Evaluation of pyrrolidine and pyrazolone derivatives as inhibitors of trypanosomal phosphodiesterase B1 (TbrPDEB1).

Human African trypanosomiasis (HAT) is a parasitic disease, caused by the protozoan pathogen Trypanosoma brucei, which affects thousands every year and which is in need of new therapeutics. Herein we report the synthesis and assessment of a series of pyrrolidine and pyrazolone derivatives of human phosphodiesterase 4 (hPDE4) inhibitors for the assessment of their activity against the trypanosomal phosphodiesterase TbrPDEB1. The synthesized compounds showed weak potency against TbrPDEB1.

Repurposing human PDE4 inhibitors for neglected tropical diseases: design, synthesis and evaluation of cilomilast analogues as Trypanosoma brucei PDEB1 inhibitors.

A medicinal chemistry exploration of the human phosphodiesterase 4 (hPDE4) inhibitor cilomilast (1) was undertaken in order to identify inhibitors of phosphodiesterase B1 of Trypanosoma brucei (TbrPDEB1). T. brucei is the parasite which causes African sleeping sickness, a neglected tropical disease that affects thousands each year, and TbrPDEB1 has been shown to be an essential target of therapeutic relevance. Noting that 1 is a weak inhibitor of TbrPDEB1, we report the design and synthesis of analogs of this compound, culminating in 12b, a sub-micromolar inhibitor of TbrPDEB1 that shows modest inhibition of T. brucei proliferation.

Synthesis and assessment of catechol diether compounds as inhibitors of trypanosomal phosphodiesterase B1 (TbrPDEB1).

Human African trypanosomiasis (HAT) is a parasitic neglected tropical disease that affects 10,000 patients each year. Current treatments are sub-optimal, and the disease is fatal if not treated. Herein, we report our continuing efforts to repurpose the human phosphodiesterase 4 (hPDE4) inhibitor piclamilast to target trypanosomal phosphodiesterase TbrPDEB1. We prepared a range of substituted heterocyclic replacements for the 4-amino-3,5-dichloro-pyridine headgroup of piclamilast, and found that these compounds exhibited weak inhibitory activity of TbrPDEB1.

Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 2. Tadalafil analogs.

In this Letter we describe our ongoing target repurposing efforts focused on discovery of inhibitors of the essential trypanosomal phosphodiesterase TbrPDEB1. This enzyme has been implicated in virulence of Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). We outline the synthesis and biological evaluation of analogs of tadalafil, a human PDE5 inhibitor currently utilized for treatment of erectile dysfunction, and report that these analogs are weak inhibitors of TbrPDEB1.

Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 1. Sildenafil analogs.

Parasitic diseases, such as African sleeping sickness, have a significant impact on the health and well-being in the poorest regions of the world. Pragmatic drug discovery efforts are needed to find new therapeutic agents. In this Letter we describe target repurposing efforts focused on trypanosomal phosphodiesterases. We outline the synthesis and biological evaluation of analogs of sildenafil (1), a human PDE5 inhibitor, for activities against trypanosomal PDEB1 (TbrPDEB1). We find that, while low potency analogs can be prepared, this chemical class is a sub-optimal starting point for further development of TbrPDE inhibitors.

Crystal structure of Leishmania major oligopeptidase B gives insight into the enzymatic properties of a trypanosomatid virulence factor.

Oligopeptidase B (OPB) is a serine peptidase with dibasic substrate specificity. It is found in bacteria, plants, and trypanosomatid pathogens, where it has been identified as a virulence factor and potential drug target. In this study we expressed active recombinant Leishmania major OPB and provide the first structure of an oligopeptidase B at high resolution. The crystallographic study reveals that OPB comprises two domains, a catalytic and a propeller domain, linked together by a hinge region. The structure has been determined in complex with the oligopeptide, protease-inhibitor antipain, giving detailed information on the enzyme active site and extended substrate binding pockets. It shows that Glu-621 plays a critical role in the S1 binding pocket and, along with Phe-603, is largely responsible for the enzyme substrate specificity in P1. In the S2 binding pocket, Tyr-499 was shown to be important for substrate stability. The structure also allowed an investigation into the function of residues highlighted in other studies including Glu-623, which was predicted to be involved in the S1 binding pocket but is found forming an inter-domain hydrogen bond. Additional important salt bridges/hydrogen bonds between the two domains were observed, highlighting the significance of the domain interface in OPB. This work provides a foundation for the study of the role of OPBs as virulence factors in trypanosomatids. It could facilitate the development of specific OPB inhibitors with therapeutic potential by exploiting its unique substrate recognition properties as well as providing a model for OPBs in general.

Structures of Leishmania major orthologues of macrophage migration inhibitory factor.

