Our research group identified CTN1122, an imidazo[1,2-a]pyrazine derivative, as a promising antileishmanial agent targeting intramacrophage amastigotes of Leishmania major and Leishmania donovani. CTN1122 selectively inhibits Leishmania casein kinase 1 (L-CK1.2) with a favorable safety profile. Docking studies based on a homology model highlighted key pharmacophoric elements: a 4-pyridyl group at C3, crucial for hydrogen bonding with leucine 90 in the ATP-binding site, and a 4-fluorophenyl moiety at C2, fitting into a hydrophobic pocket. In order to validate these findings, 14 analogs were synthesized with targeted modifications on the imidazo[1,2-a]pyrazine core structure. Three probed the C8 position, three evaluated the impact of C2 substitution, six assessed the C3 4-pyridyl group, and two combined changes at C8 and C3. The study confirmed the critical role of C2 and C3 substituents, as their absence significantly reduced L-CK1.2 inhibition and antileishmanial activity. Additionally, the nitrogen's position within the pyridine ring at C3 proved essential: compound 23, with a meta-pyridyl group, was inactive. Notably, compound 30 exhibited the highest antileishmanial in vitro potency (IC50 = 0.20 μM for L. major; 0.16 μM for L. donovani) alongside enhanced L-CK1.2 inhibition (IC50 = 0.384 μM), with no significant mammalian cytotoxicity.

Leishmania CK1.2 (L-CK1.2) is a serine/threonine protein kinase essential for the survival and immune evasion of the protozoan parasite Leishmania , the causative agent of Leishmaniasis. This study investigates the multifaceted roles of L-CK1.2, which exhibits a dynamic localization pattern and a broad spectrum of interacting partners within the parasite. Using proteomic analysis and confocal microscopy, we identified 230 L-CK1.2-associated proteins (L-CKAPs) across the parasite life stages, promastigotes, which replicate within the insect vector, and amastigotes, which proliferate inside the phagolysosomes of host macrophages. This analysis revealed the ubiquitous presence of L-CK1.2 within various cellular structures, including the cytoskeleton, basal body, and flagellum. Our data also identified 64 host proteins (L-CKAP host ) that interact with L-CK1.2, implicating it in modulating critical host biological processes such as immune response, apoptosis, and purine biosynthesis, processes regulated by Leishmania during infection. The study highlights the dual function of L-CK1.2 as it plays roles both in the parasite (cis) and within the host cell (trans), positioning this kinase as a key player in host-pathogen interactions. This work provides a comprehensive map of L-CK1.2 interactions and uncovers its importance in regulating intracellular survival, providing potential therapeutic targets for Leishmaniasis. Furthermore, given the evolutionary conservation of CK1.2 across other parasitic organisms, our findings may have broader implications for understanding and managing parasitic infections. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-13-ISV3-0009, ANR-10-LABX-62-IBEID

Our research group previously discovered CTN1122, an imidazo[1,2-a]pyrazine compound with promising antileishmanial activity against intramacrophage amastigotes of Leishmania major and L. donovani strains. CTN1122 effectively targets Leishmania casein kinase 1 (L-CK1.2) and exhibits a favorable safety profile. To further explore its chemical space, we developed a convergent strategy to modify the C2 position of the imidazo[1,2-a]pyrazine core using Suzuki-Miyaura coupling of the corresponding triflate intermediate. Among 15 newly synthesized analogs, seven derivatives featuring variously substituted phenyl rings at C2 demonstrated L-CK1.2 inhibition within micromolar to submicromolar ranges and antileishmanial activity in vitro with low cytotoxicity in macrophages. Compounds 7 d and 7 l were particularly potent, with IC50 values of 1.25 mu M and 0.92 mu M against L. major, and 1.44 mu M and 2.34 mu M against L. donovani, respectively. They showed IC50 L-CK1.2=0.30 mu M and 0.57 mu M with enhanced selectivity indices (SI=3.8 and 1.6) over the human CK1 & varepsilon; ortholog. Additionally, four C2 analogs and two C5 isomers exhibited notable antiparasitic effects without strongly inhibiting L-CK1.2, indicating a possible alternative mechanism of action. Compound 7 k displayed the highest general activity, with IC50 values of 0.31 mu M on L. major and 0.27 mu M on L. donovani, coupled with favorable selectivity indexes.

