Clofazimine for Treatment of Cryptosporidiosis in Human Immunodeficiency Virus Infected Adults: An Experimental Medicine, Randomized, Double-blind, Placebo-controlled Phase 2a Trial.

BACKGROUND: We evaluated the efficacy, pharmacokinetics (PK), and safety of clofazimine (CFZ) in patients living with human immunodeficiency virus (HIV) with cryptosporidiosis. METHODS: We performed a randomized, double-blind, placebo-controlled study. Primary outcomes in part A were reduction in Cryptosporidium shedding, safety, and PK. Primary analysis was according to protocol (ATP). Part B of the study compared CFZ PK in matched individuals living with HIV without cryptosporidiosis. RESULTS: Twenty part A and 10 part B participants completed the study ATP. Almost all part A participants had high viral loads and low CD4 counts, consistent with failure of antiretroviral (ARV) therapy. At study entry, the part A CFZ group had higher Cryptosporidium shedding, total stool weight, and more diarrheal episodes compared with the placebo group. Over the inpatient period, compared with those who received placebo, the CFZ group Cryptosporidium shedding increased by 2.17 log2 Cryptosporidium per gram stool (95% upper confidence limit, 3.82), total stool weight decreased by 45.3 g (P = .37), and number of diarrheal episodes increased by 2.32 (P = .87). The most frequent solicited adverse effects were diarrhea, abdominal pain, and malaise. One placebo and 3 CFZ participants died during the study. Plasma levels of CFZ in participants with cryptosporidiosis were 2-fold lower than in part B controls. CONCLUSIONS: Our findings do not support the efficacy of CFZ for the treatment of cryptosporidiosis in a severely immunocompromised HIV population. However, this trial demonstrates a pathway to assess the therapeutic potential of drugs for cryptosporidiosis treatment. Screening persons living with HIV for diarrhea, and especially Cryptosporidium infection, may identify those failing ARV therapy. CLINICAL TRIALS REGISTRATION: NCT03341767.

Invasion of hepatocytes by Plasmodium sporozoites requires cGMP-dependent protein kinase and calcium dependent protein kinase 4.

Invasion of hepatocytes by sporozoites is essential for Plasmodium to initiate infection of the mammalian host. The parasite's subsequent intracellular differentiation in the liver is the first developmental step of its mammalian cycle. Despite their biological significance, surprisingly little is known of the signalling pathways required for sporozoite invasion. We report that sporozoite invasion of hepatocytes requires signalling through two second-messengers - cGMP mediated by the parasite's cGMP-dependent protein kinase (PKG), and Ca2+ , mediated by the parasite's calcium-dependent protein kinase 4 (CDPK4). Sporozoites expressing a mutated form of Plasmodium berghei PKG or carrying a deletion of the CDPK4 gene are defective in invasion of hepatocytes. Using specific and potent inhibitors of Plasmodium PKG and CDPK4, we demonstrate that PKG and CDPK4 are required for sporozoite motility, and that PKG regulates the secretion of TRAP, an adhesin that is essential for motility. Chemical inhibition of PKG decreases parasite egress from hepatocytes by inhibiting either the formation or release of merosomes. In contrast, genetic inhibition of CDPK4 does not significantly decrease the number of merosomes. By revealing the requirement for PKG and CDPK4 in Plasmodium sporozoite invasion, our work enables a better understanding of kinase pathways that act in different Plasmodium stages.

Structure of aldose reductase from Giardia lamblia.

Giardia lamblia is an anaerobic aerotolerant eukaryotic parasite of the intestines. It is believed to have diverged early from eukarya during evolution and is thus lacking in many of the typical eukaryotic organelles and biochemical pathways. Most conspicuously, mitochondria and the associated machinery of oxidative phosphorylation are absent; instead, energy is derived from substrate-level phosphorylation. Here, the 1.75 Å resolution crystal structure of G. lamblia aldose reductase heterologously expressed in Escherichia coli is reported. As in other oxidoreductases, G. lamblia aldose reductase adopts a TIM-barrel conformation with the NADP(+)-binding site located within the eight ß-strands of the interior.

