Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

Development of two novel high-throughput assays to quantify ubiquitylated proteins in cell lysates: application to screening of new anti-malarials.

BACKGROUND: The ubiquitin proteasome system (UPS) is one of the main proteolytical pathways in eukaryotic cells and plays an essential role in key cellular processes such as cell cycle, stress response, signal transduction, and transcriptional regulation. Many components of this pathway have been implicated in diverse pathologies including cancer, neurodegeneration and infectious diseases, such as malaria. The success of proteasome inhibitors in clinical trials underlines the potential of the UPS in drug discovery. METHODS: Plasmodium falciparum, the malaria causative pathogen, has been used to develop two assays that allow the quantification of the parasite protein ubiquitylation levels in a high-throughput format that can be used to find new UPS inhibitors. RESULTS: In both assays tandem ubiquitin binding entities (TUBEs), also known as ubiquitin traps, have been used to capture ubiquitylated proteins from cell lysates. The primary assay is based on AlphaLISA technology, and the orthogonal secondary assay relies on a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) system. A panel of well-known proteasome inhibitors has been used to validate both technologies. An excellent correlation was obtained between these biochemical assays and the standard whole cell assay that measures parasite growth inhibition. CONCLUSIONS: The two assays presented can be used in a high-throughput format to find new UPS inhibitors for P. falciparum and could help to identify new targets within this system. This methodology is also applicable to other cellular contexts or pathologies.

Magnetic isolation of Plasmodium falciparum schizonts iRBCs to generate a high parasitaemia and synchronized in vitro culture.

BACKGROUND: The establishment of methods for an in vitro continuous culture of Plasmodium falciparum is essential for gaining knowledge into its biology and for the development of new treatments. Previously, several techniques have been used to synchronize, enrich and concentrate P. falciparum, although obtaining cultures with high parasitaemia continues being a challenging process. Current methods produce high parasitaemia levels of synchronized P. falciparum cultures by frequent changes of culture medium or reducing the haematocrit. However, these methods are time consuming and sometimes lead to the loss of synchrony. METHODS: A procedure that combines Percoll and sorbitol treatments, the use of magnetic columns, and the optimization of the in vitro culture conditions to reach high parasitaemia levels for synchronized Plasmodium falciparum cultures is described. RESULTS: A new procedure has been established using P. falciparum 3D7, combining previous reported methodologies to achieve in vitro parasite cultures that reach parasitaemia up to 40% at any intra-erythrocytic stage. High parasitaemia levels are obtained only one day after magnetic column purification without compromising the parasite viability and synchrony. CONCLUSIONS: The described procedure allows obtaining a large scale synchronized parasite culture at a high parasitaemia with less manipulations than other methods previously described.

High Throughput Screening to Identify Selective and Nonpeptidomimetic Proteasome Inhibitors As Antimalarials.

The Ubiquitin Proteasome System is the main proteolytic pathway in eukaryotic cells, playing a role in key cellular processes. The essentiality of the Plasmodium falciparum proteasome is well validated, underlying its potential as an antimalarial target, but selective compounds are required to avoid cytotoxic effects in humans. Almost 550000 compounds were tested for the inhibition of the chymotrypsin-like activity of the P. falciparum proteasome using a Proteasome-GLO luminescence assay. Hits were confirmed in an orthogonal enzyme assay using Rho110-labeled peptides, and selectivity was assessed against the human proteasome. Four nonpeptidomimetic chemical families with some selectivity for the P. falciparum proteasome were identified and characterized in assays of proteasome trypsin and caspase activities and in parasite growth inhibition assays. Target engagement studies were performed, validating our approach. Hits identified are good starting points for the development of new antimalarial drugs and as tools to better understand proteasome function in P. falciparum.

Identification of Small Molecules Disrupting the Ubiquitin Proteasome System in Malaria.

The ubiquitin proteasome system (UPS) is one of the main proteolytic pathways in eukaryotic cells, playing an essential role in key cellular processes such as cell cycling and signal transduction. Changes in some of the components of this pathway have been implicated in various conditions, including cancer and infectious diseases such as malaria. The success of therapies based on proteasome inhibitors has been shown in human clinical trials. In addition to its proven tractability, the essentiality of the Plasmodium falciparum UPS underlines its potential as a source of targets to identify new antimalarial treatments. Two assays, previously developed to quantify the parasite protein ubiquitylation levels in a high throughput format, have been used to identify compounds that inhibit parasite growth by targeting P. falciparum UPS. Among the positive hits, specific inhibitors of the P. falciparum proteasome have been identified and characterized. Hits identified using this approach may be used as starting points for development of new antimalarial drugs. They may also be used as tools to further understand proteasome function and to identify new targets in P. falciparum UPS.

High-throughput screening of the Plasmodium falciparum cGMP-dependent protein kinase identified a thiazole scaffold which kills erythrocytic and sexual stage parasites.

