Structure-Activity Relationship of a Pyrrole Based Series of PfPKG Inhibitors as Anti-Malarials.

Controlling malaria requires new drugs against Plasmodium falciparum. The P. falciparum cGMP-dependent protein kinase (PfPKG) is a validated target whose inhibitors could block multiple steps of the parasite's life cycle. We defined the structure-activity relationship (SAR) of a pyrrole series for PfPKG inhibition. Key pharmacophores were modified to enable full exploration of chemical diversity and to gain knowledge about an ideal core scaffold. In vitro potency against recombinant PfPKG and human PKG were used to determine compound selectivity for the parasite enzyme. P. berghei sporozoites and P. falciparum asexual blood stages were used to assay multistage antiparasitic activity. Cellular specificity of compounds was evaluated using transgenic parasites expressing PfPKG carrying a substituted "gatekeeper" residue. The structure of PfPKG bound to an inhibitor was solved, and modeling using this structure together with computational tools was utilized to understand SAR and establish a rational strategy for subsequent lead optimization.

Filarial nematode phenotypic screening cascade to identify compounds with anti-parasitic activity for drug discovery optimization.

Filarial diseases, including lymphatic filariasis and onchocerciasis, are considered among the most devastating of all tropical diseases, affecting over 86 million people worldwide. To control and more rapidly eliminate onchocerciasis requires treatments that target the adult stage of the parasite. Drug discovery efforts are challenged by the lack of preclinical animal models using the human-pathogenic filariae, requiring the use of surrogate parasites for Onchocerca volvulus for both ex vivo and in vivo evaluation. Herein, we describe a platform utilizing phenotypic ex vivo assays consisting of the free-living nematode Caenorhabditis elegans, microfilariae and adult filariae of the bovine filariae Onchocerca lienalis and Onchocerca gutturosa, respectively, as well as microfilariae and adult filariae of the feline filariae Brugia pahangi, the rodent filariae Litomosoides sigmodontis and the human-pathogenic filariae Brugia malayi to assess activity across various surrogate parasites. Utilization of those surrogate nematodes for phenotypic ex vivo assays in order to assess activity across various parasites led to the successful establishment of a screening cascade and identification of multiple compounds with potential macrofilaricidal activity and desirable physicochemical, MW = 200-400 and low lipophilicity, logP <4, and pharmacokinetic properties, rat and human liver S9 stability of ≥70% remaining at 60 min, and AUC exposures above 3 µM h. This platform demonstrated the successful establishment of a screening cascade which resulted in the discovery of potential novel macrofilaricidal compounds for futher drug discovery lead optimization efforts. This screening cascade identified two distinct chemical series wherein one compound produced a significant 68% reduction of adult Litomosoides sigmodontis in the mouse model. Successful demonstration of efficacy prompted lead optimization medicinal chemistry efforts for this novel series.

A novel series of putative Brugia malayi histone demethylase inhibitors as potential anti-filarial drugs.

Filariasis, caused by a family of parasitic nematodes, affects millions of individuals throughout the tropics and is a major cause of acute and chronic morbidity. Current drugs are largely used in mass drug administration programs aimed at controlling the spread of disease by killing microfilariae, larval forms of the parasite responsible for transmission from humans to humans through insect vectors with limited efficacy against adult parasites. Although these drugs are effective, in some cases there are toxic liabilities. In case of loiasis which is caused by the parasitic eyeworm Loa loa, mass drug administration is contraindicative due to severe side effects of microfilariae killing, which can be life threatening. Our screening program and medicinal chemistry efforts have led to the identification of a novel series of compounds with potent killing activity against adult filarial parasites and minimal activity against microfilariae. A structural comparison search of our compounds demonstrated a close structural similarity to a recently described histone demethylase inhibitor, GSKJ1/4 which also exhibits selective adult parasite killing. We demonstrated a modification of histone methylation in Brugia malayi parasites treated with our compounds which might indicate that the mode of drug action is at the level of histone methylation. Our results indicate that targeting B. malayi and other filarial parasite demethylases may offer a novel approach for the development of a new class of macrofilaricidal therapeutics.

Characterization of Competitive Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is an enticing antimalarial drug target. Novel chemotypes are needed because existing inhibitors have safety issues that may prevent further development. This work demonstrates isoxazole-based compounds are potent ATP competitive inhibitors of PfPKG and discloses a new analogue in this series. Isoxazoles 3 and 5 had Ki values that are comparable to a known standard, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine. They also exhibited excellent selectivity for PfPKG over the human orthologue and the gatekeeper mutant T618Q PfPKG, which mimics the less accessible binding site of the human orthologue. The human orthologue's larger binding site volume is predicted to explain the selectivity of the inhibitors for the P. falciparum enzyme.

