Acceleration of infectious disease drug discovery and development using a humanized model of drug metabolism.

A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here, we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily ("8HUM") can circumvent these problems. The pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani, and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery.

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.

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

Compounds enhancing human sperm motility identified using a high-throughput phenotypic screening platform.

STUDY QUESTION: Can a high-throughput screening (HTS) platform facilitate male fertility drug discovery? SUMMARY ANSWER: An HTS platform identified a large number of compounds that enhanced sperm motility. WHAT IS KNOWN ALREADY: Several efforts to find small molecules modulating sperm function have been performed but none have used high-throughput technology. STUDY DESIGN, SIZE, DURATION: Healthy donor semen samples were used and samples were pooled (3-5 donors per pool). Primary screening was performed singly; dose-response screening was performed in duplicate (using independent donor pools). PARTICIPANTS/MATERIALS, SETTING, METHODS: Spermatozoa isolated from healthy donors were prepared by density gradient centrifugation and incubated in 384-well plates with compounds (6.25 µM) to identify those compounds with enhancing effects on motility. Approximately 17 000 compounds from the libraries, ReFRAME, Prestwick, Tocris, LOPAC, CLOUD and MMV Pathogen Box, were screened. Dose-response experiments of screening hits were performed to confirm the enhancing effect on sperm motility. Experiments were performed in a university setting. MAIN RESULTS AND THE ROLE OF CHANCE: From our primary single concentration screening, 105 compounds elicited an enhancing effect on sperm motility compared to dimethylsulphoxide-treated wells. Confirmed enhancing compounds were grouped based on their annotated targets/target classes. A major target class, phosphodiesterase inhibitors, were identified, in particular PDE10A inhibitors as well as number of compounds not previously known to enhance human sperm motility, such as those related to GABA signalling. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although this approach provides data about the activity of the compound, it is only a starting point. For example, further substantive experiments are necessary to provide a more comprehensive picture of each compound's activity, the effect on the kinetics of the cell populations and subpopulations, and their potential mechanisms of action. Compounds have been tested with prepared donor spermatozoa, incubated under non-capacitating conditions, and only incubated with compounds for a relatively short period of time. Therefore, the effect of compounds under different conditions, for example in whole semen, for longer incubation times, or using samples from patient groups, may be different and require further study. All experiments were performed in vitro. WIDER IMPLICATIONS OF THE FINDINGS: This phenotypic screening assay identified a large number of compounds that increased sperm motility. In addition to furthering our understanding of human sperm function, for example identifying new avenues for discovery, we highlight potential compounds as promising start-point for a medicinal chemistry programme for potential enhancement of male fertility. Moreover, with disclosure of the results of screening, we present a substantial resource to inform further work in the field. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Bill and Melinda Gates Foundation, Scottish Funding Council and Scottish Universities Life Science Alliance. C.L.R.B. is Editor for RBMO. C.L.R.B. receives funding from Chief Scientists Office (Scotland), ESHRE and Genus PLC, consulting fees from Exscientia and lecture fees from Cooper Surgical and Ferring. S.M.d.S. is an Associate Editor of Human Reproduction, and an Associate Editor of Reproduction and Fertility. S.M.d.S. receives funding from Cooper Surgical and British Dietetic Society. No other authors declared a COI.

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.

Preparation, biological & cheminformatics-based assessment of N2,N4-diphenylpyrimidine-2,4-diamine as potential Kinase-targeted antimalarials.

Twenty eight new N2,N4-diphenylpyrimidine-2,4-diamines have been prepared in order to expand our understanding of the anti-malarial SAR of the scaffold. The aim of the study was to make structural modifications to improve the overall potency, selectivity and solubility of the series by varying the anilino groups attached to the 2- and 4-position. We evaluated the activity of the compounds against Plasmodium falciparum (Pf) 3D7, cytotoxicity against HepG2, % inhibition at a panel of 10 human kinases, solubility, permeability and lipophilicity, and human and rat in vitro clearance. 11 was identified as a potent anti-malarial with an IC50 of 0.66 µM at the 3D7 strain and a selectivity (SI) of ~ 40 in terms of cytotoxicity against the HepG2 cell line. It also displayed low experimental logD7.4 (2.27), reasonable solubility (124 µg/ml), good metabolic stability, but low permeability. A proteo-chemometric workflow was employed to identify putative Pf targets of the most promising compounds. Ligand-based similarity searching of the ChEMBL database led to the identification of most probable human targets. These were then used as input for sequence-based searching of the Pf proteome. Homology modelling and molecular docking were used to evaluate whether compounds could indeed bind to these targets with valid binding modes. In vitro biological testing against close human analogs of these targets was subsequently undertaken. This allowed us to identify potential Pf targets and human anti-targets that could be exploited in future development.