Structure of nitrilotriacetate monooxygenase component B from Mycobacterium thermoresistibile.

Mycobacterium tuberculosis belongs to a large family of soil bacteria which can degrade a remarkably broad range of organic compounds and utilize them as carbon, nitrogen and energy sources. It has been proposed that a variety of mycobacteria can subsist on alternative carbon sources during latency within an infected human host, with the help of enzymes such as nitrilotriacetate monooxygenase (NTA-Mo). NTA-Mo is a member of a class of enzymes which consist of two components: A and B. While component A has monooxygenase activity and is responsible for the oxidation of the substrate, component B consumes cofactor to generate reduced flavin mononucleotide, which is required for component A activity. NTA-MoB from M. thermoresistibile, a rare but infectious close relative of M. tuberculosis which can thrive at elevated temperatures, has been expressed, purified and crystallized. The 1.6 Å resolution crystal structure of component B of NTA-Mo presented here is one of the first crystal structures determined from the organism M. thermoresistibile. The NTA-MoB crystal structure reveals a homodimer with the characteristic split-barrel motif typical of flavin reductases. Surprisingly, NTA-MoB from M. thermoresistibile contains a C-terminal tail that is highly conserved among mycobacterial orthologs and resides in the active site of the other protomer. Based on the structure, the C-terminal tail may modulate NTA-MoB activity in mycobacteria by blocking the binding of flavins and NADH.

Fl-160. A surface antigen of Trypanosoma cruzi that mimics mammalian nervous tissue.

Chagas' disease, caused by Trypanosoma cruzi, is an excellent model for autoimmune disease induced by an infectious agent. Transfer of T cells, directed against crossreactive antigens of T. cruzi and nervous tissue, have been shown to reproduce pathology found in chronic Chagas' disease. We used recombinant DNA technology to characterize one of these crossreactive antigens (Fl-160). We have cloned DNA from T. cruzi, which expresses a protein corresponding to a 160-kD protein found on the surface of the trypanosome, overlying the flagellum. This clone hybridizes to a 4.5-kb poly(A)+ RNA that is distributed in a differentiation-specific manner, suggesting expression of this protein is transcriptionally controlled. Antibodies to this protein crossreact with a 48-kD mammalian nervous tissue protein found in sciatic nerve, brain, and myenteric plexi of gut. The myenteric plexi are destroyed by inflammatory infiltrates in Chagas' disease, leading to the characteristic megaesophagus and megacolon Chagas' disease pathology. Thus, this antigen is a candidate antigen for autoimmune mimicry leading to nervous tissue pathology.

The major 85-kD surface antigen of the mammalian form of Trypanosoma cruzi is encoded by a large heterogeneous family of simultaneously expressed genes.

Trypanosoma cruzi is an obligate intracellular protozoan parasite. The parasite mammalian stage surface antigens exhibit extensive antigenic diversity. We have characterized a family of T. cruzi genes that code for a polymorphic set of 85-kD surface antigens, the SA85-1 antigens. The family contains greater than 100 genes and pseudogenes, of which a minimum of nine are transcribed. The gene family is expressed in the mammalian stage only. A subset of the gene family is present in two telomere-linked copies in the genome. Telomere linkage of other expressed SA85-1 genes has not been demonstrated. We have shown that at least three members of the SA85-1 gene family encode antigens at the surface of the mammalian stage of the parasite. Interestingly, these three antigens are expressed on all the trypanosomes examined. This suggests that T. cruzi simultaneously expresses a large repertoire of similar, but diverse antigens at its surface. Thus, T. cruzi exhibits extensive antigenic diversity in a system unique from that of African trypanosomes, perhaps reflecting its intracellular niche.

FL-160 proteins of Trypanosoma cruzi are expressed from a multigene family and contain two distinct epitopes that mimic nervous tissues.

