Three Different Mutations in the DNA Topoisomerase 1B in Leishmania Infantum Contribute to Resistance to Antitumor Drug Topotecan

doi:10.21203/rs.3.rs-527648/v1

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Research Article

Three Different Mutations in the DNA Topoisomerase 1B in Leishmania Infantum Contribute to Resistance to Antitumor Drug Topotecan

https://doi.org/10.21203/rs.3.rs-527648/v1

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Background

The evolution of drug resistance is one of the biggest challenges in leishmaniasis and has prompted the need for new antileishmanial drugs. Repurposing of approved drugs is a faster and very attractive strategy that is gaining supporters worldwide. Different anticancer topoisomerase 1B (TOP1B) inhibitors have shown strong antileishmanial activity and promising selective indices, supporting the potential repurposing of these drugs. However, cancer cells and Leishmania share the ability to become rapidly resistant. The aim of this study was to complete a whole-genome exploration of the effects caused by exposure to topotecan in order to highlight the potential mechanisms deployed by Leishmania to favor its survival in the presence of a TOP1B inhibitor.

Methods

We used a combination of stepwise drug-resistance selection, whole-genome sequencing, functional validation, and theoretical approaches to explore the propensity of and potential mechanisms deployed by three independent clones of L. infantum to resist the action of TOP1B-inhibitor topotecan.

Results

We demonstrated that L. infantum is capable of becoming resistant to high concentrations of topotecan without impaired growth ability. No gene deletions or amplifications were identified from the next-generation sequencing data in any of the three resistant lines, ruling out the overexpression of efflux pumps as the preferred mechanism of topotecan resistance. We identified three different mutations in the large subunit of the leishmanial TOP1B (Top1B^F187Y, Top1B^G191A and Top1B^W232R). Overexpression of these mutated alleles in the wild-type background led to high levels of resistance to topotecan. Computational molecular dynamics simulations, in both covalent and non-covalent complexes, showed that these mutations have an effect on the arrangement of the catalytic pentad and on the interaction of these residues with surrounding amino acids and DNA. This altered architecture of the binding pocket results in decreased persistence of topotecan in the ternary complex.

Conclusions

This work helps elucidate the previously-unclear potential mechanisms of topotecan resistance in Leishmania by mutations in the large subunit of TOP1B and provides a valuable clue for the design of improved inhibitors to combat resistance in both leishmaniasis and cancer. Our data highlights the importance of including drug-resistance evaluation in drug-discovery cascades.

Parasitology

Protozoan parasites

Leishmania

DNA topoisomerases

topotecan

drug resistance

SNPs

Despite 1 million new cases of leishmaniasis being declared every year, there is still no effective vaccine available for humans. Moreover, available treatments for this major neglected tropical disease (NTD) are limited and outdated [1]. Clinical manifestations vary in severity and include cutaneous, mucocutaneous, and visceral leishmaniasis. The latter form, caused by Leishmania infantum, is fatal within two years if left untreated [2]. Leishmaniasis is also responsible for a significant health, psychosocial and economic burden around the world [3, 4]. Due to the limited pharmacopeia, most available antileishmanial drugs are used in both humans and dogs. Of note, dogs constitute the main reservoir for the zoonotic life cycle of Leishmania [5, 6].

The rapid emergence and spread of resistant parasite strains has prompted the need for new intervention pathways and antileishmanial drugs. In this way, repurposing of approved drugs has become a very attractive strategy to tackle leishmaniases and is gaining supporters worldwide [1, 7, 8]. DNA topoisomerases (TOP) have garnered attention since their discovery by James Wang in 1971 [9]. TOP are key enzymes for many essential biological functions such as DNA replication, transcription, recombination, DNA repair, and DNA segregation [10]. TOP enzymes are preferential targets against rapid-dividing, highly-proliferative eukaryotic cells, such as those responsible for various types of cancer (e.g. ovarian cancer or metastatic carcinoma of the colon) [11, 12]. Type 1B TOP (TOP1B) are ATP-independent enzymes that relax topological tensions in supercoiled DNA during replication and transcription processes mediated by DNA and RNA polymerases. Eukaryotic TOP1B are monomeric enzymes, except in kinetoplastid organisms (e.g. Leishmania) that rely on heterodimeric TOP1B enzymes, with coding genes located in different chromosomes, 34 and 4, respectively [13, 14]. The major structural and functional differences between Homo sapiens TOP1B (hTOP1B) and L. infantum TOP1B (LiTOP1B) [15, 16], coupled with Leishmania’s rapid proliferation rate, offer great potential for selective chemotherapy [13]. Different anticancer TOP1B inhibitors, including the water-soluble camptothecin derivative topotecan (TPT) currently used to treat ovarian carcinoma [11], have been successfully tested against Leishmania parasites in in vitro, ex vivo and in vivo murine models, demonstrating strong antileishmanial activity and promising selective indices, supporting the potential repurposing of these drugs [8, 17, 18]. However, tumor cells and Leishmania parasites share an undesirable and important feature in being prone to become resistant to drug treatment. L. infantum relies on DNA copy number variations (CNVs) for regulating the expression of drug targets [19, 20]) and drug-resistance genes [20, 21]. In addition to CNVs, single-nucleotide polymorphisms (SNPs) in drug target genes or in transporters can lead to drug resistance without the need for altering gene content [22, 23, 24]. Markedly, recent studies point to the need for experimental generation of drug-resistance to promising compounds in order to clinically evaluate and eventually circumvent the phenomenon [25, 26, 27].

