Identification of FTY720 and COH29 as novel topoisomerase I catalytic inhibitors by experimental and computational studies.

The development of novel topoisomerase I (TOP1) inhibitors is crucial for overcoming the drawbacks and limitations of current TOP1 poisons. Here, we identified two potential TOP1 inhibitors, namely, FTY720 (a sphingosine 1-phosphate antagonist) and COH29 (a ribonucleotide reductase inhibitor), through experimental screening of known active compounds. Biological experiments verified that FTY720 and COH29 were nonintercalative TOP1 catalytic inhibitors that did not induce the formation of DNA-TOP1 covalent complexes. Molecular docking revealed that FTY720 and COH29 interacted favorably with TOP1. Molecular dynamics simulations revealed that FTY720 and COH29 could affect the catalytic domain of TOP1, thus resulting in altered DNA-binding cavity size. The alanine scanning and interaction entropy identified Arg536 as a hotspot residue. In addition, the bioinformatics analysis predicted that FTY720 and COH29 could be effective in treating malignant breast tumors. Biological experiments verified their antitumor activities using MCF-7 breast cancer cells. Their combinatory effects with TOP1 poisons were also investigated. Further, FTY720 and COH29 were found to cause less DNA damage compared with TOP1 poisons. The findings provide reliable lead compounds for the development of novel TOP1 catalytic inhibitors and offer new insights into the potential clinical applications of FTY720 and COH29 in targeting TOP1.

Variation of Structure and Cellular Functions of Type IA Topoisomerases across the Tree of Life.

Topoisomerases regulate the topological state of cellular genomes to prevent impediments to vital cellular processes, including replication and transcription from suboptimal supercoiling of double-stranded DNA, and to untangle topological barriers generated as replication or recombination intermediates. The subfamily of type IA topoisomerases are the only topoisomerases that can alter the interlinking of both DNA and RNA. In this article, we provide a review of the mechanisms by which four highly conserved N-terminal protein domains fold into a toroidal structure, enabling cleavage and religation of a single strand of DNA or RNA. We also explore how these conserved domains can be combined with numerous non-conserved protein sequences located in the C-terminal domains to form a diverse range of type IA topoisomerases in Archaea, Bacteria, and Eukarya. There is at least one type IA topoisomerase present in nearly every free-living organism. The variation in C-terminal domain sequences and interacting partners such as helicases enable type IA topoisomerases to conduct important cellular functions that require the passage of nucleic acids through the break of a single-strand DNA or RNA that is held by the conserved N-terminal toroidal domains. In addition, this review will exam a range of human genetic disorders that have been linked to the malfunction of type IA topoisomerase.

Structures and implications of the C962R protein of African swine fever virus.

African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. Although it has been extensively studied in the past, no vaccine or other useful treatment against ASFV is available. The genome of ASFV encodes more than 170 proteins, but the structures and functions for the majority of the proteins remain elusive, which hindered our understanding on the life cycle of ASFV and the development of ASFV-specific inhibitors. Here, we report the structural and biochemical studies of the highly conserved C962R protein of ASFV, showing that C962R is a multidomain protein. The N-terminal AEP domain is responsible for the DNA polymerization activity, whereas the DNA unwinding activity is catalyzed by the central SF3 helicase domain. The middle PriCT2 and D5_N domains and the C-terminal Tail domain all contribute to the DNA unwinding activity of C962R. C962R preferentially works on forked DNA, and likely functions in Base-excision repair (BER) or other repair pathway in ASFV. Although it is not essential for the replication of ASFV, C962R can serve as a model and provide mechanistic insight into the replicative primase proteins from many other species, such as nitratiruptor phage NrS-1, vaccinia virus (VACV) and other viruses.

Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domain.

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a ß-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length and is dispensable for DNA supercoiling, the ß-bulge loop is required for supercoiling activity. It has previously been shown that the ß-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a ß-bulge loop as a minimal latch is reported. It is shown that the ß-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.

