Overview of the regulatory approval of tecovirimat intravenous formulation for treatment of smallpox: potential impact on smallpox outbreak response capabilities, and future tecovirimat development potential.

INTRODUCTION: Tecovirimat oral capsule formulation is approved in the US and Canada for treatment of smallpox and in the United Kingdom (UK) and European Union (EU) for treatment of multiple human orthopoxvirus diseases, including mpox. Smallpox is considered a serious threat, and there is currently an unprecedented global mpox outbreak. AREAS COVERED: A brief summary of the threat of smallpox, the threat of increasing mpox spread in endemic regions, and the unprecedented emergence of mpox into non-endemic regions is presented. The tecovirimat intravenous formulation clinical development program leading to USFDA approval for smallpox treatment is discussed. EXPERT OPINION: As of January 2023 tecovirimat is approved to treat mpox in the UK and EU. However, published clinical trial data evaluating tecovirimat efficacy and safety in mpox patients is pending. Increasing global prevalence of mpox highlights the potential benefits of a well-characterized, effective, and safe antiviral treatment for mpox infection. Ongoing trials in mpox patients may provide results supporting the use of tecovirimat to treat this disease. USFDA approval of tecovirimat for post-exposure prophylaxis in the event of a smallpox release, and the development of pediatric liquid formulations for patients under 13 kg, could provide additional public health benefits.

Dioxygen reactivity of a biomimetic [4Fe-4S] compound exhibits [4Fe-4S] to [2Fe-2S] cluster conversion.

Fumarate and nitrate reductase (FNR) is a gene regulatory protein that controls anaerobic to aerobic respiration in Escherichia coli, for which O2 serves as a control switch to induce a protein structural change by converting [4Fe-4S] cofactors to [2Fe-2S] clusters. Although biomimetic models can aid in understanding the complex functions of their protein counterparts, the inherent sensitivity of discrete [Fe-S] molecules to aerobic conditions poses a unique challenge to mimic the O2-sensing capability of FNR. Herein, we report unprecedented biomimetic O2 reactivity of a discrete [4Fe-4S] complex, [Fe4S4(SPhF)4]2- (1) where SPhF is 4-fluorothiophenolate, in which the reaction of 1 with O2(g) in the presence of thiolate produces its [2Fe-2S] analogue, [Fe2S2(SPhF)4]2- (2), at room temperature. The cluster conversion of 1 to 2 can also be achieved by employing disulfide as an oxidant under the same reaction conditions. The [4Fe-4S] to [2Fe-2S] cluster conversion by O2 was found to be significantly faster than that by disulfide, while the reaction with disulfide produced higher yields of 2.

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity.

Nitric oxide (NO) is an important signaling molecule that is involved in a wide range of physiological and pathological events in biology. Metal coordination chemistry, especially with iron, is at the heart of many biological transformations involving NO. A series of heme proteins, nitric oxide synthases (NOS), soluble guanylate cyclase (sGC), and nitrophorins, are responsible for the biosynthesis, sensing, and transport of NO. Alternatively, NO can be generated from nitrite by heme- and copper-containing nitrite reductases (NIRs). The NO-bearing small molecules such as nitrosothiols and dinitrosyl iron complexes (DNICs) can serve as an alternative vehicle for NO storage and transport. Once NO is formed, the rich reaction chemistry of NO leads to a wide variety of biological activities including reduction of NO by heme or non-heme iron-containing NO reductases and protein post-translational modifications by DNICs. Much of our understanding of the reactivity of metal sites in biology with NO and the mechanisms of these transformations has come from the elucidation of the geometric and electronic structures and chemical reactivity of synthetic model systems, in synergy with biochemical and biophysical studies on the relevant proteins themselves. This review focuses on recent advancements from studies on proteins and model complexes that not only have improved our understanding of the biological roles of NO but also have provided foundations for biomedical research and for bio-inspired catalyst design in energy science.

Generation of H2S from Thiol-Dependent NO Reactivity of Model [4Fe-4S] Cluster and Roussin's Black Anion.