The goal of this study was to complete a whole-genome exploration of the effects caused by prolonged exposure to TPT to highlight the different potential drug-resistance mechanisms (changes in ploidy, CNVs, SNPs, etc.) deployed by L. infantum to favor its survival in the presence of an FDA-approved TOP1B inhibitor. This information is critical to the fight against drug-resistant Leishmania parasites, furthering knowledge of the mechanisms these parasites use to persist in the presence of drugs, as well as serving as a foundation for improved drug-repurposing strategies.

Leishmania cultures

Leishmania infantum (MHOM/MA/67/ITMAP-263) wild-type (WT) and mutants resistant to > 700 µM TPT (TPT700.1, TPT700.2 and TPT700.3) generated in vitro in a stepwise manner were grown in M199 medium at 25°C supplemented with 10% fetal bovine serum, and 5 µg/mL of hemin at pH 7.0. 700 µM of TPT (Topotecan hydrochloride hydrate, Sigma-Aldrich) was also added to the media for the maintenance of the endpoint mutants. Growth curves were performed in 25 cm² cell culture flasks by seeding 1 × 10⁶ parasites/mL, and the number of parasites was determined daily – up to 7 days – by manual counting using the Neubauer hemocytometer. Growth assays were performed with at least three biological replicates from independent cultures (n = 3).

Mutant selection

Three L. infantum WT independent clones, which were obtained by plating the MHOM/MA/67/ITMAP-263 strain onto solid M199, were independently selected in 25 cm² flasks containing 5 mL M199 medium supplemented with 10% heat inactivated fetal bovine serum (FBS) and 5 µg/mL hemin in the presence of increasing TPT concentrations, as previously described [23]. Briefly, the stepwise drug selection ranged from 1× the EC₅₀ of TPT (24 µM) up to 16× the EC₅₀ of TPT (384 µM), with a 2-fold increase in drug concentration (24, 48, 96, 192, and 384 µM) every three sub-culturing passages. Topotecan hydrochloride hydrate (Sigma-Aldrich, St Louis, MO, USA) was used as the source of TPT.

Drug susceptibility assays

Antileishmanial values in promastigotes were determined by monitoring the growth of parasites after 72 h of incubation at 25°C in the presence of increasing concentrations of TPT, by measuring A₆₀₀ using a Cytation 5 multimode reader (BioTek, USA). Drug-efficacy assays were performed with at least three biological replicates from independent cultures (n = 3). EC₅₀ values were calculated based on dose-response curves analyzed by non-linear regression with GraphPad Prism 8.4.3 software (GraphPad Software, La Jolla California, USA). Statistical analyses were performed using unpaired two-tailed t-tests. A p value < 0.05 was considered statistically significant.

Whole-genome sequencing of TPT-resistant mutants

Comparative whole genome sequencing (WGS) was performed as previously described [23]. Briefly, genomic DNA was prepared from a mid-log phase clonal culture of each TPT-resistant mutant. DNA was quantified fluorometrically, and 50 ng of material was used for library preparation using Nextera™ DNA Sample Preparation Kit (Illumina) according to manufacturer's instructions. The size distribution of Nextera™ libraries was validated using an Agilent 2100 Bioanalyzer and High Sensitivity DNA chips (Agilent Technologies). Sequencing libraries were quantified with the QuantiFluor® dsDNA System and sequenced using an Illumina MiSeq platform with 250-nucleotide paired-ends reads. An average genome coverage of over 50-fold was obtained for the mutants. This approach allowed identification of SNPs when compared with the reference genome sequence of L. infantum JPCM5 (TriTrypDB v9.0) [28] and L. infantum 263 WT [29]. Sequence reads were aligned to the L. infantum JPCM5 genome and L. infantum 263 WT using the software bwa-mem [30]. The maximum number of mismatches was 4, the seed length was 32, and 2 mismatches were allowed within the seed. Read duplicates were marked using Picard (http://broadinstitute.github.io/picard), and GATK was applied for InDel realignment and SNP and InDel discovery in the three TPT mutants. PCR amplification and conventional DNA sequencing verified SNPs of interest detected by WGS. CNVs were derived from read depth coverage by comparing the coverage of uniquely mapped reads between each of the three TPT mutants and the L. infantum 263 WT in 5 kb non-overlapping genomic windows for the 36 chromosomes (normalized to the total number of uniquely mapped reads for each strain) [31]. The sequence data for the L. infantum TPT-resistant mutants is available at the NCBI BioProject (https://www.ncbi.nlm.nih.gov/bioproject/) under study accession PRJNA647847 and sample accessions SAMN15599759, SAMN15599760, SAMN15599761, corresponding to TPT-resistant clones TPT700.1, TPT700.2 and TPT700.3, respectively.