Leishmania major, an intracellular parasitic protozoon that infects, differentiates and replicates within macrophages, expresses two closely related MIF-like proteins. To ascertain the roles and potential differences of these two Leishmania proteins, recombinant L. major MIF1 and MIF2 have been produced and the structures resolved by X-ray crystallography. Each has a trimeric ring architecture similar to mammalian MIF, but with some structurally distinct features. LmjMIF1, but not LmjMIF2, has tautomerase activity. LmjMIF2 is found in all life cycle stages whereas LmjMIF1 is found exclusively in amastigotes, the intracellular stage responsible for mammalian disease. The findings are consistent with parasite MIFs modulating or circumventing the host macrophage response, thereby promoting parasite survival, but suggest the LmjMIFs have potentially different biological roles. Analysis of the Leishmania braziliensis genome showed that this species lacks both MIF genes. Thus MIF is not a virulence factor in all species of Leishmania.

Locomotor and geotactic behavior of Drosophila melanogaster over-expressing neprilysin 2.

The neprilysin (M13) family of zinc-metallopeptidases has been implicated in a variety of physiological processes, but principally the control of neuropeptide levels in a range of animal species. The over-expression of the amyloid-degrading enzyme, neprilysin, as a therapeutic strategy for Alzheimer's disease is a concept that is gaining in popularity. Here we utilize the GAL4/UAS system to over-express the Drosophila melanogaster Nep2 gene, a close homologue of neprilysin, in flies yielding an increase in NEP2 protein that is detectable by both immunoblotting and enzyme activity. This increase in NEP2 caused a behavioral phenotype manifested in abnormal climbing behavior. Wild type flies climb in a linear, vertical path, but NEP2 over-expressing (Nep2(OEX)) flies tend to climb in a spiral pattern and display an increase in grooming behavior during frequent stationary periods. Nep2(OEX) flies also perform poorly in a geotaxis maze, taking ten times as long to complete the course compared to wild type Drosophila. We hypothesize that the poor performance of the Nep2(OEX) flies in locomotor assays is due to perturbation of neuropeptide signaling and provides evidence of detrimental effects of neprilysin over-expression.

Neuropeptidases and the metabolic inactivation of insect neuropeptides.

Neuropeptidases play a key role in regulating neuropeptide signalling activity in the central nervous system of animals. They are oligopeptidases that are generally found on the surface of neuronal cells facing the synaptic and peri-synaptic space and therefore are ideally placed for the metabolic inactivation of neuropeptide transmitters/modulators. This review discusses the structure of insect neuropeptides in relation to their susceptibility to hydrolysis by peptidases and the need for specialist enzymes to degrade many neuropeptides. It focuses on five neuropeptidase families (neprilysin, dipeptidyl-peptidase IV, angiotensin-converting enzyme, aminopeptidase and dipeptidyl aminopeptidase III) that have been implicated in the metabolic inactivation of neuropeptides in the central nervous system of insects. Experimental evidence for the involvement of these peptidases in neuropeptide metabolism is reviewed and their properties are compared to similar neuropeptide inactivating peptidases of the mammalian brain. We also discuss how the sequencing of insect genomes has led to the molecular identification of candidate neuropeptidase genes.

Bioinformatic analysis of the neprilysin (M13) family of peptidases reveals complex evolutionary and functional relationships.

BACKGROUND: The neprilysin (M13) family of endopeptidases are zinc-metalloenzymes, the majority of which are type II integral membrane proteins. The best characterised of this family is neprilysin, which has important roles in inactivating signalling peptides involved in modulating neuronal activity, blood pressure and the immune system. Other family members include the endothelin converting enzymes (ECE-1 and ECE-2), which are responsible for the final step in the synthesis of potent vasoconstrictor endothelins. The ECEs, as well as neprilysin, are considered valuable therapeutic targets for treating cardiovascular disease. Other members of the M13 family have not been functionally characterised, but are also likely to have biological roles regulating peptide signalling. The recent sequencing of animal genomes has greatly increased the number of M13 family members in protein databases, information which can be used to reveal evolutionary relationships and to gain insight into conserved biological roles. RESULTS: The phylogenetic analysis successfully resolved vertebrate M13 peptidases into seven classes, one of which appears to be specific to mammals, and insect genes into five functional classes and a series of expansions, which may include inactive peptidases. Nematode genes primarily resolved into groups containing no other taxa, bar the two nematode genes associated with Drosophila DmeNEP1 and DmeNEP4. This analysis reconstructed only one relationship between chordate and invertebrate clusters, that of the ECE sub-group and the DmeNEP3 related genes. Analysis of amino acid utilisation in the active site of M13 peptidases reveals a basis for their biochemical properties. A relatively invariant S1' subsite gives the majority of M13 peptidases their strong preference for hydrophobic residues in P1' position. The greater variation in the S2' subsite may be instrumental in determining the specificity of M13 peptidases for their substrates and thus allows M13 peptidases to fulfil a broad range of physiological roles. CONCLUSION: The M13 family of peptidases have diversified extensively in all species examined, indicating wide ranging roles in numerous physiological processes. It is predicted that differences in the S2' subsite are fundamental to determining the substrate specificities that facilitate this functional diversity.

Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster.

Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. In this study we show that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, we also found strong neprilysin-like neutral endopeptidase activity in a soluble embryo fraction, which we identify as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly(7)-Leu(8)), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'(1) sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster.

Drosophila melanogaster NEP2 is a new soluble member of the neprilysin family of endopeptidases with implications for reproduction and renal function.

The mammalian neprilysin (NEP) family members are typically type II membrane endopeptidases responsible for the activation/inactivation of neuropeptides and peptide hormones. Differences in substrate specificity and subcellular localization of the seven mammalian NEPs contribute to their functional diversity. The sequencing of the Drosophila melanogaster genome has revealed a large expansion of this gene family, resulting in over 20 fly NEP-like genes, suggesting even greater diversity in structure and function than seen in mammals. We now report that one of these genes (Nep2) codes for a secreted endopeptidase with a highly restricted pattern of expression. D. melanogaster NEP2 is expressed in the specialized stellate cells of the renal tubules and in the cyst cells that surround the elongating spermatid bundles in adult testis, suggesting roles for the peptidase in renal function and in spermatogenesis. D. melanogaster NEP2 was found in vesicle-like structures in the syncytial cytoplasm of the spermatid bundles, suggesting that the protein was acquired by endocytosis of protein secreted from the cyst cells. Expression of NEP2 cDNA in D. melanogaster S2 cells confirmed that the peptidase is secreted and is only weakly inhibited by thiorphan, a potent inhibitor of human NEP. D. melanogaster NEP2 also differs from human NEP in the manner in which the peptidase cleaves the tachykinin, GPSGFYGVR-amide. Molecular modelling suggests that there are important structural differences between D. melanogaster NEP2 and human NEP in the S1' and S2' ligand-binding subsites, which might explain the observed differences in inhibitor and substrate specificities. A soluble isoform of a mouse NEP-like peptidase is strongly expressed in spermatids, suggesting an evolutionarily conserved role for a soluble endopeptidase in spermatogenesis.

Repurposing human PDE4 inhibitors for neglected tropical diseases. Evaluation of analogs of the human PDE4 inhibitor GSK-256066 as inhibitors of PDEB1 of Trypanosoma brucei.

Cyclic nucleotide phosphodiesterases (PDEs) have been identified as important enzyme targets for drug development in both humans and Trypanosoma brucei, the causative agent of human African trypanosomiasis. With this in mind, we recently reported the profiling of a range of human phosphodiesterase inhibitors, showing that human PDE4 inhibitors tend to display the best potency against the trypanosomal phosphodiesterase TbrPDEB1. Among these was GSK-256066, a potent inhibitor of human PDE4 and a weak inhibitor of TbrPDEB1. In this report, we describe the results of a structure-activity relationship study of this chemotype, leading to the discovery of analogs with improved potency against TbrPDEB1 and micromolar inhibition of T. brucei cellular growth. We rationalize the potency trends via molecular docking of the new inhibitors into a recently reported apo structure of TbrPDEB1. The studies in this article will inform future efforts in repurposing human PDE inhibitors as antitrypanosomal agents.

Pharmacological validation of Trypanosoma brucei phosphodiesterases B1 and B2 as druggable targets for African sleeping sickness.

Neglected tropical disease drug discovery requires application of pragmatic and efficient methods for development of new therapeutic agents. In this report, we describe our target repurposing efforts for the essential phosphodiesterase (PDE) enzymes TbrPDEB1 and TbrPDEB2 of Trypanosoma brucei , the causative agent for human African trypanosomiasis (HAT). We describe protein expression and purification, assay development, and benchmark screening of a collection of 20 established human PDE inhibitors. We disclose that the human PDE4 inhibitor piclamilast, and some of its analogues, show modest inhibition of TbrPDEB1 and B2 and quickly kill the bloodstream form of the subspecies T. brucei brucei . We also report the development of a homology model of TbrPDEB1 that is useful for understanding the compound-enzyme interactions and for comparing the parasitic and human enzymes. Our profiling and early medicinal chemistry results strongly suggest that human PDE4 chemotypes represent a better starting point for optimization of TbrPDEB inhibitors than those that target any other human PDEs.