DNA preference of indenoisoquinolines: a computational approach.

The human topoisomerase IB (hTopoIB) enzyme is a monomeric protein that relaxes the supercoils on double-stranded DNA by forming a covalent DNA/hTopoIB complex by introducing a nick on the DNA strand. Inhibition of hTopoIB results in cell death, which makes this protein a strong target for the treatment of various cancer types, including small-cell lung cancers and ovarian cancers. Camptothecin (CPT) and indenoisoquinoline (IQN) classes of compounds inhibit the hTopoIB activity by intercalating to nicked DNA pairs; however, these inhibitors show different preferences towards DNA bases when bound to the DNA/hTopoIB complex. Here, we investigated the affinities of CPT and one IQN derivative towards different DNA base pairs. The two inhibitors showed different stacking behaviors in the intercalation site and interaction pattern with binding pocket residues, indicating that they have different inhibition mechanisms in the binding pocket that affects the base-pair selectivity. The results obtained from this study are expected to guide researchers in designing gene-specific and more potent compounds to fight cancer through hTopoIB poisoning.

DNA topoisomerase Top3ß is impacted by early life stress in the developing female and male rat brain.

DNA topoisomerases are essential for preserving genomic integrity. DNA topoisomerases induce breakage of DNA to facilitate replication and transcription by relaxing DNA and relieving supercoiling. Aberrant expression and deletions of topoisomerases are associated with psychiatric disorders such as schizophrenia and autism. Our study investigated the effects of early life stress (ELS) on three topoisomerases, Top1, Top3α, and Top3ß in the developing rat brain. Newborn rats were exposed to a predator odor stress on postnatal days 1, 2, and 3; brain tissue was collected either 30 min after the last stressor on postnatal day 3 or during the juvenile period. We found that exposure to predator odor resulted in a decrease in Top3ß expression levels in the neonatal male amygdala and in the juvenile prefrontal cortex of males and females. These data suggest that developing males and females respond differently to predator odor-induced stress. As ELS results in lower Top3ß levels, these data suggest that ELS experienced during development may have consequences for genomic structural integrity and increased mental health risk.

Unraveling topoisomerase IA gate dynamics in presence of PPEF and its preclinical evaluation against multidrug-resistant pathogens.

Type IA topoisomerases maintain DNA topology by cleaving ssDNA and relaxing negative supercoils. The inhibition of its activity in bacteria prevents the relaxation of negative supercoils, which in turn impedes DNA metabolic processes leading to cell death. Using this hypothesis, two bisbenzimidazoles, PPEF and BPVF are synthesized, selectively inhibiting bacterial TopoIA and TopoIII. PPEF stabilizes the topoisomerase and topoisomerase-ssDNA complex, acts as an interfacial inhibitor. PPEF display high efficacy against ~455 multi-drug resistant gram positive and negative bacteria. To understand molecular mechanism of inhibition of TopoIA and PPEF, accelerated MD simulation is carried out, and results suggested that PPEF binds, stabilizes the closed conformation of TopoIA with -6Kcal/mol binding energy and destabilizes the binding of ssDNA. The TopoIA gate dynamics model can be used as a tool to screen TopoIA inhibitors as therapeutic candidates. PPEF and BPVF cause cellular filamentation and DNA fragmentation leading to bacterial cell death. PPEF and BPVF show potent efficacy against systemic and neutropenic mouse models harboring E. coli, VRSA, and MRSA infection without cellular toxicity.

Binding profile of a mixed-ligand silver(I) complex with DNA and Topoisomerase I.