Discovery of Imidazole-Based Inhibitors of Plasmodium falciparum cGMP-Dependent Protein Kinase.

The discovery of new targets for the treatment of malaria, in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this paper presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization, and cell-based antiparasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity, and cell-based antiparasitic activity against multiple Plasmodium species that appears to be correlated with the in vitro potency.

Phosphatidylserine-Targeting Monoclonal Antibodies Exhibit Distinct Biochemical and Cellular Effects on Anti-CD3/CD28-Stimulated T Cell IFN-γ and TNF-α Production.

Phosphatidylserine (PS)-targeting monoclonal Abs (mAbs) that directly target PS and target PS via ß2-gp1 (ß2GP1) have been in preclinical and clinical development for over 10 y for the treatment of infectious diseases and cancer. Although the intended targets of PS-binding mAbs have traditionally included pathogens as well as stressed tumor cells and its associated vasculature in oncology, the effects of PS-targeting mAbs on activated immune cells, notably T cells, which externalize PS upon Ag stimulation, is not well understood. Using human T cells from healthy donor PBMCs activated with an anti-CD3 + anti-CD28 Ab mixture (anti-CD3/CD28) as a model for TCR-mediated PS externalization and T cell stimulation, we investigated effects of two different PS-targeting mAbs, 11.31 and bavituximab (Bavi), on TCR activation and TCR-mediated cytokine production in an ex vivo paradigm. Although 11.31 and Bavi bind selectivity to anti-CD3/28 activated T cells in a PS-dependent manner, surprisingly, they display distinct functional activities in their effect on IFN-γ and TNF-ɑ production, whereby 11.31, but not Bavi, suppressed cytokine production. This inhibitory effect on anti-CD3/28 activated T cells was observed on both CD4+ and CD8+ cells and independently of monocytes, suggesting the effects of 11.31 were directly mediated by binding to externalized PS on activated T cells. Imaging showed 11.31 and Bavi bind at distinct focal depots on the cell membrane. Collectively, our findings indicate that PS-targeting mAb 11.31 suppresses cytokine production by anti-CD3/28 activated T cells.

Discovery of isoxazolyl-based inhibitors of Plasmodium falciparum cGMP-dependent protein kinase.

The cGMP-dependent protein kinase in Plasmodium falciparum (PfPKG) plays multiple roles in the life cycle of the parasite. As a result, this enzyme is a potential target for new antimalarial agents. Existing inhbitors, while potent and active in malaria models are not optimal. This communication describes initial optimization of a structurally distinct class of PfPKG inhibitors.

Plasmodium falciparum cGMP-Dependent Protein Kinase - A Novel Chemotherapeutic Target.

The primary effector of cGMP signaling in Plasmodium is the cGMP-dependent protein kinase (PKG). Work in human-infective Plasmodium falciparum and rodent-infective Plasmodium berghei has provided biological validation of P. falciparum PKG (PfPKG) as a drug target for treating and/or protecting against malaria. PfPKG is essential in the asexual erythrocytic and sexual cycles as well as the pre-erythrocytic cycle. Medicinal chemistry efforts, both target-based and phenotype-based, have targeted PfPKG in the past few years. This review provides a brief overview of their results and challenges.

Discovery of a Stress-Activated Protein Kinase Inhibitor for Lymphatic Filariasis.

Lymphatic filariasis infects over 120 million people worldwide and can lead to significant disfigurement and disease. Resistance is emerging with current treatments, and these therapies have dose limiting adverse events; consequently new targets are needed. One approach to achieve this goal is inhibition of parasitic protein kinases involved in circumventing host defense mechanisms. This report describes structure-activity relationships leading to the identification of a potent, orally bioavailable stress activated protein kinase inhibitor that may be used to investigate this hypothesis.

Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase.

Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s(-1), the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 µM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 µM for trimethoprim, 109 ± 34 µM for pyrimethamine, 154 ± 46 µM for 2,4-diaminoquinazoline, 771 ± 44 µM for cycloguanil, and >20,000 µM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR.

Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication.

