Faculty Bibliography

Autoantibodies targeting host antigens contribute to autoimmune disorders, frequently occur during and after infections and have been proposed to contribute to malaria-induced anemia. We measured anti-phosphatidylserine (PS) and anti-DNA antibody levels in 382 Ugandan children prospectively recruited in a study of severe malaria (SM). High antibody levels were defined as antibody levels greater than the mean plus 3 standard deviations of community children (CC). We observed increases in median levels of anti-PS and anti-DNA antibodies in children with SM compared to CC (p < 0.0001 for both). Children with severe malarial anemia were more likely to have high anti-PS antibodies than children with cerebral malaria (16.4% vs. 7.4%), p = 0.02. Increases in anti-PS and anti-DNA antibodies were associated with decreased hemoglobin (p < 0.05). A one-unit increase in anti-DNA antibodies was associated with a 2.99 (95% CI, 1.68, 5.31) increase odds of acute kidney injury (AKI) (p < 0.0001). Elevated anti-PS and anti-DNA antibodies were associated with post-discharge mortality (p = 0.031 and p = 0.042, respectively). Children with high anti-PS antibodies were more likely to have multiple hospital readmissions compared to children with normal anti-PS antibody levels (p < 0.05). SM is associated with increased autoantibodies against PS and DNA. Autoantibodies were associated with anemia, AKI, post-discharge mortality, and hospital readmission.

Zika virus infection is associated with the development of Guillain-Barré syndrome (GBS), a neurological autoimmune disorder caused by immune recognition of gangliosides and other components at nerve membranes. Using a high-throughput ELISA, we have analyzed the anti-glycolipid antibody profile, including gangliosides, of plasma samples from patients with Zika infections associated or not with GBS in Salvador, Brazil. We have observed that Zika patients that develop GBS present higher levels of anti-ganglioside antibodies when compared to Zika patients without GBS. We also observed that a broad repertoire of gangliosides was targeted by both IgM and IgG anti-self antibodies in these patients. Since Zika virus infects neurons, which contain membrane gangliosides, antigen presentation of these infected cells may trigger the observed autoimmune anti-ganglioside antibodies suggesting direct infection-induced autoantibodies as a cause leading to GBS development. Collectively, our results establish a link between anti-ganglioside antibodies and Zika-associated GBS in patients.

A high-throughput screening (HTS) campaign was carried out for Trypanosoma cruzi glucokinase (TcGlcK), a potential drug-target of the pathogenic protozoan parasite. Glycolysis and the pentose phosphate pathway (PPP) are important metabolic pathways for T. cruzi and the inhibition of the glucose kinases (i.e. glucokinase and hexokinase) may be a strategic approach for drug discovery. Glucose kinases phosphorylate d-glucose with co-substrate ATP to yield G6P, and moreover, the produced G6P enters both pathways for catabolism. The TcGlcK - HTS campaign revealed 25 novel enzyme inhibitors that were distributed in nine chemical classes and were discovered from a primary screen of 13,040 compounds. Thirteen of these compounds were found to have low micromolar IC₅₀ enzyme - inhibition values; strikingly, four of those compounds exhibited low toxicity towards NIH-3T3 murine host cells and notable in vitro trypanocidal activity. These compounds were of three chemical classes: (a) the 3-nitro-2-phenyl-2H-chromene scaffold, (b) the N-phenyl-benzenesulfonamide scaffold, and (c) the gossypol scaffold. Two compounds from the 3-nitro-2-phenyl-2H-chromene scaffold were determined to be hit-to-lead candidates that can proceed further down the early-stage drug discovery process.

Malaria is a highly inflammatory disease caused by Plasmodium infection of host erythrocytes. However, the parasite does not induce inflammatory cytokine responses in macrophages in vitro and the source of inflammation in patients remains unclear. Here, we identify oxidative stress, which is common in malaria, as an effective trigger of the inflammatory activation of macrophages. We observed that extracellular reactive oxygen species (ROS) produced by xanthine oxidase (XO), an enzyme upregulated during malaria, induce a strong inflammatory cytokine response in primary human monocyte-derived macrophages. In malaria patients, elevated plasma XO activity correlates with high levels of inflammatory cytokines and with the development of cerebral malaria. We found that incubation of macrophages with plasma from these patients can induce a XO-dependent inflammatory cytokine response, identifying a host factor as a trigger for inflammation in malaria. XO-produced ROS also increase the synthesis of pro-IL-1β, while the parasite activates caspase-1, providing the two necessary signals for the activation of the NLRP3 inflammasome. We propose that XO-produced ROS are a key factor for the trigger of inflammation during malaria.

Lapatinib, an approved epidermal growth factor receptor inhibitor, was explored as a starting point for the synthesis of new hits against Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Previous work culminated in 1 (NEU-1953), which was part of a series typically associated with poor aqueous solubility. In this report, we present various medicinal chemistry strategies that were used to increase the aqueous solubility and improve the physicochemical profile without sacrificing antitrypanosomal potency. To rank trypanocidal hits, a new assay (summarized in a cytocidal effective concentration (CEC₅₀)) was established, as part of the lead selection process. Increasing the sp³ carbon content of 1 resulted in 10e (0.19 μM EC₅₀ against T. brucei and 990 μM aqueous solubility). Further chemical exploration of 10e yielded 22a, a trypanocidal quinolinimine (EC₅₀: 0.013 μM; aqueous solubility: 880 μM; and CEC₅₀: 0.18 μM). Compound 22a reduced parasitemia 10⁹ fold in trypanosome-infected mice; it is an advanced lead for HAT drug development.