The partial sequence of a gene encoding the COOH terminus of a protein of apparent molecular weight of 160 kD associated with the flagellum of trypomastigotes of Trypanosoma cruzi (FL-160 now renamed to FL-160-1) has been previously reported. The COOH terminus of FL-160-1 has an epitope, defined by 12 amino acids, which molecularly miMics a nervous tissue antigen of 48 kD found in myenteric plexus, sciatic nerve, and a subset of cells in the central nervous system. We now report that FL-160 is a family of highly related genes. The sequence has been determined for the entire open reading frame (ORF) of one of the members of the FL-160 gene family (FL-160-2) and three other partial ORFs. Sequence analysis reveals the various members of the FL-160 gene family to be approximately 80% homologous in the predicted amino acid sequence, but all retain the 12-amino acid molecular mimicry epitope on the COOH terminus. Comparison of the sequence of FL-160-2 to other sequences demonstrates amino acid homology to bacterial sialidase (27%), members of the SA85 gene family (25-30%) and the shed acute-phase antigen/neuraminidase/trans-sialidase gene family (25-30%). Quantitative hybridization at high stringency suggests 750 copies of FL-160 are present in the DNA of each parasite. Reverse transcription and sequence analysis demonstrates that at least five of the members of the FL-160 gene family are transcribed. The NH2 terminus of one of the FL-160 gene products was expressed and antibodies prepared. Antibodies directed to either the COOH or the NH2 terminus of FL-160 bind a 160-kD T. cruzi protein. Both antibodies bind the surface membrane in the flagellar pocket of the trypomastigote. Antibodies to the NH2 terminus bind epineurium and scattered linear densities in sciatic nerve in a pattern distinct from the pattern with antibodies to the COOH terminus. Thus, there are at least two distinct molecular mimicry epitopes on the FL-160 molecule and both mimic epitopes found in nervous tissues. FL-160 may be involved in the generation of autoimmunity to nervous tissues by molecular mimicry, observed in chronic Chagas' disease.

The cutaneous infiltrates of leprosy. A transmission electron microscopy study.

The dermal lesions of 18 patients with leprosy have been examined by transmission electron microscopy. The patients exhibited a spectrum of disease from polar lepromatous to polar tuberculoid with intermediate stages in various states of therapy and relapse. The nature and quantities of inflammatory cells and bacteria have been determined by electron microscopy to supplement previous light and fluorescence microscopy studies. Lepromatous leprosy was characterized by many parasitized foam cells containing large, multibacillary vacuoles with intact, osmiophilic Mycobacterium leprae: Bacteria were embedded in an electron-lucent matrix. No extracellular bacteria were evident. Only small numbers of scattered lymphocytes were found. As one approached the borderline state, smaller numbers of bacilli were present as singlets and doublets in small vacuoles of macrophages. The more reactive forms showed increasing bacillary fragmentation, larger numbers of lymphoid cells, and an occasional epithelioid cell. At the tuberculoid end of the spectrum, clear evidence of an exuberant lymphocyte response was evident. Large numbers of T cells with extremely long and complex filipodia were closely associated with epithelioid and multinucleated giant cells. Many of the mononuclear phagocytes appeared nonviable, and areas of necrosis were evident. Bacillary remnants were scarce and the cytoplasm of the epithelioid cells contained occasional dense bodies and many stacks of endoplasmic reticulum and mitochondria. These results suggest that Leu 3a/OKT4 helper cells may be capable of driving the effector function of mononuclear phagocytes. This would lead to a significant microbicidal effect on M. leprae, perhaps through the production of toxic oxygen intermediates.

Human dendritic cells. Enrichment and characterization from peripheral blood.