A new mixed-ligand Ag(I) complex, [Ag(daf)(phen)]NO3 (daf = 4,5-diazafluoren-9-one and dian = N-(4,5-diazafluoren-9-ylidene)aniline), was synthesized. The elemental analysis, FTIR, 1HNMR, UV-Vis spectroscopy, cyclic voltammetry, and DFT (Density Functional Theory) geometry optimization method were applied in order to predict the Ag(I) complex structure which concluded to a distorted tetrahedral N4 coordination around the Ag(I) center. A detailed in silico analysis of the bioaffinity of the complex to DNA and human DNA-Topoisomerase I was conducted using molecular docking simulations and ONIOM (Our own N-layered Integrated molecular Orbital and molecular Mechanics) techniques. In this overall scenario, the results suggest the dominance of π-π stacking interactions of the heteroaromatic ligands in the intercalating pocket of DNA and the active site of the enzyme and the rational correlation between being a good intercalator and a potent Topoisomerase I inhibitor. In vitro DNA-binding experiments by spectrophotometric, spectrofluorometric, Voltammetric, and viscometric techniques at physiological pH also confirmed the computational results. The complex inhibited MCF-7 cell growth in a dose-dependent manner while being nontoxic on HUVEC normal cells.

Evolution of TOP1 and TOP1MT Topoisomerases in Chordata.

Type IB topoisomerases relax the torsional stress associated with DNA metabolism in the nucleus and mitochondria and constitute important molecular targets of anticancer drugs. Vertebrates stand out among eukaryotes by having two Type IB topoisomerases acting specifically in the nucleus (TOP1) and mitochondria (TOP1MT). Despite their major importance, the origin and evolution of these paralogues remain unknown. Here, we examine the molecular evolutionary processes acting on both TOP1 and TOP1MT in Chordata, taking advantage of the increasing number of available genome sequences. We found that both TOP1 and TOP1MT evolved under strong purifying selection, as expected considering their essential biological functions. Critical active sites, including those associated with resistance to anticancer agents, were found particularly conserved. However, TOP1MT presented a higher rate of molecular evolution than TOP1, possibly related with its specialized activity on the mitochondrial genome and a less critical role in cells. We could place the duplication event that originated the TOP1 and TOP1MT paralogues early in the radiation of vertebrates, most likely associated with the first round of vertebrate tetraploidization (1R). Moreover, our data suggest that cyclostomes present a specialized mitochondrial Type IB topoisomerase. Interestingly, we identified two missense mutations replacing amino acids in the Linker region of TOP1MT in Neanderthals, which appears as a rare event when comparing the genome of both species. In conclusion, TOP1 and TOP1MT differ in their rates of evolution, and their evolutionary histories allowed us to better understand the evolution of chordates.

Holanamine, a Steroidal Alkaloid from the Bark of Holarrhena pubescens Wall. ex G. Don Inhibits the Growth of Leishmania donovani by Targeting DNA Topoisomerase 1B.

Leishmaniasis is a group of neglected tropical diseases (NTDs) caused by about 20 species of obligate intracellular protozoan parasites of the genus Leishmania, which occurs in cutaneous, mucocutaneous, and visceral forms. Many researchers have sought to utilize natural products for novel and effective treatments to combat many infectious diseases, including leishmaniasis. Holarrhena pubescens Wall. ex G. Don (Apocynaceae) bark is a rich source of bioactive steroidal alkaloids. The total alkaloidal extract (IC50 6.12 ± 0.117 µg/mL), and the isolated alkaloid, holanamine, showed significant antileishmanial activity (IC50 2.66 ± 0.112 µM against AG83 and 3.80 ± 0.126 µM against BHU-575) against the Leishmania donovani parasite, better than miltefosine (IC50 19.61 ± 0.093 µM against AG83 and 23.20 ± 0.094 µM against BHU-575). Holanamine inhibited the L. donovani topoisomerase 1B (LdToP1B) in a non-competitive manner (IC50 2.81 ± 0.105 µM), indicating that it interacts with the free enzyme and enzyme-DNA complex without inhibiting human topoisomerase. Hydrogen bonding and hydrophobic interactions of holanamine with the N-terminal and hinge region of the large subunit of LTop1B is responsible for its potent antileishmanial activity, as shown by docking studies. Treatment with holanamine causes apoptotic-like cell death by generating cellular and mitochondrial reactive oxygen species, disrupting the mitochondrial membrane potential and inducing ultrastructural alterations in the promastigotes. Holanamine effectively clears intracellular amastigotes but minimally affects host macrophages with no significant cytotoxicity in HEK 293 and L929 cell lines. Thus, our studies show that holanamine can further be used to develop effective antileishmanial agents against evolving drug-resistant parasites.

