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.

Identification and Optimization of Novel Inhibitors of the Polyketide Synthase 13 Thioesterase Domain with Antitubercular Activity.

There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for Mycobacterium tuberculosis growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 µM) and in vitro ADMET profiles.

Male contraceptive development: A medicinal chemistry perspective.

There is a need for non-hormonal contraceptives. One area that needs further investigation is the development of male contraceptives. Comparatively little is understood about potential drug targets in men to achieve a reversible contraceptive effect. In this article, we review the need for male contraceptives and some thoughts around the characteristics of a male contraceptive and the potential development pathway. We then discuss different potential approaches to discovering male contraceptives and then highlight potential targets that have been discussed in the literature.

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.

High-throughput phenotypic screening of the human spermatozoon.

Despite recent advances in male reproductive health research, there remain many elements of male infertility where our understanding is incomplete. Consequently, diagnostic tools and treatments for men with sperm dysfunction, other than medically assisted reproduction, are limited. On the other hand, the gaps in our knowledge of the mechanisms which underpin sperm function have hampered the development of male non-hormonal contraceptives. The study of mature spermatozoa is inherently difficult. They are a unique and highly specialised cell type which does not actively transcribe or translate proteins and cannot be cultured for long periods of time or matured in vitro. One large-scale approach to both increasing the understanding of sperm function and the discovery and development of compounds that can modulate sperm function is to directly observe responses to compounds with phenotypic screening techniques. These target agnostic approaches can be developed into high-throughput screening platforms with the potential to drastically increase advances in the field. Here, we discuss the rationale and development of high-throughput phenotypic screening platforms for mature human spermatozoa and the multiple potential applications these present, as well as the current limitations and leaps in our understanding and the capabilities needed to overcome them. Further development and use of these technologies could lead to the identification of compounds which positively or negatively affect sperm cell motility or function or novel platforms for toxicology or environmental chemical testing among other applications. Ultimately, each of these potential applications is also likely to increase the understanding within the field of sperm biology.

Substituted Aminoacetamides as Novel Leads for Malaria Treatment.

Herein we describe the optimization of a phenotypic hit against Plasmodium falciparum based on an aminoacetamide scaffold. This led to N-(3-chloro-4-fluorophenyl)-2-methyl-2-{[4-methyl-3-(morpholinosulfonyl)phenyl]amino}propanamide (compound 28) with low-nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactive in a mammalian cell counter-screen up to 25 µm. Inhibition of gametes in the dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomal stability to a point at which the aminoacetamides would be suitable for in vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potency and selectivity; it could therefore serve as a suitable chemical tool for drug target identification.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Discovery of a Quinoline-4-carboxamide Derivative with a Novel Mechanism of Action, Multistage Antimalarial Activity, and Potent in Vivo Efficacy.

The antiplasmodial activity, DMPK properties, and efficacy of a series of quinoline-4-carboxamides are described. This series was identified from a phenotypic screen against the blood stage of Plasmodium falciparum (3D7) and displayed moderate potency but with suboptimal physicochemical properties and poor microsomal stability. The screening hit (1, EC50 = 120 nM) was optimized to lead molecules with low nanomolar in vitro potency. Improvement of the pharmacokinetic profile led to several compounds showing excellent oral efficacy in the P. berghei malaria mouse model with ED90 values below 1 mg/kg when dosed orally for 4 days. The favorable potency, selectivity, DMPK properties, and efficacy coupled with a novel mechanism of action, inhibition of translation elongation factor 2 (PfEF2), led to progression of 2 (DDD107498) to preclinical development.

Trisubstituted Pyrimidines as Efficacious and Fast-Acting Antimalarials.

In this paper we describe the optimization of a phenotypic hit against Plasmodium falciparum, based on a trisubstituted pyrimidine scaffold. This led to compounds with good pharmacokinetics and oral activity in a P. berghei mouse model of malaria. The most promising compound (13) showed a reduction in parasitemia of 96% when dosed at 30 mg/kg orally once a day for 4 days in the P. berghei mouse model of malaria. It also demonstrated a rapid rate of clearance of the erythrocytic stage of P. falciparum in the SCID mouse model with an ED90 of 11.7 mg/kg when dosed orally. Unfortunately, the compound is a potent inhibitor of cytochrome P450 enzymes, probably due to a 4-pyridyl substituent. Nevertheless, this is a lead molecule with a potentially useful antimalarial profile, which could either be further optimized or be used for target hunting.

