parvum, L. major, T. cruzi and T. brucei.


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Figure 7

Human diseases Toxoplasmosis Malaria Leishmaniasis Schistosomaisis Candidiasis Chagas disease Cryptosporidiosis

Livestock diseases

Plant fungal diseases

Cryptosporidiosis Cocciodiosis Babesiosis Theleriosis Histomoniasis

Phytophthora sps Puccinia sps Fusarium sps Rhizotonia sps

Figure 7. Conservation of druggable targets in pathogens that infect humans, livestock or plants allows the exploitation of common pharmacophores. Such scaffolds provide hit molecules around whom rapid and more selective drug development can be initiated.



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Page 36 of 36

For Table of Contents Use Only

Title: Structure-based targeting of orthologous pathogen proteins accelerates anti-parasitic drug discovery

Authors: Vitul Jain, Arvind Sharma, Gajinder Singh, Manickam Yogavel and Amit Sharma*

STOPP

MAD-EWTHHIKLPM-NVC-AEDKER MAEQEWTHIIKLPM-NVC-AEDGTRF MAKSEWTHIIKLPM-NVC-AEDGTED

Multiple sequence alignment

Test for parasite growth inhbiton 3D structure of target-inhibitor complex

Test enzyme-drug binding & Inhibition


Structure-Based Targeting of Orthologous ... - ACS Publications

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