Antimalarial drug resistance compels the quest for new compounds that target alternative pathways to current drugs. The Plasmodium cyclic GMP-dependent protein kinase (PKG) has essential functions in all of the major life cycle developmental stages. An imidazopyridine PKG inhibitor scaffold was previously shown to clear P. falciparum infection in a rodent model in vivo and blocked transmission to mosquitoes providing proof of concept for this target. To find new classes of PKG inhibitors to serve as alternative chemical starting points, we performed a high-throughput screen of the GSK Full Diversity Collection using recombinant P. falciparum PKG. We developed a robust enzymatic assay in a 1536-well plate format. Promising compounds were then tested for activity against P. falciparum asexual blood stage growth, selectivity and cytotoxicity. By using a scoring system we selected the 66 most promising PKG inhibitors (comprising nine clusters and seven singletons). Among these, thiazoles were the most potent scaffold with mid-nanomolar activity on P. falciparum blood stage and gamete development. Using Kinobeads profiling we identified additional P. falciparum protein kinases targeted by the thiazoles that mediate a faster speed of the kill than PKG-selective compounds. This scaffold represents a promising starting point to develop a new antimalarial.

Inhibiting the stringent response blocks Mycobacterium tuberculosis entry into quiescence and reduces persistence.

The stringent response enables Mycobacterium tuberculosis (Mtb) to shut down its replication and metabolism under various stresses. Here we show that Mtb lacking the stringent response enzyme RelMtb was unable to slow its replication rate during nutrient starvation. Metabolomics analysis revealed that the nutrient-starved relMtb -deficient strain had increased metabolism similar to that of exponentially growing wild-type bacteria in nutrient-rich broth, consistent with an inability to enter quiescence. Deficiency of relMtb increased the susceptibility of mutant bacteria to killing by isoniazid during nutrient starvation and in the lungs of chronically infected mice. We screened a pharmaceutical library of over 2 million compounds for inhibitors of RelMtb and showed that the lead compound X9 was able to directly kill nutrient-starved M. tuberculosis and enhanced the killing activity of isoniazid. Inhibition of RelMtb is a promising approach to target M. tuberculosis persisters, with the potential to shorten the duration of TB treatment.

Isolation of the Ubiquitin-Proteome from Tumor Cell Lines and Primary Cells Using TUBEs.

Tandem ubiquitin-binding entities (TUBEs) act as molecular traps to isolate polyubiquitylated proteins facilitating the study of this highly reversible posttranslational modification. We provide here sample preparation and adaptations required for TUBE-based enrichment of the ubiquitin proteome from tumor cell lines or primary cells. Our protocol is suitable to identify ubiquitin substrates, enzymes involved in the ubiquitin proteasome pathway, as well as proteasome subunits by mass spectrometry. This protocol was adapted to prepare affinity columns, reduce background, and improve the protein recovery depending on the sample source and necessities.

New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach.

Malaria, caused by Plasmodium falciparum (P. falciparum), ranks as one of the most baleful infectious diseases worldwide. New antimalarial treatments are needed to face existing or emerging drug resistant strains. Protein degradation appears to play a significant role during the asexual intraerythrocytic developmental cycle (IDC) of P. falciparum. Inhibition of the ubiquitin proteasome system (UPS), a major intracellular proteolytic pathway, effectively reduces infection and parasite replication. P. falciparum and erythrocyte UPS coexist during IDC but the nature of their relationship is largely unknown. We used an approach based on Tandem Ubiquitin-Binding Entities (TUBEs) and 1D gel electrophoresis followed by mass spectrometry to identify major components of the TUBEs-associated ubiquitin proteome of both host and parasite during ring, trophozoite and schizont stages. Ring-exported protein (REX1), a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, was found to reach a maximum level of ubiquitylation in trophozoites stage. The Homo sapiens (H. sapiens) TUBEs associated ubiquitin proteome decreased during the infection, whereas the equivalent P. falciparum TUBEs-associated ubiquitin proteome counterpart increased. Major cellular processes such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. BIOLOGICAL SIGNIFICANCE: In this work we analyze for the first time the interconnection between Plasmodium and human red blood cells ubiquitin-regulated proteins in the context of infection. We identified a number of human and Plasmodium proteins whose ubiquitylation pattern changes during the asexual infective stage. We demonstrate that ubiquitylation of REX1, a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, peaks in trophozoites stage. The ubiquitin-proteome from P. falciparum infected red blood cells (iRBCs) revealed a significant host-parasite crosstalk, underlining the importance of ubiquitin-regulated proteolytic activities during the intraerythrocytic developmental cycle (IDC) of P. falciparum. Major cellular processes defined from gene ontology such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. Given the importance of ubiquitylation in the development of infectious diseases, this work provides a number of potential drug-target candidates that should be further explored.