Scaffold-Hopping Strategy on a Series of Proteasome Inhibitors Led to a Preclinical Candidate for the Treatment of Visceral Leishmaniasis.

There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 (1) as a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a phenotypic screen was optimized to give a compound with in vivo efficacy, which was hampered by poor solubility and genotoxicity. The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving medicinal chemistry design, in silico profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure and the impact this has on our understanding of the series' structure-activity relationships.

Identification and Optimization of a Series of 8-Hydroxy Naphthyridines with Potent In Vitro Antileishmanial Activity: Initial SAR and Assessment of In Vivo Activity.

Visceral leishmaniasis (VL) is a parasitic infection that results in approximately 26 000-65 000 deaths annually. The available treatments are hampered by issues such as toxicity, variable efficacy, and unsuitable dosing options. The need for new treatments is urgent and led to a collaboration between the Drugs for Neglected Diseases initiative (DNDi), GlaxoSmithKline (GSK), and the University of Dundee. An 8-hydroxynaphthyridine was identified as a start point, and an early compound demonstrated weak efficacy in a mouse model of VL but was hampered by glucuronidation. Efforts to address this led to the development of compounds with improved in vitro profiles, but these were poorly tolerated in vivo. Investigation of the mode of action (MoA) demonstrated that activity was driven by sequestration of divalent metal cations, a mechanism which was likely to drive the poor tolerability. This highlights the importance of investigating MoA and pharmacokinetics at an early stage for phenotypically active series.

The Qi Site of Cytochrome b is a Promiscuous Drug Target in Trypanosoma cruzi and Leishmania donovani.

Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Qi active-site of cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Our studies go on to identify the Qi site as a promiscuous drug target in Leishmania donovani and Trypanosoma cruzi with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.

Identification of 6-amino-1H-pyrazolo[3,4-d]pyrimidines with in vivo efficacy against visceral leishmaniasis.

Visceral leishmaniasis (VL) affects millions of people across the world, largely in developing nations. It is fatal if left untreated and the current treatments are inadequate. As such, there is an urgent need for new, improved medicines. In this paper, we describe the identification of a 6-amino-N-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidine scaffold and its optimization to give compounds which showed efficacy when orally dosed in a mouse model of VL.

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.

Pharmacological Validation of N-Myristoyltransferase as a Drug Target in Leishmania donovani.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and L. infantum, is responsible for ∼30 000 deaths annually. Available treatments are inadequate, and there is a pressing need for new therapeutics. N-Myristoyltransferase (NMT) remains one of the few genetically validated drug targets in these parasites. Here, we sought to pharmacologically validate this enzyme in Leishmania. A focused set of 1600 pyrazolyl sulfonamide compounds was screened against L. major NMT in a robust high-throughput biochemical assay. Several potent inhibitors were identified with marginal selectivity over the human enzyme. There was little correlation between the enzyme potency of these inhibitors and their cellular activity against L. donovani axenic amastigotes, and this discrepancy could be due to poor cellular uptake due to the basicity of these compounds. Thus, a series of analogues were synthesized with less basic centers. Although most of these compounds continued to suffer from relatively poor antileishmanial activity, our most potent inhibitor of LmNMT (DDD100097, K i of 0.34 nM) showed modest activity against L. donovani intracellular amastigotes (EC50 of 2.4 µM) and maintained a modest therapeutic window over the human enzyme. Two unbiased approaches, namely, screening against our cosmid-based overexpression library and thermal proteome profiling (TPP), confirm that DDD100097 (compound 2) acts on-target within parasites. Oral dosing with compound 2 resulted in a 52% reduction in parasite burden in our mouse model of VL. Thus, NMT is now a pharmacologically validated target in Leishmania. The challenge in finding drug candidates remains to identify alternative strategies to address the drop-off in activity between enzyme inhibition and in vitro activity while maintaining sufficient selectivity over the human enzyme, both issues that continue to plague studies in this area.

Identification of GSK3186899/DDD853651 as a Preclinical Development Candidate for the Treatment of Visceral Leishmaniasis.

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.

Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis.

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.

Chemical Validation of Methionyl-tRNA Synthetase as a Druggable Target in Leishmania donovani.