DNA constructs and nucleofection

The WT allele of LinJ.34.3220 (DNA topoisomerase IB large subunit) and its three mutant variants were amplified from genomic DNA derived from either L. infantum or mutants TPT700.1, TPT700.2 and TPT700.3 (mutations T560A; G572C and T724C, respectively) using compatible primer pairs (Fw: 5’-XbaI-ATGAAGGTGGAGAA-3’; Rv: 5’-HindIII-TACACCCTCAAAGC-3’). PCR fragments were ligated into pGEM T-easy vector (Promega) to confirm the quality of the insert by standard sequencing. PCR fragments were then cloned in the Leishmania expression vector pSP72αhygα, which contains the gene hygromycin phosphotransferase (hyg), a selectable marker in Leishmania [32]. A total of 20 µg of plasmid DNA for episomal expression, either the empty vector (mock) or carrying the genes of interest, were delivered into L. infantum WT promastigotes by nucleofection, as previously described [33]. Selection was achieved in the presence of a final concentration of 300 µg/mL hygromycin.

Computational methods for the in silico study of Leishmania topoisomerase 1B

The three-dimensional structure of the L. donovani TOP1LS complex (PDB ID: 2B9S [34]) contains residues 27–456 and 221–262 of the large and small subunits, respectively. Residues missing in the crystal structure, 427–430 of the large subunit, have been modelled as reported in Roy et al., 2011 [35]. Following this procedure, a non-covalent interaction of the LiTOP1 bi-subunit with a 22 base-pair DNA substrate was modelled using the program UCSF Chimera [36] by fitting the structure of the protein backbone atoms on the coordinates of the human enzyme trapped in complex with the DNA (PDB ID 1A36). The same procedure was carried out in order to model an LiTOP1-DNA covalent interaction, using the human TOP1-DNA-topotecan ternary complex, extrapolated from the PDB structure 1K4S [37].

Once the two systems (wild-type LiTOP1-DNA covalent and non-covalent complexes) were obtained, the program UCSF Chimera [36] was used to introduce the three point mutations identified in our whole-genome sequencing experiments (F187Y, G191A, and W242R). All systems were subjected to molecular dynamics (MD) simulations aimed at understanding the structural-dynamical effect of the mutations, thus explaining their impact on TPT sensitivity. Briefly, each covalent and non-covalent complex was placed in a triclinic simulative box filled with water molecules TIP3P model [38], and the resulting systems were rendered electroneutral by the addition of sodium counterions; this step, i.e. the topology building, was performed using the amber14 all-atoms force field, highly suitable for simulating nucleic acids [39]. The systems were first subjected to a round of minimization of 10,000 steps of steepest descent followed by 10,000 steps of conjugate gradient. Relaxation of water molecules and thermalization in NPT environment were carried out for 1.2 ns at 1 fs time-step. In particular, 6 runs of 200 ps each were carried out while increasing the temperature by 50 K at each step, ranging from 50 K to 300 K.

The systems were then simulated with a 2 fs time-step for 300 ns in periodic boundary conditions, using a cut-off of 8 Å for the evaluation of short-range non-bonded interactions and the Particle Mesh Ewald method for long-range electrostatic interactions [40]. The temperature was kept constant at 300 K with Langevin dynamics [41], whereas pressure was fixed at 1 Atmosphere through the Langevin piston method [42]. The bond lengths of solute and water molecules were restrained with the SHAKE [43] and SETTLE [44] algorithms, respectively. Atomic positions were saved every 250 steps (i.e. 0.5 ps) for analysis with the Gromacs 4.6 package [45].

In vitro resistance selection and characterization of the TPT-resistant mutants

Selection for resistance to TPT was performed to evaluate the possible propensity for drug resistance to this anticancer TOP1B inhibitor if repurposed against visceral leishmaniasis. Experimental concentration-response assays with L. infantum WT promastigotes revealed a TPT-sensitive phenotype within the low micromolar range (Fig. 1A; EC₅₀ = 25 µM). Selection of independent TPT-resistant mutants began at 1× EC₅₀ up to 16× EC₅₀ of the WT parental line. The selection procedure was fast (3 subsequent passages per drug concentration), and cells rapidly adapted to growing concentrations of TPT. All three clones reached the final selection step at 16× EC₅₀ (passage 3). At this point, clones were re-evaluated in terms of growth ability and sensitivity to TPT (Fig. 1A). All three clones were able to survive concentrations higher than 700 µM (ca. 28× EC₅₀ of the WT), demonstrating very high EC₅₀ values (612, 519 and 602 µM, for TPT700.1, TPT700.2 and TPT700.3, respectively) compared to the parental WT strain (approximately 25 µM). Once the final selection step was reached, the growth rate of each TPT mutant was determined in order to verify their fitness compared to the WT parent line. As depicted in Fig. 1B, the TPT-resistant phenotype experimentally induced in promastigotes did not lead to any significant difference in the growth rate of the parasite when compared to the WT cells (Fig. 1B). Resistant phenotypes were stable after growth in media lacking TPT for over 60 passages (Fig. 1C).