Iron-sulfur clusters (Fe-S) have been well established as a target for nitric oxide (NO) in biological systems. Complementary to protein-bound studies, synthetic models have provided a platform to study what iron nitrosylated products and byproducts are produced depending on a controlled reaction environment. We have previously shown a model [2Fe-2S] system that produced a dinitrosyl iron complex (DNIC) upon nitrosylation along with hydrogen sulfide (H2S), another important gasotransmitter, in the presence of thiol, and hypothesized a similar reactivity pattern with [4Fe-4S] clusters which have largely produced inconsistent reaction products across biological and synthetic systems. Roussin's black anion (RBA), [Fe4(µ3-S)3(NO)7]-, is a previously established reaction product from synthetic [4Fe-4S] clusters with NO. Here, we present a new reactivity for the nitrosylation of a synthetic [4Fe-4S] cluster in the presence of thiol and thiolate. [Et4N]2[Fe4S4(SPh)4] (1) was nitrosylated in the presence of excess PhSH to generate H2S and an "RBA-like" intermediate that when further reacted with [NEt4][SPh] produced a {Fe(NO)2}9 DNIC, [Et4N][Fe(NO)2(SPh)2] (2). This "RBA-like" intermediate proved difficult to isolate but shares striking similarities to RBA in the presence of thiol based on IR υ(NO) stretching frequencies. Surprisingly, the same reaction products were produced when the reaction started with RBA and thiol. Similar to 1/NO, RBA in the presence of thiol and thiolate generates stoichiometric amounts of DNIC while releasing its bridging sulfides as H2S. These results suggest not only that RBA may not be the final product of [4Fe-4S] + NO but also that RBA has unprecedented reactivity with thiols and thiolates which may explain current challenges around identifying biological nitrosylated Fe-S clusters.

An overview of tecovirimat for smallpox treatment and expanded anti-orthopoxvirus applications.

INTRODUCTION: Tecovirimat (TPOXX®; ST-246) was approved for the treatment of symptomatic smallpox by the USFDA in July of 2018 and has been stockpiled by the US government for use in a smallpox outbreak. While there has not been a reported case of smallpox since 1978 it is still considered a serious bioterrorism threat. AREAS COVERED: A brief history of smallpox from its proposed origins as a human disease through its eradication in the late 20th century is presented. The current smallpox threat and the current public health response plans are described. The discovery, and development of tecovirimat through NDA submission and subsequent approval for treatment of smallpox are discussed. Google Scholar and PubMed were searched over all available dates for relevant publications. EXPERT OPINION: Approval of tecovirimat to treat smallpox represents an important milestone in biosecurity preparedness. Incorporating tecovirimat into the CDC smallpox response plan, development of pediatric liquid and intravenous formulations, and approval for post-exposure prophylaxis would provide additional health security benefit.Tecovirimat shows broad efficacy against orthopoxviruses in vitro and in vivo and could be developed for use against emerging orthopoxvirus diseases such as monkeypox, vaccination-associated adverse events, and side effects of vaccinia oncolytic virus therapy.

Zinc Photocages with Improved Photophysical Properties and Cell Permeability Imparted by Ternary Complex Formation.

Photocaged complexes can control the availability of metal ions to interrogate cellular signaling pathways. We describe a new photocage, {bis[(2-pyridyl)methyl]amino}(9-oxo-2-xanthenyl)acetic acid (XDPAdeCage, 1), which utilizes a 2-xanthone acetic acid group to mediate a photodecarboxylation reaction. XDPAdeCage photolyzes with a quantum yield of 27%, and binds Zn2+ with 4.6 pM affinity, which decreases by over 4 orders of magnitude after photolysis. For comparison to our previous approach to Zn2+ release via photodecarboxylation, the analogous photocage {bis[(2-pyridyl)methyl]amino}(m-nitrophenyl)acetic acid (DPAdeCage, 2), which uses a m-nitrobenzyl chromophore, was also prepared and characterized. The advantages of the 2-xanthone acetic acid chromophore include red-shifted excitation and a higher extinction coefficient at the preferred uncaging wavelength. The neutral ternary complex of [Zn(XDPAdeCage)]+ with the anionic ligand pyrithione is membrane permeable, which circumvents the need to utilize invasive techniques to introduce intracellular Zn2+ fluctuations. Using fluorescent imaging, we have confirmed transport of Zn2+ across membranes; in addition, RT-PCR experiments demonstrate changes in expression of Zn2+-responsive proteins after photolysis.

Treatment with the smallpox antiviral tecovirimat (ST-246) alone or in combination with ACAM2000 vaccination is effective as a postsymptomatic therapy for monkeypox virus infection.

The therapeutic efficacies of smallpox vaccine ACAM2000 and antiviral tecovirimat given alone or in combination starting on day 3 postinfection were compared in a cynomolgus macaque model of lethal monkeypox virus infection. Postexposure administration of ACAM2000 alone did not provide any protection against severe monkeypox disease or mortality. In contrast, postexposure treatment with tecovirimat alone or in combination with ACAM2000 provided full protection. Additionally, tecovirimat treatment delayed until day 4, 5, or 6 postinfection was 83% (days 4 and 5) or 50% (day 6) effective.