Methionyl-tRNA synthetase (MetRS) has been chemically validated as a drug target in the kinetoplastid parasite Trypanosoma brucei. In the present study, we investigate the validity of this target in the related trypanosomatid Leishmania donovani. Following development of a robust high-throughput compatible biochemical assay, a compound screen identified DDD806905 as a highly potent inhibitor of LdMetRS (Ki of 18 nM). Crystallography revealed this compound binds to the methionine pocket of MetRS with enzymatic studies confirming DDD806905 displays competitive inhibition with respect to methionine and mixed inhibition with respect to ATP binding. DDD806905 showed activity, albeit with different levels of potency, in various Leishmania cell-based viability assays, with on-target activity observed in both Leishmania promastigote cell assays and a Leishmania tarentolae in vitro translation assay. Unfortunately, this compound failed to show efficacy in an animal model of leishmaniasis. We investigated the potential causes for the discrepancies in activity observed in different Leishmania cell assays and the lack of efficacy in the animal model and found that high protein binding as well as sequestration of this dibasic compound into acidic compartments may play a role. Despite medicinal chemistry efforts to address the dibasic nature of DDD806905 and analogues, no progress could be achieved with the current chemical series. Although DDD806905 is not a developable antileishmanial compound, MetRS remains an attractive antileishmanial drug target.

Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity.

Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.

A newly classified vertebrate calpain protease, directly ancestral to CAPN1 and 2, episodically evolved a restricted physiological function in placental mammals.

The most studied members of the calpain protease superfamily are CAPN1 and 2, which are conserved across vertebrates. Another similar family member called mu/m-CAPN has been identified in birds alone. Here, we establish that mu/m-CAPN shares one-to-one orthology with CAPN11, previously described only in eutherians (placental mammals). We use the name CAPN11 for this family member and identify orthologues across vertebrate lineages, which form a monophyletic phylogenetic clade directly ancestral to CAPN1 and 2. In lineages branching before therians (live-bearing mammals), the CAPN11 coding region has evolved under strong purifying selection, with low nonsynonymous (d(N)) versus synonymous (d(S)) substitution rates (d(N)/d(S) = 0.076 across pretherians), and its transcripts were detected widely across different tissues. These characteristics are present in CAPN1 and 2 across vertebrate lineages and indicate that pretherian CAPN11 likewise has conserved a wide physiological function. However, an approximately 7-fold elevation in d(N)/d(S) is evident along the CAPN11 branch splitting eutherians from platypus, paralleled by a shift to "testis-specific" gene regulation. Estimates of d(N)/d(S) in eutherians were approximately 3-fold elevated compared with pretherians and coding and transcriptional-level evidence suggests that CAPN11 is functionally absent in marsupials. Many CAPN11 sites are functionally constrained in eutherians to conserve a residue with radically different biochemical properties to a fixed state shared between pretherian CAPN11 and CAPN1 and 2. Protein homology modeling demonstrated that many such eutherian-specific residue replacements modify or ablate interactions with the calpain inhibitor calpastatin that are observed in both pretherian orthologues and CAPN1/2. We propose a model akin to the Dykhuizen-Hartl effect, where inefficient purifying selection and increased genetic drift associated with a reduction in effective population size, drove the fixation of mutations in regulatory and coding regions of CAPN11 of a common marsupial-eutherian ancestor. A subset of these changes had a cumulative adaptive advantage in a eutherian ancestor because of lineage-specific aspects of sperm physiology, whereas in marsupials, no advantage was realized and the gene was disabled. This work supports that functional divergence among gene family member orthologues is possible in the absence of widespread positive selection.

Regulation of plant gene expression by alternative splicing.

AS (alternative splicing) is a post-transcriptional process which regulates gene expression through increasing protein complexity and modulating mRNA transcript levels. Regulation of AS depends on interactions between trans-acting protein factors and cis-acting signals in the pre-mRNA (precursor mRNA) transcripts, termed 'combinatorial' control. Dynamic changes in AS patterns reflect changes in abundance, composition and activity of splicing factors in different cell types and in response to cellular or environmental cues. Whereas the SR protein family of splicing factors is well-studied in plants, relatively little is known about other factors influencing the regulation of AS or the consequences of AS on mRNA levels and protein function. To address fundamental questions on AS in plants, we are exploiting a high-resolution RT (reverse transcription)-PCR system to analyse multiple AS events simultaneously. In the present paper, we describe the current applications and development of the AS RT-PCR panel in investigating the roles of splicing factors, cap-binding proteins and nonsense-mediated decay proteins on AS, and examining the extent of AS in genes involved in the same developmental pathway or process.