Previous studies demonstrated that lymphoid tissues of mice and rats contain small numbers (less than 1 percent of nucleated cells) of dendritic cells (DC) with special cytologic, surface, and functional properties. We show here that similar DC represent 0.1-0.5 percent of human peripheral blood mononuclear cells. DC can be enriched to 20-60 percent purity by a multistep procedure analogous to that used in mice. Adherent peripheral blood mononuclear cells are cultured overnight, and the released cells are depleted of monocytes and B cells by readherence to plastic, rosetting with erythrocytes coated with anti-human IgG, and centrifugation in dense albumin columns. Enriched DC have similar cytologic features to rodent DC by light and electron microscopy. DC express HLA, and HLA-DR and the leukocyte-common antigens. They lack phagocytic capacity, receptors for antibody-coated and neuraminidase-treated erythrocytes, surface and intracellular Ig, esterase, peroxidase, and azurophilic granules. DC do not react with several monoclonal antibodies directed to phagocytes (OKM 1, "mac-1," 63D3, and 61D3) and T cells (OKT 3, 6, 8). Unlike the mouse, human DC express complement receptors. When maintained in culture for 4 d, human DC did not give rise to either B cells or monocytes. Therefore, DC identified by cytologic criteria are distinct from other leukocytes. Enriched populations of DC have been compared to fractions enriched in monocytes, B cells, and T cells in three functional assays: stimulation of the primary allogeneic mixed leukocyte reaction, stimulation of the primary syngeneic MLR, and accessory function for the proliferation of periodate- modified T cells. In each case, the DC fraction was 10-fold or more active than other cell fractions. We conclude that DC circulate in man, and represent the principal cell type required for the initiation of several immune responses.

Specific antimononuclear phagocyte monoclonal antibodies. Application to the purification of dendritic cells and the tissue localization of macrophages.

3C10 and 1D9 are two related monoclonal antibodies that specifically identify human mononuclear phagocytes in a large number of sites, including blood monocytes, alveolar macrophages, and macrophages in tissue sections of spleen, lymph node, and skin. The antigen persists on monocytes cultured for greater than 4 wk, but it is not found on giant cells. The 3C10-1D9 determinant is carried by a 55 kD polypeptide, is expressed at approximately 40,000 copies per monocyte, and is protease sensitive. The antigen is clearly different from HLA-class II or Ia-like antigens that have been studied with a new monoclonal 9.3F10. The 9.3F10 antigen is found on B cells, dendritic cells and monocytes; is protease resistant, and occurs on a 33-29 kD doublet typical of class II products. The 3C10 monoclonal provides a clear distinction between human mononuclear phagocytes and dendritic cells. First, monocytes and lymphocytes can be eliminated from plastic-adherent mononuclear cells using 3C10, complement, and two previously described cytotoxic antibodies, BA-1 (anti-B cell) and Leu-1 (anti-T cell). As a result, the trace dendritic cell component of blood can be enriched to considerable purity (65-75%) and yield. Second, immunocytochemical staining of tissue sections reveals that 3C10+ macrophages are anatomically segregated from dendritic cells. Large numbers of 3C10+ cells are found in red pulp of spleen and in regions surrounding lymphatic channels of lymph node. However, 3C10+ macrophages are scarce in white pulp of spleen and the lymphocyte-rich cortex of node that are the sites where dendritic cells are localized. 3C10+ cells in skin are found in the dermis, particularly in leprosy infiltrates, but the Langerhans' cells of epidermis are 3C10-. The distinctive localization of macrophages and dendritic cells is consistent with their respective functions as effector and accessory cells in the immune response.

Relative efficacy of human monocytes and dendritic cells as accessory cells for T cell replication.