Leishmania CK1.2 (L-CK1.2) is a serine/threonine protein kinase essential for the survival of the protozoan parasite Leishmania , the causative agent of Leishmaniasis. This study investigates the dynamic localization pattern of L-CK1.2 and the broad spectrum of its interacting partners within the parasite. Using proteomic analysis and confocal microscopy, we identified 230 L-CK1.2-associated proteins across the parasite life stages, promastigotes (in the insect vector), and amastigotes, (in the phagolysosomes of host macrophages). This analysis revealed the ubiquitous presence of L-CK1.2 in various cellular structures, including the cytoskeleton, basal body, and flagellum. Using an in vitro system, sixty-four host L-CK1.2-associated proteins, involved in critical host biological processes such as immune response, apoptosis, and purine biosynthesis, were shown to interact with L-CK1.2. These processes are known to be regulated by Leishmania during infection. The study highlights the dual function of L-CK1.2, in the parasite (cis) and within the host cell (trans), positioning this kinase as a key player in host-pathogen interactions. This work provides a comprehensive map of L-CK1.2 interactions and suggest its potential importance in regulating intracellular Leishmania survival, providing potential therapeutic targets for Leishmaniasis. Furthermore, given the evolutionary conservation of CK1.2 across other parasitic organisms, our findings may have broader implications for understanding and managing parasitic infections.

Our research group identified CTN1122, an imidazo[1,2-a]pyrazine derivative, as a promising antileishmanial agent targeting intramacrophage amastigotes of Leishmania major and Leishmania donovani. CTN1122 selectively inhibits Leishmania casein kinase 1 (L-CK1.2) with a favorable safety profile. Docking studies based on a homology model highlighted key pharmacophoric elements: a 4-pyridyl group at C3, crucial for hydrogen bonding with leucine 90 in the ATP-binding site, and a 4-fluorophenyl moiety at C2, fitting into a hydrophobic pocket. In order to validate these findings, 14 analogs were synthesized with targeted modifications on the imidazo[1,2-a]pyrazine core structure. Three probed the C8 position, three evaluated the impact of C2 substitution, six assessed the C3 4-pyridyl group, and two combined changes at C8 and C3. The study confirmed the critical role of C2 and C3 substituents, as their absence significantly reduced L-CK1.2 inhibition and antileishmanial activity. Additionally, the nitrogen's position within the pyridine ring at C3 proved essential: compound 23, with a meta-pyridyl group, was inactive. Notably, compound 30 exhibited the highest antileishmanial in vitro potency (IC50 = 0.20 mu M for L. major; 0.16 mu M for L. donovani) alongside enhanced L-CK1.2 inhibition (IC50 = 0.384 mu M), with no significant mammalian cytotoxicity.

Leishmaniasis constitutes a severe public health problem, with an estimated prevalence of 12 million cases. This potentially fatal disease has a worldwide distribution and in 2012, the fatal Visceral Leishmaniasis (VL) was declared as new emerging disease in Europe, mainly due to global warming, with expected important public health impact. The available treatments are toxic, costly or lead to parasite resistance, thus there is an urgent need for new drugs with new mechanism of action. Previously, we reported the discovery of CTN1122, a potent imidazo[1,2-a]pyrazine-based antileishmanial hit compound targeting L-CK1.2 at low micromolar ranges. Here, we described structurally related, safe and selective compounds endowed with antiparasitic properties, better than miltefosine, the reference therapy by oral route. L-CK1.2 homology model gave the first structural explanations of the role of 4-pyridyl (CTN1122) and 2-aminopyrimidin-4-yl (compound 21) moieties, at the position 3 of the central core, in the low micromolar to nanomolar L-CK1.2 inhibition, whereas N-methylpyrazole derivative 11 remained inactive against the parasite kinase.

ABSTRACT Existing therapies for leishmaniases present significant limitations, such as toxic side effects, and are rendered inefficient by parasite resistance. It is of utmost importance to develop novel drugs targeting Leishmania that take these two limitations into consideration. We thus chose a target-based approach using an exoprotein kinase, Leishmania casein kinase 1.2 (LmCK1.2) that was recently shown to be essential for intracellular parasite survival and infectivity. We developed a four-step pipeline to identify novel selective antileishmanial compounds. In step 1, we screened 5,018 compounds from kinase-biased libraries with Leishmania and mammalian CK1 in order to identify hit compounds and assess their specificity. For step 2, we selected 88 compounds among those with the lowest 50% inhibitory concentration to test their biological activity on host-free parasites using a resazurin reduction assay and on intramacrophagic amastigotes using a high content phenotypic assay. Only 75 compounds showed antileishmanial activity and were retained for step 3 to evaluate their toxicity against mouse macrophages and human cell lines. The four compounds that displayed a selectivity index above 10 were then assessed for their affinity to LmCK1.2 using a target deconvolution strategy in step 4. Finally, we retained two compounds, PP2 and compound 42, for which LmCK1.2 seems to be the primary target. Using this four-step pipeline, we identify from several thousand molecules, two lead compounds with a selective antileishmanial activity.