Comparative WGS

Whole-genome sequencing was performed to compare the three independent TPT-resistant strains to the WT line to pinpoint and further explore potential mechanisms underlying the resistant phenotype. WGS was conducted by Illumina next-generation sequencing on the three independent L. infantum TPT700 resistant lines selected at 16× EC₅₀, as well as the isogenic L. infantum WT counterpart line. For all strains, this produced genome assemblies of 31 Mb with a coverage depth of at least 50-fold.

No significant deletions or amplifications were identified; however, the TPT-resistant lines demonstrated aneuploidy compared to WT parental line. Two cases of reduction in ploidy were observed in the TPT-resistant mutants (Fig. 2A and Dataset S1). These losses affected chromosome 12 in clone TPT700.3 (Fig. 2A and 2B) and chromosome 32 in the three resistant mutants (Fig. 2C). The log₂ TPT700.3/WT read ratio for chromosome 12 (Fig. 2B) was close to -0.5, which should represent a loss of one allele compared to WT parasites (going from 4 to 3 chromosome copies). In the same way, the log₂ TPT/WT read ratio (Fig. 2C) for chromosome 32 was close to -1, pinpointing a trisomic-to-disomic shift in all three TPT-resistant mutants (Fig. 2C), which could correlate with reduced TPT sensitivity. No cases of supernumerary chromosomes were observed in the TPT-resistant mutants (Fig. 2A and Dataset S1).

A search for point mutations revealed several homozygous and heterozygous SNPs in the different TPT-resistant mutants (Dataset S2). Nineteen common genes were mutated in the three TPT-resistant mutants (Table 1). In order to find potential SNP candidates, the different clones were examined for SNPs occurring in the same ORF but at different positions (to avoid mutations that are most likely to occur as natural polymorphisms in the parental strain). SNPs fulfilling these characteristics occurred in eight genes, including two coding for hypothetical proteins (LinJ.19.1680 and LinJ.19.1690), three proteophosphoglycan-coding genes (LinJ.35.0490, LinJ.35.0510 and LinJ.35.0550) often found mutated in our various sequencing screens, LinJ.24.1120 (encoding a putative pre-mRNA splicing factor), LinJ.34.0710 (encoding a putative flagellar attachment zone protein) and the gene coding for the large subunit of the heterodimeric LiTOP1B (LinJ.34.3220). The three different mutations identified in the LiTOP1B ORF by next-generation sequencing (NGS) and confirmed by targeted PCR were homozygous mutations top1B^F187Y and top1B^W232R for TPT700.1 and TPT700.3 mutants, respectively, and heterozygous SNP top1B^G191A for TPT700.2.

Table 1

**Overview of the common mutated genes in the three TPT-resistant mutants.** The information depicting all Reference/Mutant nucleotide changes is contained in Dataset S2. (Het: heterozygous; Hom: homozygous; na: not applicable due to frameshift).
Mutant	Gene ID	Mutation Position	Allel frequency	Ref/Mut AA	Gene function
TPT700.1 TPT700.2 TPT700.3	LinJ.01.0420	1073 1073 1073	Het. Het. Het.	A/E A/E A/E	Hypothetical protein conserved
TPT700.1 TPT700.2 TPT700.3	LinJ.09.0680	1388 1388 1388	Hom. Hom. Hom.	na na na	Hypothetical protein conserved
TPT700.1 TPT700.2 TPT700.3	LinJ.09.0950	17 17 17	Hom. Hom. Hom.	na na na	Polyubiquitin
TPT700.1 TPT700.2 TPT700.3	LinJ.12.0662	425 425 425	Het. Het. Het.	S/T S/T S/T	Surface antigen protein putative
TPT700.1 TPT700.2 TPT700.3	LinJ.19.1680	3792 3665 2940	Het. Het. Hom.	na V/A E/D	Hypothetical protein
TPT700.1 TPT700.2 TPT700.3	LinJ.19.1690	3956 3389 3956	Het. Het. Het.	S/N G/A S/N	Hypothetical protein
TPT700.1 TPT700.2 TPT700.3	LinJ.24.1120	771 776 771	Het. Het. Het.	S/R S/F S/R	pre-mRNA splicing factor putative
TPT700.1 TPT700.2 TPT700.3	LinJ.27.0870	2572 2572 2572	Het. Het. Het.	R/G R/G R/G	Hypothetical protein conserved
TPT700.1 TPT700.2 TPT700.3	LinJ.28.2320	856 856 856	Hom. Hom. Hom.	N/Y N/Y N/Y	Hypothetical protein conserved
TPT700.1 TPT700.2 TPT700.3	LinJ.28.2390	1321 1321 1321	Hom. Hom. Hom.	na na na	Cyclin-dependent kinase-binding protein putative
TPT700.1 TPT700.2 TPT700.3	LinJ.28.2450	3758 3758 3758	Hom. Hom. Hom.	na na na	DNA topoisomerase ii
TPT700.1 TPT700.2 TPT700.3	LinJ.29.1450	242 242 242	Hom. Het. Hom.	A/V A/V A/V	Amastin-like protein
TPT700.1 TPT700.2 TPT700.3	LinJ.29.2240	1267 1267 1267	Hom. Hom. Hom.	L/F L/F L/F	Hypothetical protein conserved
TPT700.1 TPT700.2 TPT700.3	LinJ.31.1470	1088 1088 1109	Hom. Hom. Het.	na na na	Hypothetical protein unknown function
TPT700.1 TPT700.2 TPT700.3	LinJ.34.0710	2129 76 2843	Het. Het. Het.	G/D A/P D/G	Flagellar attachment zone protein putative
TPT700.1 TPT700.2 TPT700.3	LinJ.34.3220	560 572 724	Hom. Het. Hom.	F/Y G/A W/R	DNA topoisomerase IB large subunit
TPT700.1 TPT700.2 TPT700.3	LinJ.35.0490	16451 1992 16285	Hom. Het. Hom.	R/H na V/I	Proteophosphoglycan ppg4
TPT700.1 TPT700.2 TPT700.3	LinJ.35.0510	7549 5278 2437	Het. Het. Het.	A/T V/L G/S	Proteophosphoglycan ppg4
TPT700.1 TPT700.2 TPT700.3	LinJ.35.0550	1699 2471 2471	Het. Het. Hom.	A/T na na	Proteophosphoglycan ppg1