We recently demonstrated that human p38 mitogen-activated protein kinase (MAPK) inhibitors reduced in vitro and in vivo replication of the protozoan parasites Toxoplasma gondii and Encephalitozoon cuniculi. In this study, we assessed the efficacy of five p38 MAPK inhibitors to block the replication of Plasmodium falciparum in human erythrocytes cultured ex vivo and demonstrate that the pyridinylimidazole RWJ67657 and the pyrrolobenzimidazole RWJ68198 reduced P. falciparum replication, yielded trophozoites that were greatly diminished in size at 24h, and that these two agents interfered with stage differentiation. Interestingly, the chloroquine-resistant strain W2 was significantly more sensitive to these drugs than was the chloroquine-sensitive strain HB3. These results suggest that pyridinylimidazoles and pyrrolobenzimidazoles designed to inhibit human p38 MAPK activation can be developed to treat malaria.

The role of a Brugia malayi p38 MAP kinase ortholog (Bm-MPK1) in parasite anti-oxidative stress responses.

Filariasis, caused by thread-like nematode worms, affects millions of individuals throughout the tropics and is a major cause of acute and chronic morbidity. Filarial nematodes effectively evade host immunological responses and are long lived within their hosts. Recently an emphasis has been placed on enzymatic and non-enzymatic anti-oxidant systems which counteract the generation of reactive oxygen species (ROS) by macrophages and granulocytes, a first line of defense against parasites. We have characterized an anti-oxidant pathway in the filarial parasite Brugia malayi related to the evolutionarily conserved human mitogen-activated p38 protein kinase and the Caenorhabditis elegans PMK-1 protein kinase stress pathways. We have expressed a recombinant p38/PMK-1 ortholog from B. malayi (Bm-MPK1) and have successfully activated the kinase with mammalian upstream kinases. In addition, we have demonstrated inhibition of Bm-MPK1 activity using a panel of known p38 inhibitors. Using the potent and highly selective allosteric p38 inhibitor, BIRB796, we have implicated Bm-MPK1 in a pathway which offers B. malayi protection from the effects of ROS. Our results, for the first time, describe a stress-activated protein kinase pathway within the filarial parasite B. malayi which plays a role in protecting the parasite from ROS. Inhibition of this pathway may have therapeutic benefit in treating filariasis by increasing the sensitivity of filarial parasites to ROS and other reactive intermediates.

Apicomplexa, trypanosoma and parasitic nematode protein kinases as antiparasitic therapeutic targets.

Parasitic infections caused by Plasmodium, Trypanosoma, Leishmania, Toxoplasma and parasitic nematodes affect hundreds of millions of individuals worldwide and are the cause of significant mortality and morbidity, particularly in developing countries. These diseases also have an impact on individuals from developed countries; for example, some US troops in Iraq and Afghanistan have been infected with Leishmania. The annual mortality associated with parasitic infections is estimated to be 1.5 million deaths. The socioeconomic impact of the morbidity associated with parasitic infections is significant, and the development of new drugs, aimed at novel targets, is urgently needed to develop effective treatments for these diseases. The small-molecule inhibitors discussed in this review constitute useful tools with which to explore the relevance of kinase inhibition in inducing antiparasitic activity. The aim of recent target-based approaches used in the development of parasite kinase inhibitors is to identify novel antiparasitic agents with therapeutic potential.

Tranilast: a pharmaceutical candidate for reduction of adhesions using a novel approach.

Postsurgical adhesion formation has numerous deleterious side effects in a wide variety of surgical settings. Physical barriers used together with laparoscopy were developed in hopes of reducing the tissue trauma seen with open procedures and separating tissues during the critical time of healing to reduce adhesion formation. Despite meticulous techniques by surgeons and the availability of barriers, adhesion formation remains a serious problem, with more than $1 billion spent annually on complications arising from adhesions. Our laboratories have combined a previously marketed drug, Tranilast, with a gel to provide a locally delivered medicated device that can reduce adhesion formation. This article will review the role of Tranilast in the key pathways involved in adhesion formation.

Drugs designed to inhibit human p38 mitogen-activated protein kinase activation treat Toxoplasma gondii and Encephalitozoon cuniculi infection.

We recently showed that the pyridinylimidazoles SB203580 and SB202190, drugs designed to block human p38 mitogen-activated protein kinase (MAPK) activation, also inhibited replication of the medically important intracellular parasite Toxoplasma gondii in cultured human fibroblasts through a direct effect on the parasite. We now show that additional pyridinylimidazole and imidazopyrimidine p38 MAPK inhibitors inhibit intracellular T. gondii replication in vitro and protect mice against fatal T. gondii infection. Mice surviving infection following treatment with p38 MAPK inhibitors were resistant to subsequent T. gondii challenge, demonstrating induction of protective immunity. Thus, drugs originally developed to block human p38 MAPK activation are useful for treating T. gondii infection without inducing significant immunosuppression. MAPK inhibitors combined with either of the approved anti-Toxoplasma drugs sulfadiazine and pyrimethamine resulted in improved survival among mice challenged with a fatal T. gondii inoculum. A MAPK inhibitor also treated mice infected with the Microsporidium parasite Encephalitozoon cuniculi, suggesting that MAPK inhibitors represent a novel class of agents that may have a broad spectrum of antiparasitic activity. Preliminary studies implicate a T. gondii MAPK homologue as the target of drug action, suggesting possibilities for more-selective agents.