Monocyte-specific monoclonal antibodies (7) were used to compare the efficacy of monocytes and dendritic cells as accessory or stimulator cells for human T cell replication. Both unfractionated and plastic-adherent mononuclear cells were first treated with a cytolytic antimonocyte antibody that kills greater than 95% of monocytes but not dendritic cells. When tested as stimulators of the mixed leukocyte reaction (MLR) and of oxidative mitogenesis (the proliferation of T cells modified with sodium periodate), the monocyte-depleted cells had normal or enhanced stimulatory capacity. Monocyte-depleted mononuclear cells also proliferated normally to soluble antigens (Candida albicans, tetanus toxoid), even under limiting conditions of cell dose, antigen dose, and culture time. Adherent blood mononuclear cells were next separated into monocyte-enriched and -depleted components using fluoresceinated antimonocyte antibody and the cell sorter. The depleted fraction (less than 2% monocytes by esterase staining and by cytology) contained the dendritic cells and exhibited at least 75% of the accessory activity. The monocyte-rich fraction (approximately 97% esterase positive) stimulated the MLR and oxidative mitogenesis weakly, and was comparable in potency to nonadherent cells. Cell-specific antibodies and complement were also used to prepare dendritic cells that were thoroughly depleted of monocytes and lymphocytes. The dendritic cells (70-80% pure) were potent stimulators of the allogeneic MLR, syngeneic MLR, and tetanus toxoid response, being active at stimulator to responder ratios of 1:100 or less. Taken together with previous studies (1, 2), these experiments indicate that the dendritic cell is the major stimulator of T cell replication in man. The contribution of class II products of the major histocompatibility complex (7) was then evaluated with a new monoclonal, 9.3F10. Accessory function was dramatically inhibited if cells bearing class II antigens were killed with 9.3F10 and complement, or if class II molecules were blocked by the addition of 9.3F10 Fab to the culture medium. The expression of 9.3F10 class II products was therefore studied on purified monocytes and dendritic cells. Most if not all cells in both populations reacted with 9.3F10, and each population exhibited approximately 150,000 125I-Fab 9.3F10 binding sites per cell. Since Ia+ dendritic cells are active accessory cells, but Ia+ monocytes are not, class II products are necessary but not sufficient for the stimulation of T cell proliferation in man.

Treponema pallidum major sheath protein homologue Tpr K is a target of opsonic antibody and the protective immune response.

We have identified a family of genes that code for targets for opsonic antibody and protective immunity in T. pallidum subspecies pallidum using two different approaches, subtraction hybridization and differential immunologic screening of a T. pallidum genomic library. Both approaches led to the identification of a polymorphic multicopy gene family with predicted amino acid homology to the major sheath protein of Treponema denticola. One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium. Reverse transcription polymerase chain reaction analysis of T. pallidum reveals that Tpr K is preferentially transcribed in the Nichols strain of T. pallidum. Antibodies directed to purified recombinant variable domain of Tpr K can opsonize T. pallidum, Nichols strain, for phagocytosis, supporting the hypothesis that this portion of the protein is exposed at the surface of the treponeme. Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum. This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.

Virulence in Trypanosoma cruzi infection correlates with the expression of a distinct family of sialidase superfamily genes.

The overall success of Trypanosoma cruzi depends on its ability to invade the host and establish a long-term infection. Little is known of the genetic factors responsible for observed differences in virulence from strain to strain in T. cruzi. A virulent T. cruzi line was derived from an attenuated parental line by two passages through mice. To identify virulence genes a subtraction library was constructed and screened for cDNA expressed exclusively in the virulent line. One cDNA hybridized to 3.5 and 4.5 Kb RNA present in virulent trypomastigotes but absent in attenuated trypomastigotes. Sequence analysis showed the cDNA to encode an 85 kDa protein with homology to members of the sialidase/trans-sialidase superfamily and has been designated vp85.1. The highest amino acid sequence similarity was to a previously described T. cruzi sialidase-homologue pseudogene [Takle, G.B., O'Conner, J., Young, A.J. and Cross, G.A.M. (1992) Mol. Biochem. Parasitol. 56, 117-128]. The vp85.1 amino acid sequence has higher homology to members of the 160 kDa flagellar-associated antigen family, FL-160, than to other 85 kDa expressed sialidase superfamily members. Southern blot analysis of virulent and attenuated lines demonstrated a complex hybridization pattern consistent with a multiple gene copy family that was identical in both lines. Antibody directed against recombinant vp85.1 peptide recognized proteins between 95 and 115 kDa in total virulent parasite lysates which were absent in attenuated lysates. Peptide N-glycosidase F treatment reduced the high molecular weight bands to 85 kDa, indicating vp85 is an N-linked glycoprotein. Immunofluorescence with anti-vp85.1 demonstrated surface localization of vp85.1 on virulent, but not attenuated, trypomastigotes. We postulate this new subfamily of trans-sialidases may play a role in virulence.