Impact of LiTOP1B mutant variants in L. infantum TPT drug resistance

Because top1B is an essential gene in Leishmania, our preferred method for studying the role of TOP1B mutations was episomal transfection of wild-type and mutated forms of the gene in the WT background (Fig. 3). To this end, the WT copy as well as the three mutated forms of top1B were recovered by PCR, confirmed by Sanger sequencing, and episomally overexpressed in the WT background. Overexpression of the leishmanial top1B^WT allele did not confer TPT resistance in the WT cell line when compared with the mock control. However, as depicted in Fig. 3, overexpression of all three mutated variants of the top1B gene resulted in a significant decrease in TPT sensitivity in the WT: top1B^F187Y (2.62-fold; p < 0.0001), top1B^G191A (1.96-fold; p = 0.0006) and top1B^W232R (2.28-fold; p = 0.0001).

Structural effect of the SNPs and impact on TPT sensitivity

The mechanism of inhibition exerted by TPT against TOP1B is strictly dependent on the enzyme catalytic cycle. TOP1B is able to relax the DNA substrate in a five-step reaction: i) DNA binding; ii) DNA cleavage and formation of a transient protein-DNA covalent interaction; iii) DNA controlled rotation to release the torsional stress; iv) DNA re-ligation; v) DNA release. Once the DNA cleavage has occurred, TPT is able to intercalate the DNA bases and stabilize the protein-DNA covalent complex, thus blocking the cycle and stalling the enzyme. If one of the catalytic steps is compromised, this can affect TPT sensitivity.

In order to evaluate the impact of the point mutations on the structure of the protein and its interaction with the DNA substrate for a correct progression of the catalytic cycle, we performed a molecular dynamics (MD) simulations of the WT and mutants enzymes (F187Y, W232R and G191A). In particular, we carried out MD simulations both of the WT and mutant non-covalent and covalent TOP1B-DNA complexes (for a total of eight systems), allowing detection of the mutations’ effect in any step of the two phases of the catalytic cycle, i.e. cleavage and religation. This gives important insight into the mechanism(s) underlying resistance due to the fact that these mutations are located in close proximity to the catalytic pentad and to the TPT binding site (Fig. 4).

In both groups of simulations (covalent and non-covalent complexes), the mutation of each residue has an effect on the arrangement of the catalytic pentad (R314, K352, R410 and H453 of the large subunit and Y222 of the small subunit) (Figs. 5 and 6) and on the interaction of these residues with the surrounding amino acids and the DNA substrate (Tables 2 and 3). This likely alters the cleavage/religation equilibrium, which is at the basis of an altered drug sensitivity. Of note, the profile of interaction at the cleavage site (-1/+1 bases) of the protein-DNA in the non-covalent complex is hardly affected in all mutants, making us hypothesise that due to the improper arrangement of the catalytic pentad, the reaction rate is affected. Similarly, the catalytic pentad arrangement is impacted, in particular at the level of residue K352, which is crucial for the religation reaction to proceed and for the stabilization of TPT in the DNA binding pocket