Inhibitors of unactivated p38 MAP kinase.

Inhibition of the p38 map kinase pathway has been shown to be beneficial in the treatment of inflammatory diseases. The first class of potent p38 kinase inhibitors was the pyridinylimidazole compounds from SKB. Since then several pyridinylimidazole-based compounds have been shown to inhibit activated p38 kinase in vitro and in vivo. We have developed a novel series of pyridinylimidazole-based compounds, which potently inhibit the p38 pathway by binding to unactivated p38 kinase and only weakly inhibiting activated p38 kinase activity in vitro.

A multiplexed proteomics approach to differentiate neurite outgrowth patterns.

We report here a method for proteomics pattern discovery by utilizing a self-organizing map approach to analyze data obtained from a novel multiplex iTRAQ proteomics method. Through the application of this technique, we were able to delineate the early molecular events preceding dorsal root ganglia neurite outgrowth induced by either nerve growth factor (NGF) or an immunophilin ligand, JNJ460. Following pattern analysis we discovered that each neurotrophic agent promoted mostly distinct increases in protein expression with few overlapping patterns. In the NGF-treated group, proteins possessing "biosynthesis function" (p < 0.002) and "ribosome localization" (p < 0.0003) were increased, while proteins promoting "organogenesis" (p < 0.004) and related "signal transduction" (p < 0.008) functions were notably increased in the JNJ460-treated group. This study suggests that the properties of neurite outgrowth triggered by NGF and JNJ460 can be distinguished at the proteome level. Multiplexed proteomics analysis, along with pattern discovery bioinformatics tools, has the capability to differentiate subtle neuroproteomics patterns.

Effects of drug combinations on smooth muscle cell proliferation: an isobolographic analysis.

Although sirolimus is a potent inhibitor of vascular smooth muscle cell (VSMC) proliferation and is effective at preventing restenosis in the majority of clinical revascularization procedures employing sirolimus-eluting stents, some VSMC may escape the antiproliferative effects of sirolimus. The present study examines the effects of combining sirolimus with other known cell cycle-specific antiproliferative agents (cladribine, topotecan or etoposide) on cultured coronary artery VSMC proliferation and utilizes a novel isobolographic approach to determine whether sirolimus/antiproliferative agent combinations produce subadditive, additive or supraadditive potentiation of antiproliferative activity. All agents were found to inhibit coronary artery VSMC proliferation in a dose-dependent manner. Cladribine was found to potentiate the antiproliferative activity of sirolimus in either an additive or supraadditive manner, depending upon the cladribine concentration. Topotecan potentiated the sirolimus antiproliferative activity by simple additivity while etoposide yielded subadditive potentiation. The present results demonstrate the utility of isobolographic analysis for identifying and optimizing antiproliferative drug combinations.

Drug-eluting stents: sirolimus and paclitaxel differentially affect cultured cells and injured arteries.

Sirolimus and paclitaxel eluted from stents inhibit cell proliferation and other cellular processes by dramatically different mechanisms. In this study, the effects of sirolimus and paclitaxel on cultured human coronary artery smooth muscle and endothelial cell function or cell cycle changes in balloon-injured arteries were directly compared. Both sirolimus and paclitaxel inhibited smooth muscle and endothelial cell proliferation. However, only paclitaxel inhibited smooth muscle and endothelial cell migration at low (nM) concentrations. Sirolimus arrested smooth muscle and endothelial cells in the G0/G1 phase of the cell cycle without inducing apoptosis while paclitaxel produced apoptosis in both cell types at low nanomolar concentrations. Although both agents blocked neointimal formation, sirolimus applied locally to injured rat carotid arteries increased the percentage of cycling vascular cells in G0/G1 without inducing apoptosis while paclitaxel increased the percentage of cycling cells in S and G2/M phases while inducing apoptosis. These results suggest that sirolimus reduces neointimal hyperplasia through a cytostatic mechanism while paclitaxel produces apoptotic cell death.