Structural and biochemical basis for DNA and RNA catalysis by human Topoisomerase 3ß.

In metazoans, topoisomerase 3ß (TOP3B) regulates R-loop dynamics and mRNA translation, which are critical for genome stability, neurodevelopment and normal aging. As a Type IA topoisomerase, TOP3B acts by general acid-base catalysis to break and rejoin single-stranded DNA. Passage of a second DNA strand through the transient break permits dissipation of hypernegative DNA supercoiling and catenation/knotting. Additionally, hsTOP3B was recently demonstrated as the human RNA topoisomerase, required for normal neurodevelopment and proposed to be a potential anti-viral target upon RNA virus infection. Here we elucidate the biochemical mechanisms of human TOP3B. We delineate the roles of divalent metal ions, and of a conserved Lysine residue (K10) in the differential catalysis of DNA and RNA. We also demonstrate that three regulatory factors fine-tune the catalytic performance of TOP3B: the TOP3B C-terminal tail, its protein partner TDRD3, and the sequence of its DNA/RNA substrates.

A tale of topoisomerases and the knotty genetic material in the backdrop of Plasmodium biology.

The untangling or overwinding of genetic material is an inevitable part of DNA replication, repair, recombination, and transcription. Topoisomerases belong to a conserved enzyme family that amends DNA topology during various processes of DNA metabolism. To relax the genetic material, topoisomerases transiently break the phosphodiester bond on one or both DNA strands and remain associated with the cleavage site by forming a covalent enzyme-DNA intermediate. This releases torsional stress and allows the broken DNA to be re-ligated by the enzyme. The biological function of topoisomerases ranges from the separation of sister chromatids following DNA replication to the aiding of chromosome condensation and segregation during mitosis. Topoisomerases are also actively involved in meiotic recombination. The unicellular apicomplexan parasite, Plasmodium falciparum, harbors different topoisomerase subtypes, some of which have substantially different sequences and functions from their human counterparts. This review highlights the biological function of each identified Plasmodium topoisomerase along with a comparative analysis of their orthologs in human or other model organisms. There is also a focus on recent advancements towards the development of topoisomerase chemical inhibitors, underscoring the druggability of unique topoisomerase subunits that are absent in humans. Plasmodium harbors three distinct genomes in the nucleus, apicoplast, and mitochondria, respectively, and undergoes non-canonical cell division during the schizont stage of development. This review emphasizes the specific developmental stages of Plasmodium on which future topoisomerase research should focus.

A Yeast-Based Screening System for Differential Identification of Poisons and Suppressors of Human Topoisomerase I.