Table 2

**Analysis of the hydrogen-bond network in covalent complex simulations.** (PTR stands for phosphotyrosine while “s” following the residue number indicates that residue belongs to the small subunit).
	WT	F187Y	G191A	W232R
Active site pentad and surrounding residues
ARG314:PTR222s CYS329:ARG314 LYS352:ASP353 ILE355:LYS352 ARG410:ALA406 ARG410:PTR222s ARG410:ALA312 ARG410:CYS451 ALA414:ARG410 HIS453:ALA202s HIS453:PTR222s SER214s:HIS453 PTR222s:SER218s	97.78 98.46 60.41 78.65 44.55 99.72 85.33 90.37 85.22 97.64 93.57 76.09	98.26 98.33 77.83 99.79 97.66 41.72 90.21 82.20 90.28 93.87 65.74	97.97 98.38 78.79 99.67 91.83 85.86 82.39 96.79 87.36 66.94	98.82 98.12 86.33 99.55 97.50 48.01 89.70 80.23 90.86 92.65 67.68
Residues of point mutations and surrounding residues
PHE(Y)187:LYS198 GLY189:PHE(Y)187 GLY197:PHE187 HIS193:GLY(A)191 TRP(R)242:LYS225	99.05 93.63 96.20	99.37 80.85 99.31 65.35 84.25	99.40 69.95 99.53 98.36	99.53 64.26 99.56 65.47 95.50

Table 3

**Analysis of the hydrogen-bond network in non-covalent complex simulations.** (“s” following the residue number indicates that residue belongs to the small subunit).
	WT	F187Y	G191A	W232R
Active site pentad and surrounding residues and DNA bases
ARG314:ASN452 CYS329: ARG314 LYS352:ASP353 ILE355:LYS352 ARG410:ALA406 ARG410:ALA312 ARG410:TYR222s ARG410:CYS451 ALA414:ARG410 HIS453:ALA202s SER214s:HIS453 TYR222s:SER218s ARG314:THY + 1 LYS352::THY − 1 LYS352:THY + 1 ARG410:THY + 1 HIS453:THY + 1 TYR222s:THY − 1 TYR222s:THY + 1	98.76 70.96 57.14 44.32 90.58 59.80 89.56 79.59 85.85 68.45 87.60 75.18 94.79 65.22 55.33	97.96 68.41 73.93 61.96 98.60 83.62 45.97 89.25 85.58 93.02 73.21 98.50 52.21 86.34 96.96 80.60	69.42 98.37 88.26 96.42 97.86 75.04 96.27 90.06 76.47 78.77 73.82 68.58 55.49	97.98 75.04 90.59 90.98 86.04 88.41 64.46 88.02 53.88 74.12 87.59 43.97 70.77
Residues of point mutations and surrounding residues
PHE(Y)187:LYS198 GLY189:PHE(Y)187 GLY197:PHE187 HIS193:GLY(A)191 TRP(R)242:LYS225 TRP(R)242:LYS182	99.25 92.48 98.45	99.14 99.16 98.33	99.33 99.29 46.89 98.03	99.48 44.74 99.52 61.69 94.67 73.40

Topotecan is a semi-synthetic, water-soluble analogue of camptothecin (CPT), as well as the first FDA-approved oral TOP1B inhibitor for the treatment of several types of cancer. CPT derivatives have been repeatedly suggested as a good source of repurposed drugs for the treatment of a variety of infectious diseases caused by protozoan parasites [1, 13, 46]. With the alarming decrease in effectiveness of first-line drugs in areas where L. infantum is endemic, repurposed drugs could represent a faster solution at lower cost [47]. However, the predisposition of Leishmania to develop drug resistance should be addressed when repurposing a drug [1, 25, 27]. In this study, we used a combination of stepwise drug-resistance selection, whole-genome sequencing and theoretical approaches to explore the propensity of and potential mechanisms deployed by three independent clones of L. infantum to resist the activity of the TOP1B-inhibitor TPT. One of the major strengths of this approach is that both the parent cell line and three directly derived drug-resistant lines are studied together, and thus, any confounding factor derived from strain-related heterogeneity is excluded from the analysis [23, 48].

Firstly, we demonstrated that L. infantum is able to become resistant to high concentrations of TPT. While the mechanisms involved in TPT resistance have not been fully elucidated in tumor cells, several studies have shown the implication of different drug transporters, such as multidrug resistance-associated protein 1 (ABCC1/MRP1) or the ABCG2 transporter [49, 50]. Likewise, Leishmania parasites rely on the amplification of ABC transporter MRPA (ABCC) and inactivation of the aquaglyceroporin 1 (AQP1) gene to counter the action of antimonial drugs [21, 23, 51]. As gene expression in Leishmania is regulated predominantly by gene dosage [20, 21], we proceeded to search for large-scale copy number variations (deletions and duplications) in chromosomes of the three selected clones for comparison with the unselected parental line. In the past, different ABCG and ABCC efflux-pump gene clusters were identified in L. infantum as part of chromosomes 6, 23 and 31 [52]. In addition, overexpression of the ABCG6 transporter is known to be involved in CPT resistance in Leishmania parasites [53]. Of note, none of these regions was found amplified in any of the three TPT-resistant clones in our whole-genome comparative analysis, thus indicating a different mechanism of drug resistance when compared with CPT.