Macrophages can reprogram their metabolism in response to the surrounding stimuli, which affects their capacity to kill intracellular pathogens. We have investigated the metabolic and immune status of human macrophages after infection with the intracellular trypanosomatid parasites Leishmania donovani, L. amazonensis and T. cruzi and their capacity to respond to a classical polarizing stimulus (LPS and IFN-γ). We found that macrophages infected with Leishmania preferentially upregulate oxidative phosphorylation, which could be contributed by both host cell and parasite, while T. cruzi infection did not significantly increase glycolysis or oxidative phosphorylation. Leishmania and T. cruzi infect macrophages without triggering a strong inflammatory cytokine response, but infection does not prevent a potent response to LPS and IFN-γ. Infection appears to prime macrophages, since the cytokine response to activation with LPS and IFN-γ is more intense in infected macrophages compared to uninfected ones. Metabolic polarization in macrophages can influence infection and immune evasion of these parasites since preventing macrophage cytokine responses would help parasites to establish a persistent infection. However, macrophages remain responsive to classical inflammatory stimuli and could still trigger inflammatory cytokine secretion by macrophages.

Cerebral malaria (CM) is a clinical syndrome involving irreversible and lethal signs of brain injury associated to infection by parasites of the genus Plasmodium. The pathogenesis of CM derives from infection-induced proinflammatory cytokines associated with cytoadherence of parasitized red blood cells to brain microvasculature. Glycoconjugates are very abundant in the surface of Plasmodium spp., and are critical mediators of parasite virulence in host-pathogen interactions. Herein, we show that 6-Diazo-5-oxo-L-norleucine (DON) therapeutically used for blocking hexosamine biosynthetic pathway leads to recovery in experimental murine cerebral malaria. DON-induced protection was associated with decreased parasitism, which severely reduced Plasmodium transmission to mosquitoes. These findings point to a potential use of DON in combination therapies against malaria.

Human African trypanosomiasis, Chagas disease, and leishmaniasis are human infections caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania. Besides their severity and global impact, treatments are still challenging. Currently available drugs have important limitations, highlighting the urgent need to develop new drugs. Phosphoglucose isomerase (PGI) is considered a promising target for the development of antiparasitic drugs, as it acts on two essential metabolic pathways, glycolysis and gluconeogenesis. Herein, we describe the identification of new nonphosphorylated inhibitors of Leishmania mexicana PGI ( LmPGI), with the potential for the development of antiparasitic drugs. A fluorescence-based high-throughput screening (HTS) assay was developed by coupling the activities of recombinant LmPGI with glucose-6-phosphate dehydrogenase and diaphorase. This coupled assay was used to screen 42,720 compounds from ChemBridge and TimTec commercial libraries. After confirmatory assays, selected LmPGI inhibitors were tested against homologous Trypanosoma cruzi and humans. The PGI hits are effective against trypanosomatid PGIs, with IC₅₀ values in the micromolar range, and also against the human homologous enzyme. A computational analysis of cavities present on PGI's crystallographic structure suggests a potential binding site for the proposed mixed-type inhibition mechanism.

We recently reported the medicinal chemistry re-optimization of a series of compounds derived from the human tyrosine kinase inhibitor, lapatinib, for activity against Plasmodium falciparum. From this same library of compounds, we now report potent compounds against Trypanosoma brucei brucei (which causes human African trypanosomiasis), T. cruzi (the pathogen that causes Chagas disease), and Leishmania spp. (which cause leishmaniasis). In addition, sub-micromolar compounds were identified that inhibit proliferation of the parasites that cause African animal trypanosomiasis, T. congolense and T. vivax. We have found that this set of compounds display acceptable physicochemical properties and represent progress towards identification of lead compounds to combat several neglected tropical diseases.

Discovery of new chemotherapeutic lead agents can be accelerated by optimizing chemotypes proven to be effective in other diseases to act against parasites. One such medicinal chemistry campaign has focused on optimizing the anilinoquinazoline drug lapatinib (1) and the alkynyl thieno[3,2-d]pyrimidine hit GW837016X (NEU-391, 3) into leads for antitrypanosome drugs. We now report the structure-activity relationship studies of 3 and its analogs against Trypanosoma brucei, which causes human African trypanosomiasis (HAT). The series was also tested against Trypanosoma cruzi, Leishmania major, and Plasmodium falciparum. In each case, potent antiparasitic hits with acceptable toxicity margins over mammalian HepG2 and NIH3T3 cell lines were identified. In a mouse model of HAT, 3 extended life of treated mice by 50%, compared to untreated controls. At the cellular level, 3 inhibited mitosis and cytokinesis in T. brucei. Thus, the alkynylthieno[3,2-d]pyrimidine chemotype is an advanced hit worthy of further optimization as a potential chemotherapeutic agent for HAT.