ABSTRACT Protein kinase inhibitors have emerged as new drugs in various therapeutic areas, including leishmaniasis, an important parasitic disease. Members of the Leishmania casein kinase 1 (CK1) family represent promising therapeutic targets. Leishmania casein kinase 1 isoform 2 (CK1.2) has been identified as an exokinase capable of phosphorylating host proteins, thus exerting a potential immune-suppressive action on infected host cells. Moreover, its inhibition reduces promastigote growth. Despite these important properties, its requirement for intracellular infection and its chemical validation as a therapeutic target in the disease-relevant amastigote stage remain to be established. In this study, we used a multidisciplinary approach combining bioinformatics, biochemical, and pharmacological analyses with a macrophage infection assay to characterize and define Leishmania CK1.2 as a valid drug target. We show that recombinant and transgenic Leishmania CK1.2 (i) can phosphorylate CK1-specific substrates, (ii) is sensitive to temperature, and (iii) is susceptible to CK1-specific inhibitors. CK1.2 is constitutively expressed at both the promastigote insect stage and the vertebrate amastigote stage. We further demonstrated that reduction of CK1 activity by specific inhibitors, such as D4476, blocks promastigote growth, strongly compromises axenic amastigote viability, and decreases the number of intracellular Leishmania donovani and L. amazonensis amastigotes in infected macrophages. These results underline the potential role of CK1 kinases in intracellular survival. The identification of differences in structure and inhibition profiles compared to those of mammalian CK1 kinases opens new opportunities for Leishmania CK1.2 antileishmanial drug development. Our report provides the first chemical validation of Leishmania CK1 protein kinases, required for amastigote intracellular survival, as therapeutic targets.

Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition.

Leishmania parasites cause important human morbidity and mortality. Essential Leishmania genes escape genetic assessment by loss-of-function analyses due to lethal null mutant phenotypes, even though these genes and their products are biologically most significant and represent validated drug targets. Here we overcome this limitation using a facilitated null mutant approach applied for the functional genetic analysis of the MAP kinase LmaMPK4. This system relies on the episomal expression of the target gene from vector pXNG that expresses the Herpes simplex virus thymidine kinase gene thus rendering transgenic parasites susceptible for negative selection using the antiviral drug ganciclovir. Using this system we establish the genetic proof of LmaMPK4 as essential kinase in promastigotes. LmaMPK4 structure/function analysis by plasmid shuffle allowed us to identify regulatory kinase sequence elements relevant for chemotherapeutic intervention. A partial null mutant, expressing an MPK4 derivative with altered ATP-binding properties, showed defects in metacyclogenesis, establishing a first link of MPK4 function to parasite differentiation. The approaches presented here are broadly applicable to any essential gene in Leishmania thus overcoming major bottlenecks for their functional genetic analysis and their exploitation for structure-informed drug development.

Trypanosomatid parasites of the genus Leishmania cause severe human diseases collectively termed leishmaniasis. Parasite ATP-binding proteins have emerged as potent targets for chemotherapeutic intervention. However, many parasite-specific ATP-binding proteins may escape current efforts in drug target identification, validation and deconvolution due to the lack of sequence conservation and functional annotation of these proteins in early branching, eukaryotic trypanosomatids. Here, we selectively enriched for ATP-binding proteins from Leishmania donovani axenic promastigote and amastigote total protein extracts utilizing a Capture Compound (TM) (CC) linked to the ATP-competitive inhibitor staurosporine. As judged by in-gel kinase activity assay and competitive inhibition with free staurosporine, the CC specifically enriched for parasite phosphotransferases. Comparative nanoLC-MSn analysis identified 70 captured proteins, including 24 conserved protein kinases, and 32 hypothetical proteins with potential ATP-binding function. We identified conserved signature sequence motifs characteristic for staurosporine-binding protein kinases, and identified the hypothetical proteins LinJ.20.0280 and LinJ.09.1630 as novel ATP-binding proteins. Thus, functional enrichment procedures such as described here, combined with bio-informatics analyses and activity assays, provide powerful tools for the discovery of parasite-specific ATP-binding proteins that escape homology-based identification, which can be subsequently targeted for pharmacological intervention.Biological significanceFunctional enrichment using a Capture Compound (TM) linked to the ATP-competitive inhibitor staurosporine provides a powerful new tool for the discovery of parasite-specific ATP-binding proteins that escape homology-based identification, which can be subsequently targeted for pharmacological intervention. (C) 2013 Elsevier B.V. All rights reserved.