The absence of significant amplifications, coupled with the very unusual fact that no prominent changes in ploidy were observed for any of the studied clones [54], led us to suspect the potential implication of SNPs and small nucleotide insertions or deletions (indels) in the TPT-resistant phenotypes. Several SNPs have been shown to contribute to drug resistance (e.g. miltefosine, antimonial drugs, etc.) in Leishmania parasites by altering the activity of specific transporters or modifying different detoxification pathways [22, 23, 29, 55, 56]. Here we focused on SNPs and indels present in the three TPT-resistant clones and, at the same time, occurring in the same ORF but at different positions. Among the 8 genes fulfilling these criteria, we identified the ORF coding for the large subunit of the DNA topoisomerase IB, which is the main target of CPT-derivatives once bound to the DNA during cell replication [10, 11]. Clones TPT700.1 and TPT700.3 displayed homozygous mutations in the top1B gene, while the SNP identified in TPT700.2 was heterozygous. Although previously observed in diploid Leishmania parasites, homozygous mutations are rare because of their ‘non-reversible’ nature. The homozygous mutations in clones TPT700.1 and TPT700.3 may have originated from loss of heterozygosity, a well described phenomenon in Leishmania [57, 58]. Importantly, these results reinforce previous works demonstrating the possibility that, although rare, Leishmania can generate SNPs associated with drug resistance without the need for alteration of its genomic architecture and gene expression [59].

Due to the impossibility of generating a null mutant, our preferred way for studying the role of mutated variants of top1B consisted of episomal transfection of the mutated forms into a wild-type strain [23]. As expected, since transfected parasites still carry the top1B^WT, we were able to only partly recreate the resistance observed in the TPT700 mutants. However, the relative strength of each mutation followed the same drug-resistance trend in both the original mutants (TPT700.1 > TPT700.3 > TPT700.2) and the episomal transfectants (top1B^F187Y > top1B^W232R > top1B^G191A).

To better understand the potential contribution of these three SNPs to TPT resistance in the mutants, we performed several MD simulations. All three residues identified in the TPT700 mutants (F187, G191 and W232) were conserved between the human and parasitic enzyme and can be structurally aligned (Fig. 4). They were located in proximity of the TPT binding site, in close proximity to residues found to be crucial in the human enzyme for the interaction and stabilization of TPT with residues R364 and N722 once intercalated between DNA bases [60], corresponding to leishmanial residues R190 and N221. Thus, we can hypothesize that a change in one of the residues of this cluster may influence the arrangement of the TPT binding site. Although these residues are in proximity of the catalytic pentad [11, 14, 61], the mutations identified in this study are likely affecting the ability of binding of TPT to the DNA-TOP1B complex without altering the global catalytic function of the enzyme, but potentially accelerating the re-ligation step. Indeed, it has been very well established that a malfunction of cleavage/re-ligation reactions will be reflected through altered protein drug sensitivity [11, 60]. Of note, two of the three SNPs identified in the TPT700 mutants (F187Y and G191A) were located within the conserved region corresponding to amino acids 361–365 in the hTOP1B enzyme. These results confirm the findings of Rubin et al. (1993) showing that a substitution of residue F361 can induce high levels of resistance against a CPT derivate (e.g. 9-nitro-20(S)camptothecin) in human U-937 myeloid leukemia cells [62]. Likewise, Li et al. (1997) showed that certain substitutions in the 361–364 region affect DNA cleavage/ligation by the enzyme, as well as contribute to resistance against CPT since they may be included in the CPT-binding domain [63]. These results suggest that these mutations are able to modify the architecture of the binding site, decreasing the persistence of TPT in the binding pocket, as well indicate that CPT and TPT may share binding sites in the LiTOP1B–DNA complex.

Furthermore, in the covalent complexes, K352 and R410 demonstrate a changed profile of interaction in all three TPT700 mutants. In particular, the hydrogen bond between K352 and D353 is lost. This interaction is crucial for the correct position of K352 (known to be a key player in the re-ligation reaction), and when incorrectly positioned affects the re-ligation rate and thus TPT sensitivity [15, 64]. Moreover, D353 is itself involved in the network of residues and TPT interaction. As such, the lack of the K352-D353 hydrogen bond and side chains orientation may be a main cause for rearrangement of the TPT binding site and lowered stabilization of the drug in the binding pocket, further explaining the observed resistance.

Importantly, the three TPT700 mutants became resistant to TPT without impairing their ability to proliferate in vitro. The process of becoming drug resistant can lead to different evolutionary disadvantages (‘fitness cost’), such as reduced survival [65]. However, this concept remains controversial in Leishmania and is highly dependent on the parasite’s genetic and environmental context [48]. Likewise, drug-resistant cancer cell lines exhibit different fitness-cost profiles, including subpopulations with increased fitness when compared to their sensitive counterparts [66]. The absence of fitness cost in vitro in the TPT700 mutants could be due in part to the fact that these cells do not use ATP-dependent drug efflux pumps to resist treatment (e.g. MRPA in antimony resistance), for which they would have to divert energy away from proliferation towards running of the pumps. Moreover, the absence of a cost in terms of growth would also explain why the TPT700 mutants did not return to sensitivity once the TPT was withdrawn. Mutants of this type have the potential to become a major risk for the spread of drug resistance into an environment devoid of antileishmanial drugs. However, at this point, whether this phenomenon would be stable in vivo or in the vector remains to be evaluated.

