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

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Tetrahedron Letters 56 (2015): 2832-2835, doi:10.1016/j.tetlet.2015.04.061.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.We acknowledge funding from the National Institutes of Health (R01AI082577)

A target repurposing approach identifies N-myristoyltransferase as a new candidate drug target in filarial nematodes

Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC50 values of 2.5-10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis

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

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Bioorganic & Medicinal Chemistry Letters 23 (2013): 5971-5974, doi:10.1016/j.bmcl.2013.08.057.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 head group of piclamilast, and found that these compounds exhibited weak inhibitory activity of TbrPDEB1.We acknowledge funding from the National Institutes of Health (R01AI082577)

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

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Bioorganic & Medicinal Chemistry Letters 24 (2014): 4084-4089, doi:10.1016/j.bmcl.2014.07.063.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.This work was funded by the National Institutes of Health (R01AI082577)

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

Author Posting. © The Author(s), 2012. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Bioorganic & Medicinal Chemistry Letters 22 (2012): 2582-2584, doi:10.1016/j.bmcl.2012.01.118.In this report 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.This work was supported by the National Institutes of Health (R01AI082577), Boston University and Northeastern University

A chemical screen identifies small molecules that regulate hepcidin expression

AbstractHepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Bone morphogenic protein and Stat3 signaling regulate Hepcidin's transcription. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 2.7kb of the human Hepcidin promoter upstream of a firefly reporter gene. We used high throughput methods to screen 10,169 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1 standard deviation below the mean of the other chemicals (z-score >3 or <1), while not adversely affecting cell viability, quantified by fluorescence assay. Following validation assays, we identified 16 chemicals in a broad range of functional classes that promote Hepcidin expression. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 or Stat3

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

Author Posting. © The Author(s), 2012. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Bioorganic & Medicinal Chemistry Letters 22 (2012): 2579-2581, doi:10.1016/j.bmcl.2012.01.119.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 report 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.This work was supported by the National Institutes of Health (R01AI082577), Boston University and Northeastern University

Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability

This paper combines two techniques—mass spectrometry and protein charge ladders—to examine the relationship between the surface charge and hydrophobicity of a protein (bovine carbonic anhydrase II; BCA II) and its rate of amide hydrogen-deuterium (H/D) exchange. Mass spectrometric analysis indicated that the sequential acetylation of surface lysine-$\epsilon$-NH$_{3}$$^{+}$ groups—a type of modification that increases the net negative charge and hydrophobicity of the surface of BCA II without affecting its 2° or 3° structure—resulted in a linear increase in the total number of backbone amide hydrogen that are protected from exchange with solvent (2 h, pD 7.4, 15 ºC). Each successive acetylation produced BCA II proteins with one additional hydrogen protected after two hours in deuterated buffer (pD 7.4, 15 ºC). NMR spectroscopy demonstrated that these protected hydrogen atoms were not located on the side chain of the acetylated lysine residues (i.e., lys-$\epsilon$-NHCOCH$_{3}$). The decrease in rate of exchange associated with acetylation paralleled a decrease in thermostability: the most slowly exchanging rungs were the least thermostable (as measured by differential scanning calorimetry). The fact that the rates of H/D exchange were similar for perbutyrated BCA II (e.g., [lys-$\epsilon$-NHCO(CH$_{2}$)$_{2}$CH$_{3}$]$_{18}$) and peracetylated BCA II (e.g., [lys-$\epsilon$-NHCOCH$_{3}$]$_{18}$) suggests that the charge is more important than the hydrophobicity of surface groups in determining the rate of H/D exchange. These kinetic electrostatic effects could complicate the interpretation of experiments in which H/D exchange methods are used to probe the structural effects of non-isoelectric perturbations to proteins (i.e., phosphorylation, acetylation, or the binding of the protein to an oligonucleotide or another charged ligand or protein).Chemistry and Chemical Biolog

Calcium-Dependent Protein Kinase 5 Is Required for Release of Egress-Specific Organelles in Plasmodium falciparum

ABSTRACT The human malaria parasite Plasmodium falciparum requires efficient egress out of an infected red blood cell for pathogenesis. This egress event is highly coordinated and is mediated by several signaling proteins, including the plant-like P. falciparum calcium-dependent protein kinase 5 (PfCDPK5). Knockdown of PfCDPK5 results in an egress block where parasites are trapped inside their host cells. The mechanism of this PfCDPK5-dependent block, however, remains unknown. Here, we show that PfCDPK5 colocalizes with a specialized set of parasite organelles known as micronemes and is required for their discharge, implicating failure of this step as the cause of the egress defect in PfCDPK5-deficient parasites. Furthermore, we show that PfCDPK5 cooperates with the P. falciparum cGMP-dependent kinase (PfPKG) to fully activate the protease cascade critical for parasite egress. The PfCDPK5-dependent arrest can be overcome by hyperactivation of PfPKG or by physical disruption of the arrested parasite, and we show that both treatments facilitate the release of the micronemes required for egress. Our results define the molecular mechanism of PfCDPK5 function and elucidate the complex signaling pathway of parasite egress

Target repurposing for neglected diseases

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Future Science for personal use, not for redistribution. The definitive version was published in Future Medicinal Chemistry 3 (2011): 1307-1315, doi:10.4155/fmc.11.92.Infectious diseases are an enormous burden to global health, and since drug discovery is costly, those infectious diseases that affect the developing world are often not pursued by commercial drug discovery efforts. Therefore, pragmatic means by which new therapeutics can be discovered are needed. One such approach is target repurposing, where pathogen targets are matched with homologous human targets that have been pursued for drug discovery for other indications. In many cases, the medicinal chemistry, structural biology, and biochemistry knowledge around these human targets can be directly repurposed to launch and accelerate new drug discovery efforts against the pathogen targets. This article describes the overarching strategy of target repurposing as a tool for initiating and prosecuting neglected disease drug discovery programs, highlighting this approach with three case studies.Support from the National Institutes of Health (R01 AI082577) is gratefully acknowledged.2012-08-0