Validation of N-myristoyltransferase as Potential Chemotherapeutic Target in Mammal-Dwelling Stages of Trypanosoma cruzi.

BACKGROUND: Trypanosoma cruzi causes Chagas disease, an endemic and debilitating illness in Latin America. Lately, owing to extensive population movements, this neglected tropical disease has become a global health concern. The two clinically available drugs for the chemotherapy of Chagas disease have rather high toxicity and limited efficacy in the chronic phase of the disease, and may induce parasite resistance. The development of new anti-T. cruzi agents is therefore imperative. The enzyme N-myristoyltransferase (NMT) has recently been biochemically characterized, shown to be essential in Leishmania major, Trypanosoma brucei, and T. cruzi¸ and proposed as promising chemotherapeutic target in these trypanosomatids. METHODOLOGY/PRINCIPAL FINDINGS: Here, using high-content imaging we assayed eight known trypanosomatid NMT inhibitors, against mammal-dwelling intracellular amastigote and trypomastigote stages and demonstrated that three of them (compounds 1, 5, and 8) have potent anti-proliferative effect at submicromolar concentrations against T. cruzi, with very low toxicity against human epithelial cells. Moreover, metabolic labeling using myristic acid, azide showed a considerable decrease in the myristoylation of proteins in parasites treated with NMT inhibitors, providing evidence of the on-target activity of the inhibitors. CONCLUSIONS/SIGNIFICANCE: Taken together, our data point out to the potential use of NMT inhibitors as anti-T. cruzi chemotherapy.

A novel multiple-stage antimalarial agent that inhibits protein synthesis.

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

Lead optimization of a pyrazole sulfonamide series of Trypanosoma brucei N-myristoyltransferase inhibitors: identification and evaluation of CNS penetrant compounds as potential treatments for stage 2 human African trypanosomiasis.

Trypanosoma brucei N-myristoyltransferase (TbNMT) is an attractive therapeutic target for the treatment of human African trypanosomiasis (HAT). From previous studies, we identified pyrazole sulfonamide, DDD85646 (1), a potent inhibitor of TbNMT. Although this compound represents an excellent lead, poor central nervous system (CNS) exposure restricts its use to the hemolymphatic form (stage 1) of the disease. With a clear clinical need for new drug treatments for HAT that address both the hemolymphatic and CNS stages of the disease, a chemistry campaign was initiated to address the shortfalls of this series. This paper describes modifications to the pyrazole sulfonamides which markedly improved blood-brain barrier permeability, achieved by reducing polar surface area and capping the sulfonamide. Moreover, replacing the core aromatic with a flexible linker significantly improved selectivity. This led to the discovery of DDD100097 (40) which demonstrated partial efficacy in a stage 2 (CNS) mouse model of HAT.

Substituted 2-phenylimidazopyridines: a new class of drug leads for human African trypanosomiasis.

A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of human African trypanosomiasis, led to the identification of substituted 2-(3-aminophenyl)oxazolopyridines as a starting point for hit-to-lead medicinal chemistry. A total of 110 analogues were prepared, which led to the identification of 64, a substituted 2-(3-aminophenyl)imidazopyridine. This compound showed antiparasitic activity in vitro with an EC50 of 2 nM and displayed reasonable druglike properties when tested in a number of in vitro assays. The compound was orally bioavailable and displayed good plasma and brain exposure in mice. Compound 64 cured mice infected with Trypanosoma brucei when dosed orally down to 2.5 mg/kg. Given its potent antiparasitic properties and its ease of synthesis, compound 64 represents a new lead for the development of drugs to treat human African trypanosomiasis.

Dialkylimidazole inhibitors of Trypanosoma cruzi sterol 14α-demethylase as anti-Chagas disease agents.

New dialkylimidazole based sterol 14α-demethylase inhibitors were prepared and tested as potential anti-Trypanosoma cruzi agents. Previous studies had identified compound 2 as the most potent and selective inhibitor against parasite cultures. In addition, animal studies had demonstrated that compound 2 is highly efficacious in the acute model of the disease. However, compound 2 has a high molecular weight and high hydrophobicity, issues addressed here. Systematic modifications were carried out at four positions on the scaffold and several inhibitors were identified which are highly potent (EC50 <1 nM) against T. cruzi in culture. The halogenated derivatives 36j, 36k, and 36p, display excellent activity against T. cruzi amastigotes, with reduced molecular weight and lipophilicity, and exhibit suitable physicochemical properties for an oral drug candidate.