This study represents the first whole-genome characterization of Leishmania parasites repeatedly exposed to a TOP1B inhibitor. Unlike current antileishmanial agents, TPT resistance did not impact leishmanial genomic organization or gene expression, and did not lead to amplification of efflux pumps; a phenomenon previously reported for CPT analogues in cancer cells. Of note, we found that these mutations could decrease the binding of TPT to the DNA-TOP1B binary complex, as well as lower the stabilization of the drug in the binding pocket of the leishmanial heterodimeric enzyme. Altogether, this work helps elucidate the previously-unclear potential mechanisms of TPT resistance in Leishmania by mutations in the large subunit of TOP1B, and provides a valuable clue for the design of improved inhibitors to combat drug resistance. Due to the conserved nature of the mutated amino acids, this knowledge could also provide important means of overcoming resistance to TOP1B-specific drugs and developing diagnostic tools to detect TPT-resistant tumor cells. Finally, this study supports and expands the importance of including drug-resistance assessments in drug-discovery and drug-repurposing cascades before proposing a molecule as a potential prototype for treatment of parasitic diseases.

A₆₀₀: absorbance at 600 nm

ABC: ATP Binding Cassette

AQP1: aquaglyceroporin 1

CNVs: DNA copy number variations

CPT: camptothecin

dsDNA: double-stranded DNA

EC₅₀: half maximal effective concentration

FDA: US Food and Drug Administration

hyg: hygromycin phosphotransferase gene

hTOP1B: Homo sapiens DNA topoisomerase 1B

InDel: insertion-deletion

LiTOP1B: Leishmania donovani infantum DNA topoisomerase 1B

MD: Molecular dynamics

MRP: Multidrug Resistance-Associated Protein

NGS: next-generation sequencing

NTD: Neglected Tropical Disease

ORF: open reading frame

SNP: single nucleotide polymorphism

TOP: DNA topoisomerase

TOP1B: Type 1B DNA topoisomerase

TPT: topotecan

WGS: whole-genome sequencing

WT: wild-type

Acknowledgements

We would like to thank to Professor Marc Ouellette for his outstanding support with WGS experiments. We would like also to thank Dr. Aida Minguez-Menendez for her help with the conception and creation of the graphical abstract and figures.

Authors' contributions

CRT, VW, AC, IDA, PL performed experiments. CRT, IDA, PL, RMM, CFP analyzed data. CRT, VW, CFP drafted the manuscript and performed manuscript preparation. CFP, RMN supervised and performed the collection of materials and all other laboratory experiments. CFP conceived the idea and coordinated the project. All authors read and approved the final manuscript.

Funding

This research was funded by the Fonds de Recherche du Québec-Nature et Technologies (FRQNT) through its New University Researchers Start-up Program, grant number 2019-NC-253631; the Canada foundation for Innovation (www.innovation.ca), grant number 37324; and by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN-2017-04480 awarded to CFP. CRT, VW and AC were supported by the FRQNT and the NSERC studentship programs. RMN is a CNPq (Brazilian National Council for Scientific and Technological Development) Research Fellow (#310640/2017-2).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files. The sequence data for L. infantum TPT-resistant mutants is available at the NCBI BioProject (https://www.ncbi.nlm.nih.gov/bioproject/) under study accession PRJNA647847 and sample accessions SAMN15599759, SAMN15599760, SAMN15599761, corresponding to TPT-resistant clones TPT700.1, TPT700.2 and TPT700.3, respectively.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no financial or non-financial competing interests.

Author details

¹Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Québec, Canada. ²The Research Group on Infectious Diseases in Production Animals (GREMIP), Faculté de Médecine Vétérinaire, Université de Montréal, Québec, Canada. ³Medtronic EMEA, Study & Scientific Solutions, Milan, Italy. ⁴Centre de Recherche en Infectiologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec, Université Laval, Canada. ⁵Research Group on Biotechnology Applied to Pathogens – Instituto René Rachou – Fundação Oswaldo Cruz/Fiocruz Minas, Belo Horizonte, Brazil. ⁶Department of Microbiology and Immunology, Faculty of Medicine, McGill University, Montréal, Québec, Canada.

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Three Different Mutations in the DNA Topoisomerase 1B in Leishmania Infantum Contribute to Resistance to Antitumor Drug Topotecan

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Version 1

Abstract

Figures

Background

Methods

Mutant selection

Drug susceptibility assays

Whole-genome sequencing of TPT-resistant mutants

DNA constructs and nucleofection

Results

Comparative WGS

Structural effect of the SNPs and impact on TPT sensitivity

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1