Expression of Trypanosoma cruzi surface antigen FL-160 is controlled by elements in the 3' untranslated, the 3' intergenic, and the coding regions.

The FL-160 surface antigen gene family of T. cruzi consists of hundreds of members of 160 kDa glycoproteins expressed in trypomastigotes, but not in epimastigotes. Steady-state levels of FL-160 mRNA were 80 to 100-fold higher in trypomastigotes than in epimastigotes, yet transcription rates were equivalent between the lifecycle stages. Luciferase reporter constructs demonstrated that the 3' untranslated region (UTR) and intergenic region (IR) following the coding sequence of FL-160 was sufficient to generate 8-fold higher luciferase expression in trypomastigotes compared with epimastigotes. Transfection of 3' UTR/IR deletion constructs revealed cis-acting elements which conferred a trypomastigote-specific expression pattern similar to that of FL-160. Parasites treated with translation and transcription inhibitors, cyclohexamide and Actinomycin D, respectively, displayed a stage-specific pattern of FL-160 mRNA degradation. Epimastigotes, but not trypomastigotes, treated with the inhibitors accumulated a 1.4 Kb FL-160 cleavage product. The cleavage site mapped to a 31 base poly-purine tract in the FL-160 coding region. The first 526 aa of FL-160, containing the 31 base poly-purine tract and several smaller tracts, were fused to green fluorescent protein (GFP) and expressed from the T. cruzi tubulin locus. Stable transformants expressed 4-fold more FL-160:GFP fusion mRNA and 12-fold more fusion protein in the trypomastigote stage than in the epimastigote stage suggesting post-transcriptional and translational control elements. These data reveal at least two distinct control mechanisms for trypomastigote-specific expression of FL-160 surface glycoproteins, one involving the 3' UTR/IR and one involving the coding region of FL-160.

Expression of mammalian cytokines by Trypanosoma cruzi indicates unique signal sequence requirements and processing.

A vector based upon the calmodulin-ubiquitin 2.65 locus of Trypanosoma cruzi has enabled the expression and secretion of the murine cytokines interleukin-2 (IL-2) and gamma-interferon (gamma-IFN) by transfected T. cruzi. The T. cruzi-derived cytokines were bioactive and produced by both epimastigotes and mammalian forms. The native coding sequence of IL-2 was sufficient to cause secretion of the protein, but the gamma-IFN signal sequence had to be replaced by the IL-2 signal sequence (IL-2/gamma-IFN) to allow efficient secretion of gamma-IFN. The amino acid sequences at the N-termini of the secreted T. cruzi-derived cytokines were different from the expected murine secreted protein. The secreted IL-2 was cleaved six amino acids downstream from the murine signal sequence cleavage site, and the hybrid IL-2/gamma-IFN molecule was cleaved three amino acids downstream from the predicted signal cleavage site in the IL-2/gamma-IFN molecule. These apparent differences in signal peptide sequence requirements and cleavage sites most likely indicate that the signal sequence processing in trypanosomes is distinct from that of higher eukaryotes.

The effects of protein farnesyltransferase inhibitors on trypanosomatids: inhibition of protein farnesylation and cell growth.