Exploring the Trypanosoma brucei Hsp83 potential as a target for structure guided drug design.

Human African trypanosomiasis is a neglected parasitic disease that is fatal if untreated. The current drugs available to eliminate the causative agent Trypanosoma brucei have multiple liabilities, including toxicity, increasing problems due to treatment failure and limited efficacy. There are two approaches to discover novel antimicrobial drugs--whole-cell screening and target-based discovery. In the latter case, there is a need to identify and validate novel drug targets in Trypanosoma parasites. The heat shock proteins (Hsp), while best known as cancer targets with a number of drug candidates in clinical development, are a family of emerging targets for infectious diseases. In this paper, we report the exploration of T. brucei Hsp83--a homolog of human Hsp90--as a drug target using multiple biophysical and biochemical techniques. Our approach included the characterization of the chemical sensitivity of the parasitic chaperone against a library of known Hsp90 inhibitors by means of differential scanning fluorimetry (DSF). Several compounds identified by this screening procedure were further studied using isothermal titration calorimetry (ITC) and X-ray crystallography, as well as tested in parasite growth inhibitions assays. These experiments led us to the identification of a benzamide derivative compound capable of interacting with TbHsp83 more strongly than with its human homologs and structural rationalization of this selectivity. The results highlight the opportunities created by subtle structural differences to develop new series of compounds to selectively target the Trypanosoma brucei chaperone and effectively kill the sleeping sickness parasite.

Discovery of a novel class of orally active trypanocidal N-myristoyltransferase inhibitors.

N-Myristoyltransferase (NMT) represents a promising drug target for human African trypanosomiasis (HAT), which is caused by the parasitic protozoa Trypanosoma brucei. We report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which has potent activity against the enzyme (IC(50) = 2 nM) and T. brucei (EC(50) = 2 nM) in culture. The compound has good oral pharmacokinetics and cures rodent models of peripheral HAT infection. This compound provides an excellent tool for validation of T. brucei NMT as a drug target for HAT as well as a valuable lead for further optimization.

Total synthesis of (+/-)-alpha-isosparteine, (+/-)-beta-isosparteine, and (+/-)-sparteine from a common tetraoxobispidine intermediate.

The three title alkaloids were separately prepared in stereocontrolled fashion from a common tetraoxobispidine precursor, 3,7-diallyl-2,4,6,8-tetraoxo-3,7-diazabicyclo[3.3.1]nonane (16). Bisimide 16 was generated from malonate via acid promoted cyclization of the Knoevenagel condensation adduct 1,1,3,3-propanetetracarboxamide. (+/-)-alpha-Isosparteine (dl-2) was elaborated from 16 in 28% overall yield by a two-directional synthetic sequence composed of four reactions: double addition of allylmagnesium bromide, ring-closing olefin metathesis (RCM), hydrogenation, and borane mediated reduction. (+/-)-beta-Isosparteine (dl-3) was targeted along similar lines by a strategic reversal in allylation and reduction operations on the core synthon. Thus, 16 was advanced to dl-3 in five steps and 12% overall yield by a reaction sequence commencing with sodium borohydride mediated reduction and followed by double Sakurai-type allylation of the resulting bishemiaminal. The synthesis of dl-3 was concluded by RCM and then global reduction (H2, Pd/C; LiAlH4). The final target, (+/-)-sparteine (dl-1), was secured in six steps and 11% overall yield from 16 by monoreduction and Sakurai allylation, followed by allyl Grignard addition and then RCM and global reduction as before. Reasons for the inherent C2-type regioselectivity of net double nucleophilic additions to tetraoxobispidines are discussed and enantioselective oxazaborolidine mediated reduction of the N,N'-dibenzyl congener of 16 is reported.

A practical synthesis of (+/-)-alpha-isosparteine from a tetraoxobispidine core.

[reaction: see text] The title alkaloid was synthesized in racemic form from 3,7-diallyl-2,4,6,8-tetraoxo-3,7-diazabicyclo[3.3.1]nonane (7) by a regioselective diallylation reaction followed by double ring-closing olefin metathesis and exhaustive reduction. Tetraoxobispidine 7 was itself prepared in three simple operations from dimethyl malonate. The entire sequence to alpha-isosparteine was conducted on a multigram scale and proceeded without recourse to chromatography.