A Systematic Study of the In Vitro Pharmacokinetics and Estimated Human In Vivo Clearance of Indole and Indazole-3-Carboxamide Synthetic Cannabinoid Receptor Agonists Detected on the Illicit Drug Market.

In vitro pharmacokinetic studies were conducted on enantiomer pairs of twelve valinate or tert-leucinate indole and indazole-3-carboxamide synthetic cannabinoid receptor agonists (SCRAs) detected on the illicit drug market to investigate their physicochemical parameters and structure-metabolism relationships (SMRs). Experimentally derived Log D7.4 ranged from 2.81 (AB-FUBINACA) to 4.95 (MDMB-4en-PINACA) and all SCRAs tested were highly protein bound, ranging from 88.9 ± 0.49% ((R)-4F-MDMB-BINACA) to 99.5 ± 0.08% ((S)-MDMB-FUBINACA). Most tested SCRAs were cleared rapidly in vitro in pooled human liver microsomes (pHLM) and pooled cryopreserved human hepatocytes (pHHeps). Intrinsic clearance (CLint) ranged from 13.7 ± 4.06 ((R)-AB-FUBINACA) to 2944 ± 95.9 mL min-1 kg-1 ((S)-AMB-FUBINACA) in pHLM, and from 110 ± 34.5 ((S)-AB-FUBINACA) to 3216 ± 607 mL min-1 kg-1 ((S)-AMB-FUBINACA) in pHHeps. Predicted Human in vivo hepatic clearance (CLH) ranged from 0.34 ± 0.09 ((S)-AB-FUBINACA) to 17.79 ± 0.20 mL min-1 kg-1 ((S)-5F-AMB-PINACA) in pHLM and 1.39 ± 0.27 ((S)-MDMB-FUBINACA) to 18.25 ± 0.12 mL min-1 kg-1 ((S)-5F-AMB-PINACA) in pHHeps. Valinate and tert-leucinate indole and indazole-3-carboxamide SCRAs are often rapidly metabolised in vitro but are highly protein bound in vivo and therefore predicted in vivo CLH is much slower than CLint. This is likely to give rise to longer detection windows of these substances and their metabolites in urine, possibly as a result of accumulation of parent drug in lipid-rich tissues, with redistribution into the circulatory system and subsequent metabolism.

Host-parasite co-metabolic activation of antitrypanosomal aminomethyl-benzoxaboroles.

Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in Trypanosoma brucei. We describe an oxidative enzymatic pathway composed of host semicarbazide-sensitive amine oxidase and a trypanosomal aldehyde dehydrogenase TbALDH3. Two sequential reactions through this pathway serve as the key underlying mechanism for activating a series of 4-aminomethylphenoxy-benzoxaboroles as potent trypanocides; the methylamine parental compounds as pro-drugs are transformed first into intermediate aldehyde metabolites, and further into the carboxylate metabolites as effective forms. Moreover, comparative biochemical and crystallographic analyses elucidated the catalytic specificity of TbALDH3 towards the benzaldehyde benzoxaborole metabolites as xenogeneic substrates. Overall, this work proposes a novel drug activation mechanism dependent on both host and parasite metabolism of primary amine containing molecules, which contributes a new perspective to our understanding of the benzoxaborole MoA, and could be further exploited to improve the therapeutic index of antimicrobial compounds.

The Anthelmintic Drug Niclosamide and Its Analogues Activate the Parkinson's Disease Associated Protein Kinase PINK1.

Mutations in PINK1, which impair its catalytic kinase activity, are causal for autosomal recessive early-onset Parkinson's disease (PD). Various studies have indicated that the activation of PINK1 could be a useful strategy in treating neurodegenerative diseases, such as PD. Herein, it is shown that the anthelmintic drug niclosamide and its analogues are capable of activating PINK1 in cells through the reversible impairment of the mitochondrial membrane potential. With these compounds, for the first time, it is demonstrated that the PINK1 pathway is active and detectable in primary neurons. These findings suggest that niclosamide and its analogues are robust compounds for the study of the PINK1 pathway and may hold promise as a therapeutic strategy in PD and related disorders.