BACKGROUND: Inhibition of human topoisomerase I (TOP1) by camptothecin and topotecan has been shown to reduce excessive transcription of PAMP (Pathogen-Associated Molecular Pattern)-induced genes in prior studies, preventing death from sepsis in animal models of bacterial and SARS-CoV-2 infections. The TOP1 catalytic activity likely resolves the topological constraints on DNA that encodes these genes to facilitate the transcription induction that leads to excess inflammation. The increased accumulation of TOP1-DNA covalent complex (TOP1cc) following DNA cleavage is the basis for the anticancer efficacy of the TOP1 poisons developed for anticancer treatment. The potential cytotoxicity and mutagenicity of TOP1 targeting cancer drugs pose serious concerns for employing them as therapies in sepsis prevention. METHODS: In this study we set up a novel yeast-based screening system that employs yeast strains expressing wild-type or a dominant lethal mutant recombinant human TOP1. The effect of test compounds on growth is monitored with and without overexpression of the recombinant human TOP1. RESULTS: This yeast-based screening system can identify human TOP1 poisons for anticancer efficacy as well as TOP1 suppressors that can inhibit TOP1 DNA binding or cleavage activity in steps prior to the formation of the TOP1cc. CONCLUSIONS: This yeast-based screening system can distinguish between TOP1 suppressors and TOP1 poisons. The assay can also identify compounds that are likely to be cytotoxic based on their effect on yeast cell growth that is independent of recombinant human TOP1 overexpression.

Discovery of novel rigid analogs of 2-naphthol with potent anticancer activity through multi-target topoisomerase I & II and tyrosine kinase receptor EGFR & VEGFR-2 inhibition mechanism.

A novel oxygen-containing heterocyclic linked 1H-benzo[f]chromene moieties (4a-g) and (6a-g) with anti-proliferative activity against cancer cell lines was designed, synthesized, and established on the basis of spectral data. All the prepared compounds were evaluated in vitro for their anti-proliferative activity against MCF-7, HCT-116, HepG-2 cell lines and normal cell lines HFL-1, WI-38. Compounds 4a, 4b, and 6e exhibited good activity against MCF-7, HCT-116, and HepG-2 cell lines, comparable to that of Vinblastine and Doxorubicin, and weak active against normal cell lines. Moreover, the potential mechanisms of the cytotoxic activity of the promising compounds 4a, 4b, and 6e on the more sensitive cell line MCF-7 were studied. We found that compounds 4a, 4b, and 6e induce cell cycle arrest at G2/M phases for MCF-7 treated cells compared to untreated cells, which causes apoptosis and inhibits both the topoisomerase I and II enzymes. In addition, compounds 4a and 4b exhibited comparable inhibitory activity on tyrosine kinase receptors EGFR and VEGFR-2 kinases to that of the reference protein kinases inhibitor Sorafenib. The in silico molecular docking of the most active compounds into the active sites of EGFR kinase and Topo I & II enzymes provides us with a reasonable clarification of the interpreted biological data.

Topoisomerase I (TOP1) dynamics: conformational transition from open to closed states.

Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.

Higher detection of JC polyomavirus in colorectal cancerous tissue after pretreatment with topoisomerase I enzyme; colorectal tissue serves as a JCPyV persistence site.

BACKGROUND: The JC polyomavirus has been blamed to contribute in colorectal cancer (CRC), however, the topic is still controversial. Varying detection rate of JCPyV genome has been reported mainly due to technical reasons. Here, we provide summative data on the topic, with emphasize on technical issues. METHODS: Formalin-fixed paraffin-embedded tissue samples from 50 patients with CRC, consisting of tumoral and non-cancerous marginal tissue (totally 100 samples) were included in the study. After DNA extraction, specific JCPyV T-Ag sequences were targeted using Real-time PCR. To unwind the supercoiled JCPyV genome, pretreatment with topoisomerase I, was applied. Immunohistochemical (IHC) staining was performed using an anti-T-Ag monoclonal antibody. RESULTS: In the first attempts, no samples were found to be positive in Real-time PCR assays. However, JCPyV sequences were found in 60% of CRC tissues and 38% of non-cancerous colorectal mucosa after application of pre-treatment step with topoisomerase I enzyme (P = 0.028). T-Ag protein was found in the nuclear compartment of the stained cells in IHC assays. CONCLUSIONS: The presence of JCPyV in CRC tissues, as well as T-Ag localization in the nucleolus, where its oncogenic effect takes place, may provide supporting evidence for JCPyV involvement in CRC development. The study highlights the importance of using topoisomerase I to enhance JCPyV genome detection. Also, colorectal tissue is one of the permissive human tissue for JC resistance after preliminary infection.

PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation.

Poly(ADP)-ribosylation (PARylation) regulates chromatin structure and recruits DNA repair proteins. Using single-molecule fluorescence microscopy to track topoisomerase I (TOP1) in live cells, we found that sustained PARylation blocked the repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) in a similar fashion as inhibition of the ubiquitin-proteasome system (UPS). PARylation of TOP1-DPC was readily revealed by inhibiting poly(ADP-ribose) glycohydrolase (PARG), indicating the otherwise transient and reversible PARylation of the DPCs. As the UPS is a key repair mechanism for TOP1-DPCs, we investigated the impact of TOP1-DPC PARylation on the proteasome and found that the proteasome is unable to associate with and digest PARylated TOP1-DPCs. In addition, PARylation recruits the deubiquitylating enzyme USP7 to reverse the ubiquitylation of PARylated TOP1-DPCs. Our work identifies PARG as repair factor for TOP1-DPCs by enabling the proteasomal digestion of TOP1-DPCs. It also suggests the potential regulatory role of PARylation for the repair of a broad range of DPCs.

In Vitro and In Silico Characterization of an Antimalarial Compound with Antitumor Activity Targeting Human DNA Topoisomerase IB.

Human DNA topoisomerase IB controls the topological state of supercoiled DNA through a complex catalytic cycle that consists of cleavage and religation reactions, allowing the progression of fundamental DNA metabolism. The catalytic steps of human DNA topoisomerase IB were analyzed in the presence of a drug, obtained by the open-access drug bank Medicines for Malaria Venture. The experiments indicate that the compound strongly and irreversibly inhibits the cleavage step of the enzyme reaction and reduces the cell viability of three different cancer cell lines. Molecular docking and molecular dynamics simulations suggest that the drug binds to the human DNA topoisomerase IB-DNA complex sitting inside the catalytic site of the enzyme, providing a molecular explanation for the cleavage-inhibition effect. For all these reasons, the aforementioned drug could be a possible lead compound for the development of an efficient anti-tumor molecule targeting human DNA topoisomerase IB.

The Mode of SN38 Derivatives Interacting with Nicked DNA Mimics Biological Targeting of Topo I Poisons.

The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered for advancement to the preclinical development stage. To gain better insights into the molecular mechanism with the biological target, here, we conducted an investigation into their interactions with model nicked DNA (1) using different techniques. In this work, we observed the complexity of the mechanism of action of the compounds 2 and 3, in addition to their decomposition products: compound 4 and SN38. Using DOSY experiments, evidence of the formation of strongly bonded molecular complexes of SN38 derivatives with DNA duplexes was provided. The molecular modeling based on cross-peaks from the NOESY spectrum also allowed us to assign the geometry of a molecular complex of DNA with compound 2. Confirmation of the alkylation reaction of both compounds was obtained using MALDI-MS. Additionally, in the case of 3, alkylation was confirmed in the recording of cross-peaks in the 1H/13C HSQC spectrum of 13C-enriched compound 3. In this work, we showed that the studied compounds-parent compounds 2 and 3, and their potential metabolite 4 and SN38-interact inside the nick of 1, either forming the molecular complex or alkylating the DNA nitrogen bases. In order to confirm the influence of the studied compounds on the topoisomerase I relaxation activity of supercoiled DNA, the test was performed based upon the measurement of the fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The presented results confirmed that studied SN38 derivatives effectively block DNA relaxation mediated by Topo I, which means that they stop the machinery of Topo I activity.

Discovery of Leishmania donovani topoisomerase IB selective inhibitors by targeting protein-protein interactions between the large and small subunits.

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 µM; LRL-TP-94: 2.9 µM; and LRL-TP-101: 35.3 µM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 µM in extracellular promastigote assay and 34.0, 53.7, and 11.4 µM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.