Mitogen-activated protein kinases (MAPKs) are involved in environmental signal sensing. They are thus expected to play key roles in the biology of Trypanosomatid parasites, which display complex life cycles and use extracellular cues to modulate cell differentiation. Despite their relevance, structural data of Trypanosomatid MAPKs is lacking. We have now determined the crystal structure of Leishmania major LmaMPK10, a stage-specifically activated MAPK, both alone and in complex with SB203580. LmaMPK10 was observed to be more similar to p38 than to other human MAPKs. However, significant differences could be identified in the catalytic pocket, as well as in potentially regulatory sites in the N-terminal lobe. The modified pocket architecture in LmaMPK10 precludes DFG-in/DFG-out regulatory flipping as observed in mammalian MAPKs. LmaMPK10-nucleotide association was also studied, revealing a potential C-terminal autoinhibitory mechanism. Overall, these data should speed the discovery of molecules interfering with LmaMPK10 functions, with relevance for antileishmanial drug development strategies.

The protozoan parasite Leishmania donovani undergoes various developmental transitions during its infectious cycle that are triggered by environmental signals encountered inside insect and vertebrate hosts. Intracellular differentiation of the pathogenic amastigote stage is induced by pH and temperature shifts that affect protein kinase activities and downstream protein phosphorylation. Identification of parasite proteins with phosphotransferase activity during intracellular infection may reveal new targets for pharmacological intervention. Here we describe an improved protocol to trace this activity in L. donovani extracts at high resolution combining in-gel kinase assay and two-dimensional gel electrophoresis. This 2D procedure allowed us to identify proteins that are associated with amastigote ATP-binding, ATPase, and phosphotransferase activities. The 2D in-gel kinase assay, in combination with recombinant phospho-protein substrates previously identified by phospho-proteomics analyses, provides a novel tool to establish specific protein kinase–substrate relationships thus improving our understanding of Leishmania signal transduction with relevance for future drug development.

BACKGROUND:Cyclosporin A (CsA) has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs) as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development.METHODOLOGY/PRINCIPAL FINDINGS:Multiple sequence alignment and cluster analysis identified 17 Leishmania CyPs with significant sequence differences to human CyPs, but with highly conserved functional residues implicated in PPIase function and CsA binding. CsA treatment of promastigotes resulted in a dose-dependent inhibition of cell growth with an IC50 between 15 and 20 microM as demonstrated by proliferation assay and cell cycle analysis. Scanning electron microscopy revealed striking morphological changes in CsA treated promastigotes reminiscent to developing amastigotes, suggesting a role for parasite CyPs in Leishmania differentiation. In contrast to promastigotes, CsA was highly toxic to amastigotes with an IC50 between 5 and 10 microM, revealing for the first time a direct lethal effect of CsA on the pathogenic mammalian stage linked to parasite thermotolerance, independent from host CyPs. Structural modeling, enrichment of CsA-binding proteins from parasite extracts by FPLC, and PPIase activity assays revealed direct interaction of the inhibitor with LmaCyP40, a bifunctional cyclophilin with potential co-chaperone function.CONCLUSIONS/SIGNIFICANCE:The evolutionary expansion of the Leishmania CyP protein family and the toxicity of CsA on host-free amastigotes suggest important roles of PPIases in parasite biology and implicate Leishmania CyPs in key processes relevant for parasite proliferation and viability. The requirement of Leishmania CyP functions for intracellular parasite survival and their substantial divergence form host CyPs defines these proteins as prime drug targets.

Somatic hypermutation (SHM) of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C) to deoxyuridine (U), catalysed by the activation induced deaminase (AID). Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue), followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR) pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.