Activation of Bicyclic Nitro-drugs by a Novel Nitroreductase (NTR2) in Leishmania.

Drug discovery pipelines for the "neglected diseases" are now heavily populated with nitroheterocyclic compounds. Recently, the bicyclic nitro-compounds (R)-PA-824, DNDI-VL-2098 and delamanid have been identified as potential candidates for the treatment of visceral leishmaniasis. Using a combination of quantitative proteomics and whole genome sequencing of susceptible and drug-resistant parasites we identified a putative NAD(P)H oxidase as the activating nitroreductase (NTR2). Whole genome sequencing revealed that deletion of a single cytosine in the gene for NTR2 that is likely to result in the expression of a non-functional truncated protein. Susceptibility of leishmania was restored by reintroduction of the wild-type gene into the resistant line, which was accompanied by the ability to metabolise these compounds. Overexpression of NTR2 in wild-type parasites rendered cells hyper-sensitive to bicyclic nitro-compounds, but only marginally to the monocyclic nitro-drugs, nifurtimox and fexinidazole sulfone, known to be activated by a mitochondrial oxygen-insensitive nitroreductase (NTR1). Conversely, a double knockout NTR2 null cell line was completely resistant to bicyclic nitro-compounds and only marginally resistant to nifurtimox. Sensitivity was fully restored on expression of NTR2 in the null background. Thus, NTR2 is necessary and sufficient for activation of these bicyclic nitro-drugs. Recombinant NTR2 was capable of reducing bicyclic nitro-compounds in the same rank order as drug sensitivity in vitro. These findings may aid the future development of better, novel anti-leishmanial drugs. Moreover, the discovery of anti-leishmanial nitro-drugs with independent modes of activation and independent mechanisms of resistance alleviates many of the concerns over the continued development of these compound series.

Discovery of super soft-drug modulators of sphingosine-1-phosphate receptor 1.

The oral S1PR1 agonist ponesimod demonstrated substantial efficacy in a phase II clinical trial of psoriasis. Unfortunately, systemic side effects were observed, which included lymphopenia and transient bradycardia. We sought to develop a topical soft-drug S1PR1 agonist with an improved therapeutic index. By modifying ponesimod, we discovered an ester series of S1PR agonists. To increase metabolic instability in plasma we synthesised esters described as specific substrates for paraoxonase and butyrylcholinesterases, esterases present in human plasma.

2,4-Diamino-6-methylpyrimidines for the potential treatment of Chagas' disease.

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, affects 8-10 million people across the Latin American population and is responsible for around 12,500 deaths per annum. The current frontline treatments, benznidazole and nifurtimox, are associated with side effects and lack efficacy in the chronic stage of the disease, leading to an urgent need for new treatments. A high throughput screening campaign against the physiologically relevant intracellular form of the parasite identified a series of 2,4-diamino-6-methylpyrimidines. Demonstrating the series did not work through the anti-target TcCYP51, and was generally cytocidal, confirmed its suitability for further development. This study reports the optimisation of selectivity and metabolic stability of the series and identification of a suitable lead for further optimisation.

Automated design of ligands to polypharmacological profiles.

The clinical efficacy and safety of a drug is determined by its activity profile across many proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to design drugs rationally a priori against profiles of several proteins would have immense value in drug discovery. Here we describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain-penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein-coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed to be correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads when multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology.

N-myristoyltransferase inhibitors as new leads to treat sleeping sickness.

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for approximately 30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target-T. brucei N-myristoyltransferase-leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N-myristoyltransferase as a promising therapeutic target for human African trypanosomiasis.

C-reactive protein is essential for innate resistance to pneumococcal infection.

No deficiency of human C-reactive protein (CRP), or even structural polymorphism of the protein, has yet been reported so its physiological role is not known. Here we show for the first time that CRP-deficient mice are remarkably susceptible to Streptococcus pneumoniae infection and are protected by reconstitution with isolated pure human CRP, or by anti-pneumococcal antibodies. Autologous mouse CRP is evidently essential for innate resistance to pneumococcal infection before antibodies are produced. Our findings are consistent with the significant association between clinical pneumococcal infection and non-coding human CRP gene polymorphisms which affect CRP expression. Deficiency or loss of function variation in CRP may therefore be lethal at the first early-life encounter with this ubiquitous virulent pathogen, explaining the invariant presence and structure of CRP in human adults.

In vitro and in vivo studies of trypanocidal activity of dietary isothiocyanates.