Attachment of the prenyl groups farnesyl and geranylgeranyl to specific eukaryotic cell proteins by protein prenyltransferases is required for the functioning of a number of cellular processes including signal transduction. In this study it was found that previously reported inhibitors of mammalian protein farnesyltransferase (PFT) [those that mimic the substrate farnesyl pyrophosphate and those that mimic the protein acceptor of the farnesyl group (CaaX mimetic)] inhibit in vitro farnesylation catalyzed by partially purified Trypanosoma brucei (T. brucei) PFT. The most potent PFT inhibitors at concentrations of 3-10 microM inhibit the growth of insect (procyclic) and bloodstream forms of T. brucei. One of the PFT inhibitors was found to block the incorporation of radiolabeled mevalonic acid (the precursor of prenyl groups) into specific T. brucei proteins. This study also shows that protein prenylation occurs in the protozoan parasites Trypanosoma cruzi (T. cruzi) and Leishmania mexicana (L. mexicana). The growth of T. cruzi intracellular form (amastigote) is also sensitive to PFT inhibitors, whereas the insect form (epimastigote) is considerably more resistant to inhibition of protein farnesylation. On the other hand, growth of 3T3 fibroblast cells (host cells for amastigote growth) was not affected by up to 100 microM PFT inhibitors. The growth of L. mexicana insect form (promastigote) is modestly inhibited by protein farnesyltransferase inhibitors. These results suggest the potential for the development of PFT inhibitors for treating trypanosomiasis and leishmaniasis.

Adenosine analogues as inhibitors of Trypanosoma brucei phosphoglycerate kinase: elucidation of a novel binding mode for a 2-amino-N(6)-substituted adenosine.

As part of a project aimed at structure-based design of adenosine analogues as drugs against African trypanosomiasis, N(6)-, 2-amino-N(6)-, and N(2)-substituted adenosine analogues were synthesized and tested to establish structure-activity relationships for inhibiting Trypanosoma brucei glycosomal phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycerol-3-phosphate dehydrogenase (GPDH). Evaluation of X-ray structures of parasite PGK, GAPDH, and GPDH complexed with their adenosyl-bearing substrates led us to generate a series of adenosine analogues which would target all three enzymes simultaneously. There was a modest preference by PGK for N(6)-substituted analogues bearing the 2-amino group. The best compound in this series, 2-amino-N(6)- [2''(p-hydroxyphenyl)ethyl]adenosine (46b), displayed a 23-fold improvement over adenosine with an IC(50) of 130 microM. 2-[[2''-(p-Hydroxyphenyl)ethyl]amino]adenosine (46c) was a weak inhibitor of T. brucei PGK with an IC(50) of 500 microM. To explore the potential of an additive effect that having the N(6) and N(2) substitutions in one molecule might provide, the best ligands from the two series were incorporated into N(6),N(2)-disubstituted adenosine analogues to yield N(6)-(2''-phenylethyl)-2-[(2'' -phenylethyl)amino]adenosine (69) as a 30 microM inhibitor of T. brucei PGK which is 100-fold more potent than the adenosine template. In contrast, these series gave no compounds that inhibited parasitic GAPDH or GPDH more than 10-20% when tested at 1.0 mM. A 3.0 A X-ray structure of a T. brucei PGK/46b complex revealed a binding mode in which the nucleoside analogue was flipped and the ribosyl moiety adopted a syn conformation as compared with the previously determined binding mode of ADP. Molecular docking experiments using QXP and SAS program suites reproduced this "flipped and rotated" binding mode.

Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of Trypanosomatidae via structure-based drug design.

In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH's provided details of how the adenosyl moiety of NAD(+) interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH's. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N(6)-(1-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (6e), N(6)-(substituted-naphthalenemethyl)-2'-deoxy-2'-(substituted-benzamido)adenosine analogues were investigated. N(6)-(1-Naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (6m), N(6)-[1-(3-hydroxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (7m), N(6)-[1-(3-methoxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (9m), N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (11e), and N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2- to 3-fold improvement over 6e and a 7100- to 25000-fold improvement over the adenosine template. IC(50)'s of these compounds were in the range 2-12 microM for T. brucei, T. cruzi, and L. mexicana GAPDH's, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure-activity relationships of this class of compounds, a library of 240 N(6)-(substituted)-2'-deoxy-2'-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N(6) and C2' substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH's in the low micromolar range. We also explored adenosine analogues containing 5'-amido substituents and found that 2',5'-dideoxy-2'-(3,5-dimethoxybenzamido)-5'-(diphenylacetamido)adenosine (49) displays an IC(50) of 60-100 microM against the three parasite GAPDH's.