Characterisation of capn1, capn2-like, capn3 and capn11 genes in Atlantic halibut (Hippoglossus hippoglossus L.): Transcriptional regulation across tissues and in skeletal muscle at distinct nutritional states.

The typical calpain proteases are a subset of a wider superfamily and regulate a broad spectrum of physiological processes. Here we characterised Atlantic halibut complete-coding orthologues of calpain-1, calpain-2-like, "muscle-specific" calpain-3, plus calpain-11, a recently recognised vertebrate-wide family member. Phylogenetic analysis established the relationship of each sequence within a comprehensive framework of vertebrate calpains, including teleost paralogues. This approach provided significant insight into the evolution of teleost calpains. For example, teleost sequences considered calpain-2 orthologues formed a monophyletic clade external to sister clades for tetrapod calpain-2 and vertebrate calpain-8. Thus, teleost "calpain-2" is likely not directly orthologous to tetrapod calpain-2 and represents a calpain-2-like protein. The characteristic domain structure of typical calpains was observed in each halibut sequence, although calpain-3, as for other teleosts, retained only one (IS2) of three further domains found in human calpain-3 (NS, IS1 and IS2). Transcripts for capn1, capn2-like and capn11 were widely detected across eleven halibut tissues, whereas capn3 was detected in striated muscles, spleen and ovary, but absent or relatively less abundant in other tissues. We assessed the transcript expression of each calpain gene in fast-twitch skeletal muscle where nutritional state was altered with 60days feed restriction, followed by 60days satiation refeeding. Measured by quantitative real-time PCR, capn1 transcript levels were highest during maximal fasting and then steadily decreased with refeeding, where muscle was in net positive protein balance. Conversely capn2-like showed little response, whereas capn3 and capn11 transcript levels were lowest at maximal fasting before being strongly constitutively upregulated with subsequent refeeding. Halibut capn3 transcript abundance was on average 6.5, 23.7 and 5.9 fold greater than capn1, capn2-like and capn11 respectively in skeletal muscle across nutritional states. In turn, transcript levels of capn1 and capn11 were invariably higher than capn2-like, but were dependent on nutritional state compared to each other. The differential regulation of these genes in response to nutritional status suggests distinct roles for typical calpain family members in regulating the balance between catabolism and growth in teleost skeletal muscle.

Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei.

We recently suggested a novel site-specific N-glycosylation mechanism in Trypanosoma brucei whereby some protein N-glycosylation sites selectively receive Man9GlcNAc2 from Man9GlcNAc2-PP-Dol while others receive Man5GlcNA(2 from Man5GlcNAc2-PP-Dol. In this paper, we test this model by creating procyclic and bloodstream form null mutants of TbALG3, the gene that encodes the alpha-mannosyltransferase that converts Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol. The procyclic and bloodstream form TbALG3 null mutants grow with normal kinetics, remain infectious to mice and tsetse flies, respectively, and have normal morphology. However, both forms display aberrant N-glycosylation of their major surface glycoproteins, procylcin, and variant surface glycoprotein, respectively. Specifically, procyclin and variant surface glycoprotein N-glycosylation sites that are modified with Man9GlcNAc2 and processed no further than Man5GlcNAc2 in the wild type are glycosylated less efficiently but processed to complex structures in the mutant. These data confirm our model and refine it by demonstrating that the biantennary glycan transferred from Man5GlcNAc2-PP-Dol is the only route to complex N-glycans in T. brucei and that Man9GlcNAc2-PP-Dol is strictly a precursor for oligomannose structures. The origins of site-specific Man5GlcNAc2 or Man9GlcNAc2 transfer are discussed and an updated model of N-glycosylation in T. brucei is presented.

Plasticity of muscle fibre number in seawater stages of Atlantic salmon in response to photoperiod manipulation.