Six dietary isothiocyanates, allyl-isothiocyanate, benzyl-isothiocyanate, phenylethyl-isothiocyanate, sulforaphane, erucin, and iberin, were tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of Trypanosoma brucei. All isothiocyanates showed a dose-dependent effect on the growth of trypanosomes. Five compounds displayed MIC values of 10 µM and GI50 values of around 1.5 µM, while allyl-isothiocyanate exhibited values of 100 and 11 µM, respectively. The compounds showed similar cytotoxic activities against human HL-60 cells with GI50 values of 1-4 µM and MIC values of 10-100 µM. Short-term experiments revealed that, with the exception of allyl-isothiocyanate, isothiocyanates at a concentration of 10 µM kill trypanosomes within 1-4 h of incubation. In contrast, HL-60 cells were not affected by any of the compounds in short-term incubation experiments. Sulforaphane, the most intensively studied isothiocyanate, was also investigated for its in vivo trypanocidal activity. However, administration of 50 mg/kg sulforaphane orally or intraperitoneally for four days had no effect on the parasitaemia in mice infected with T. brucei compared to control animals treated with vehicle alone.

Sperm Toolbox-A selection of small molecules to study human spermatozoa.

Male contraceptive options and infertility treatments are limited, and almost all innovation has been limited to updates to medically assisted reproduction protocols and methods. To accelerate the development of drugs that can either improve or inhibit fertility, we established a small molecule library as a toolbox for assay development and screening campaigns using human spermatozoa. We have profiled all compounds in the Sperm Toolbox in several automated high-throughput assays that measure stimulation or inhibition of sperm motility or the acrosome reaction. We have assayed motility under non-capacitating and capacitating conditions to distinguish between pathways operating under these different physiological states. We also assayed cell viability to ensure any effects on sperm function are specific. A key advantage of our studies is that all compounds are assayed together in the same experimental conditions, which allows quantitative comparisons of their effects in complementary functional assays. We have combined the resulting datasets to generate fingerprints of the Sperm Toolbox compounds on sperm function. The data are included in an on-line R-based app for convenient querying.

Rational Exploration of 2,4-Diaminopyrimidines as DHFR Inhibitors Active against Mycobacterium abscessus and Mycobacterium avium, Two Emerging Human Pathogens.

Nontuberculous mycobacteria (NTM) are emerging human pathogens linked to severe pulmonary diseases. Current treatments involve the prolonged use of multiple drugs and are often ineffective. Bacterial dihydrofolate reductase (DHFR) is a key enzyme targeted by antibiotics in Gram-negative bacterial infections. However, existing DHFR inhibitors designed for Gram-negative bacteria often fail against mycobacterial DHFRs. Here, we detail the rational design of NTM DHFR inhibitors based on P218, a malarial DHFR inhibitor. We identified compound 8, a 2,4-diaminopyrimidine exhibiting improved pharmacological properties and activity against purified DHFR, and whole cell cultures of two predominant NTM species: Mycobacterium avium and Mycobacterium abscessus. This study underscores the potential of compound 8 as a promising candidate for the in vivo validation of DHFR as an effective treatment against NTM infections.

Cryptosporidium lysyl-tRNA synthetase inhibitors define the interplay between solubility and permeability required to achieve efficacy.

Cryptosporidiosis is a diarrheal disease caused by infection with Cryptosporidium spp. parasites and is a leading cause of death in malnourished children worldwide. The only approved treatment, nitazoxanide, has limited efficacy in this at-risk patient population. Additional safe therapeutics are urgently required to tackle this unmet medical need. However, the development of anti-cryptosporidial drugs is hindered by a lack of understanding of the optimal compound properties required to treat this gastrointestinal infection. To address this knowledge gap, a diverse set of potent lysyl-tRNA synthetase inhibitors was profiled to identify optimal physicochemical and pharmacokinetic properties required for efficacy in a chronic mouse model of infection. The results from this comprehensive study illustrated the importance of balancing solubility and permeability to achieve efficacy in vivo. Our results establish in vitro criteria for solubility and permeability that are predictive of compound efficacy in vivo to guide the optimization of anti-cryptosporidial drugs. Two compounds from chemically distinct series (DDD489 and DDD508) were identified as demonstrating superior efficacy and prioritized for further evaluation. Both compounds achieved marked parasite reduction in immunocompromised mouse models and a disease-relevant calf model of infection. On the basis of these promising data, these compounds have been selected for progression to preclinical safety studies, expanding the portfolio of potential treatments for this neglected infectious disease.