Therapy and prophylaxis of systemic protozoan infections.

This article summarises current therapy and prophylaxis for Pneumocystis carinii, Toxoplasma gondii, Leishmania species, African trypanosomes (Trypanosoma brucei gambiense and T. b. rhodesiense), and American trypanosome (Trypanosoma cruzi) infections. Each agent and the disease it causes is briefly reviewed, and current data on the structure, mode of action, indications for treatment, dosage, administration, duration of therapy, efficacy, toxicity, and necessary monitoring during therapy are discussed for each drug. Drugs considered include cotrimoxazole (trimethoprim + sulfamethoxazole), pentamidine, dapsone (diaphenylsulfone), trimetrexate, eflornithine (DFMO), and primaquine/clindamycin and pyrimethamine/sulphonamide combinations for Pneumocystis pneumonia; pyrimethamine/sulfadiazine, spiramycin, and clindamycin for toxoplasmosis; pentavalent antimonials ('Pentostam' and 'Glucantime'), pentamidine, amphotericin B, allopurinol, ketoconazole, and itraconazole for leishmaniasis; suramin, pentamidine, melarsoprol, tryparsamide, Mel W, berenil, and eflornithine (DFMO) for African trypanosomiasis; and nifurtimox, benznidazole and gentian violet for American trypanosomiasis.

Humoral and cellular immune response of adults from northeastern Brazil with chronic Trypanosoma cruzi infection: depressed cellular immune response to T. cruzi antigen among Chagas' disease patients with symptomatic versus indeterminate infection.

Infection with Trypanosoma cruzi can cause chronic Chagas' disease manifestations (cardiac, gastrointestinal), although most persons with chronic infection have no ill effects (indeterminate form). Cell-mediated immunity (CMI) responses are believed to be intrinsically important in the containment of T. cruzi and in the pathogenesis of Chagas' disease. Humoral and CMI responses were investigated in 70 T. cruzi-infected persons from an endemic area in northeastern Brazil and in 30 uninfected controls. An epidemiologic survey, physical examination, and blood evaluation were conducted for each subject. The 70 chronically infected persons were subclassified into three clinical groups: indeterminate, cardiac, and gastrointestinal. Serum was tested for antibodies to T. cruzi by hemagglutination assay, indirect immunofluorescent assay, and enzyme-linked immunosorbent assay, and for autoantibodies to tubulin. Serum levels of soluble interleukin-2 receptor (sIL-2R), albumin, and C-reactive protein (CRP) were also measured to assess one parameter each of immunosuppression, nutritional status, and inflammation. The proliferative response of peripheral blood mononuclear cells (PBMC) to T. cruzi antigens, mitogen (phytohemagglutinin), and antigen-free controls was also assessed. Our data did not reveal any significant differences in serum levels of antibodies to T. cruzi, antibodies to tubulin, albumin, CRP, or sIL-2R among the subgroups of infected individuals. The data demonstrate differences in CMI responses. Trypanosoma cruzi trypomastigote lysate stimulated proliferation of PBMC from infected persons, but not uninfected controls. Patients with symptomatic Chagas' disease (cardiac and gastrointestinal groups) had decreased cellular responses to T. cruzi lysate (median proliferation index [PI] = 3), compared with those in the indeterminate group (median PI = 9; P < 0.005). Further investigations of the mechanism of this reduced CMI response in those with chronic disease may yield insights into the pathogenesis of Chagas' disease.