Atlantic salmon (Salmo salar L.) were fed to satiety and reared from approximately 60 g to 5000 g at ambient seawater temperatures. The effect of photoperiod manipulation on muscle growth was investigated from the start of the first sea winter. Continuous light treatment in winter/spring (1 November to 18 June) improved growth performance in fish, resulting in a 30% increase in mean body mass relative to the ambient photoperiod fish by 12 August, but had no effect on sexual maturation. Significant increases in body mass in the continuous light groups were observed after 126 days (P<0.01). The number of fast muscle fibres per trunk cross-section was determined in a subset of the fish and was 28.5% higher in the continuous light (799 x 10(3)) than the natural day length (644 x 10(3)) groups after only 40 days, corresponding to the period of decreasing natural day length. Subsequent rates of fibre recruitment were similar between treatments. At the end of the fibre recruitment phase of growth (combined June and August samples), the maximum number of fast muscle fibres was 23% higher in fish from the cages receiving continuous light (881 x 10(3)+/-32 x 10(3); N=19) than in the ambient photoperiod cages (717 x 10(3)+/-15 x 10(3); N=20) (P<0.001). Continuous light treatment was associated with a shift in the distribution of fibre diameters, reflecting the altered patterns of fibre recruitment. However, the mean rate of fibre hypertrophy showed no consistent difference between treatments. There was a linear relationship between the myonuclear content of isolated single fibres and fibre diameter. On average, there were 27% more myonuclei in 150 microm-diameter fibres in the continuous light (3118 myonuclei cm(-1)) than the ambient photoperiod (2448 myonuclei cm(-1)) fish. After 40 days, continuous light treatment resulted in a transient increase in the density of myogenic progenitor cells, identified using a c-met antibody, to a level 70% above that of fish exposed to natural light. It is suggested that short days inhibited the proliferation of myogenic progenitor cells and that this was overcome by transferring fish to continuous light, causing an increase in the number of times the myogenic precursor cells divided and/or a decrease in cell cycle time. The net increase in myogenic progenitor cells resulted in proportional increases in the number and myonuclear content of fibres. The subsequent hypertrophy of these additional fibres can explain the delayed increase in body mass observed with continuous light treatment.

Freshwater environment affects growth rate and muscle fibre recruitment in seawater stages of Atlantic salmon (Salmo salar L.).

The influence of freshwater environment on muscle growth in seawater was investigated in an inbred population of farmed Atlantic salmon (Salmo salar L.). The offspring from a minimum of 64 families per group were incubated at either ambient temperature (ambient treatment) or in heated water (heated treatment). Growth was investigated using a mixed-effect statistical model with repeated measures, which included terms for treatment effect and random fish effects for individual growth rate (alpha) and the instantaneous growth rate per unit change in temperature (gamma). Prior to seawater transfer, fish were heavier in the heated (61.6+/-1.0 g; N=298) than in the ambient (34.1+/-0.4 g; N=206) treatments, reflecting their greater growth opportunity: 4872 degree-days and 4281 degree-days, respectively. However, the subsequent growth rate of the heated group was lower, such that treatments had a similar body mass (3.7-3.9 kg) after approximately 450 days in seawater. The total cross-sectional area of fast muscle and the number (FN) and size distribution of the fibres was determined in a subset of the fish. We tested the hypothesis that freshwater temperature regime affected the rate of recruitment and hypertrophy of muscle fibres. There were differences in FN between treatments and a significant age x treatment interaction but no significant cage effect (ANOVA). Cessation of fibre recruitment was identified by the absence of fibres of <10 micro m diameter. The maximum fibre number was 22.4% more in the ambient (9.3 x 10(5)+/-2.0 x 10(4) than in the heated (7.6 x 10(5)+/-1.5 x 10(4)) treatments (N=44 and 40 fish, respectively; P<0.001). For fish that had completed fibre recruitment, there was a significant correlation between FN and individual growth rate, explaining 35% of the total variation. The density of myogenic progenitor cells was quantified using an antibody to c-met and was approximately 2-fold higher in the ambient than in the heated group, equivalent to 2-3% of the total muscle nuclei. The number of myonuclei in isolated fibre segments showed a linear relationship with fibre diameter. On average, there were 20.6% more myonuclei in 200-microm-diameter fibres isolated from the ambient (3734 myonuclei cm(-1)) than from the heated (3097 myonuclei cm(-1)) treatments. The maximum fibre diameter was greater in heated than in ambient groups, whereas the age x treatment interaction was not significantly different (ANCOVA). There were also no consistent differences in the rate of hypertrophy of muscle fibres between treatments. It was concluded that freshwater temperature regime affected fibre number and the nuclear content of fast muscle in seawater but not the rate of fibre hypertrophy. The mechanisms and life history consequences of developmental plasticity in fibre number are discussed.