Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Here we report the generation of a HS mutant mouse lung endothelial cell library by systematic deletion of HS genes expressing in the cell. We applied this library to answer several fundamental questions about HS biology including: 1) determining that strictly defined fine structure of HS, not its overall sulfation degree, is more important for FGF2-FGFR1 signaling; 2) defining the epitope features of commonly used anti-HS phage display antibodies; and 3) delineating the fine inter-regulation networks of HS modification and chain length by HS genes in mammalian cells and at a cell type specific level. Our mutant cell library will enable robust and systematic interrogation of the roles and related structures of HS in a cellular context.

The effect of a series of 2-alkylaminoethyl-1,1-bisphosphonic acids against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii has been studied. Most of these drugs exhibited an extremely potent inhibitory action against the intracellular form of T. cruzi, exhibiting IC 50 values at the low micromolar level. This cellular activity was associated with a strong inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas' disease chemotherapy. Compound 17 was an effective agent against amastigotes exhibiting an IC 50 value of 0.84 μM, while this compound showed an IC 50 value of 0.49 μM against the target enzyme TcFPPS. Interestingly, compound 19 was very effective against both T. cruzi and T. gondii exhibiting IC 50 values of 4.1 μM and 2.6 μM, respectively. In this case, 19 inhibited in at least two different enzymes of T. cruzi (TcFPPS and solanesyl diphosphate synthase (TcSPPS); 1.01 μM and 0.25 μM, respectively), while it inhibited TgFPPS in T. gondii. In general, this family of drugs was less effective against the activity of T. cruzi SPPS and against T. gondii growth in vitro. As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their potential low toxicity makes them good candidates to control tropical diseases.

As part of our efforts aimed at searching for new antiparasitic agents, 2-alkylmercaptoethyl-1,1-bisphosphonate derivatives were synthesized and evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease, and Toxoplasma gondii, the responsible agent for toxoplasmosis. Many of these sulfur-containing bisphosphonates were potent inhibitors against the intracellular form of T. cruzi, the clinically more relevant replicative form of this parasite, and tachyzoites of T. gondii targeting T. cruzi or T. gondii farnesyl diphosphate synthases (FPPSs), which constitute valid targets for the chemotherapy of these parasitic diseases. Interestingly, long chain length sulfur-containing bisphosphonates emerged as relevant antiparasitic agents. Taking compounds 37, 38, and 39 as representative members of this class of drugs, they exhibited ED50 values of 15.8 μM, 12.8 μM, and 22.4 μM, respectively, against amastigotes of T. cruzi. These cellular activities matched the inhibition of the enzymatic activity of the target enzyme (TcFPPS) having IC50 values of 6.4 μM, 1.7 μM, and 0.097 μM, respectively. In addition, these compounds were potent anti-Toxoplasma agents. They had ED50 values of 2.6 μM, 1.2 μM, and 1.8 μM, respectively, against T. gondii tachyzoites, while they exhibited a very potent inhibitory action against the target enzyme (TgFPPS) showing IC50 values of 0.024 μM, 0.025 μM, and 0.021 μM, respectively. Bisphosphonates bearing a sulfoxide unit at C-3 were also potent anti-Toxoplasma agents, particularly those bearing long aliphatic chains such as 43–45, which were also potent antiproliferative drugs against tachyzoites of T. gondii. These compounds inhibited the enzymatic activity of the target enzyme (TgFPPS) at the very low nanomolar range. These bisphosphonic acids have very good prospective not only as lead drugs but also as potential chemotherapeutic agents.

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

The effect of long chain 2-alkylaminoethyl-1,1-bisphosphonates against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii was investigated. Particularly, compound 26 proved to be an extremely potent inhibitor against the intracellular form of T. cruzi, exhibiting IC 50 values at the nanomolar range. This cellular activity was associated with a strong inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas' disease chemotherapy. Compound 26 was an effective agent against T. cruzi (amastigotes) exhibiting an IC 50 value of 0.67 μM, while this compound showed an IC 50 value of 0.81 μM against the target enzyme TcFPPS. This drug was less effective against the enzymatic activity of T. cruzi solanesyl diphosphate synthase TcSPPS showing an IC 50 value of 3.2 μM. Interestingly, compound 26 was also very effective against T. gondii (tachyzoites) exhibiting IC 50 values of 6.23 μM. This cellular activity was also related to the inhibition of the enzymatic activity towards the target enzyme TgFPPS (IC 50 = 0.29 μM) As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their potential low toxicity makes them good candidates to control different tropical diseases.The hemoflagellated protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease or American trypanosomiasis, which is an endemic disease widespread from southern United States to southern Argentina. It has been estimated that close to 18 million people are infected and over 40 million are at risk of infection by T. cruzi. 1 The central nervous system is the most frequently affected site in AIDS patients, with meningoencephalitis occurring approximately in 75% of cases. The next normally affected organ is the heart with myocarditis.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBioorg Med Chem. Author manuscript; available in PMC 2012 April 1. In addition, since people migrate from endemic areas, the possibility for cases in developed nations will also escalate. Chemotherapy for Chagas' disease still remains unsatisfactory due to limited efficacy and common side effects of the currently available drugs such as nifurtimox (1), now discontinued, and benznidazole (2), which present toxicity associated with their continued use. [2][3][4] The parasite has a complex life cycle involving blood-sucking Reduv...