Preparation and characterization of iron oxide nanoparticles coated with chitosan for removal of Cd(II) and Cr(VI) from aqueous solution.

This study investigated the applicability of magnetite Fe(3)O(4) nanoparticles coated with chitosan (CMNs) for the removal of some toxic heavy metals from simulated wastewater. Magnetic nanomaterials were synthesized using the co-precipitation method and characterized by transmission electron microscope, scanning electron microscope, X-ray diffraction, and Fourier transformer infrared spectroscopy. The magnetic properties of the prepared magnetic nanoparticles were determined by a vibrating-sample magnetometer. Batch experiments were carried out to determine the adsorption kinetics of Cr(VI) and Cd(II) by magnetic nanoparticles. It is noteworthy that CMNs show a highly efficient adsorption capacity for low concentration Cr(VI) and Cd(II) ions solution, which can reach 98% within 10 min.

TOXIC ACTIVITY AND DELAYED EFFECTS OF FIVE BOTANICAL OILS ON THE FOLLOWING GENERATIONS OF AGROTIS IPSILON (HUFNAGEL) (INSECTA: LEPIDOPTERA: NOCTUIDAE) AFTER PARENTS TREATMENT.

The present study is carried out to evaluate the toxic efficiency and delayed effects of five botanical oils on the greasy cut worm Agrotis ipsilon (Lepidoptera: Noctuidae), as a trial for the attainment of a possible use of an alternative safe and effective phytochemicals against the insect-pest. So as to minimize or prevent the repeated usage of conventional insecticides, then reduce the environmental pollution as well as the occurring hazards to man and domestic animal due to the use of the pesticides alone. Four tested concentrations (0.5, 1.0, 1.5 and 2.5% v/v) from each of camphor, red basil, menthol, rose and anise oils, were bioassayed by treating the offered castor oil bean leaves, to the 4th instar larvae along 48h, under the laboratory higrothermic conditions of 25±2 °C and 65±5% R.H. The obtained results showed that the five tested oils were found to have more or less toxic activity and drastic effects on the inspected parameters of fitness components of the treated parent generation of the insect, in particular, pupae, emerged adult moths and laid eggs/female. In this respect camphor and red basil oils were highly effective, followed by menthol oil, anise oil and the least effective one was rose oil. Moreover, the assessed unprofitable delayed effects on the going on of the biological performance within the treated insects showed the adverse effects on the fitness components of the consequent generations (fs) post (p) one treatment with each of the bioassyed oils. The prevalence of adverse effects and disturbance in the going on biological performance through the period of (p) generation; which is followed by the distinct failure of insect development in (f1) generation were recorded for each of the tested menthol oil at 0.5 and 1.5% (v/v); camphor oil at 1.5 and 2.5% and red basil oil at 2.5% (v/v). While anise and rose oils were somewhat less efficient causing the distinct failure of the following generations up to the 3rd and/or the 6th ones. That observed distinct failure of the insect development could be attributed to the rapid or/and slow cumulative effect of the induced recessive lethal genes in both influenced sexes along the interval of the following developed generations (fs) after (p) one treatment, causing apparent adverse disturbance of the normal biological performance, which finally appears at the beginning of the failed generation.

Pharmacokinetic and pharmacodynamic modeling to determine the dose of ST-246 to protect against smallpox in humans.

Although smallpox has been eradicated, the United States government considers it a "material threat" and has funded the discovery and development of potential therapeutic compounds. As reported here, the human efficacious dose for one of these compounds, ST-246, was determined using efficacy studies in nonhuman primates (NHPs), together with pharmacokinetic and pharmacodynamic analysis that predicted the appropriate dose and exposure levels to provide therapeutic benefit in humans. The efficacy analysis combined the data from studies conducted at three separate facilities that evaluated treatment following infection with a closely related virus, monkeypox virus (MPXV), in a total of 96 NHPs. The effect of infection on ST-246 pharmacokinetics in NHPs was applied to humans using population pharmacokinetic models. Exposure at the selected human dose of 600 mg is more than 4-fold higher than the lowest efficacious dose in NHPs and is predicted to provide protection to more than 95% of the population.

Safety and pharmacokinetics of the anti-orthopoxvirus compound ST-246 following a single daily oral dose for 14 days in human volunteers.

ST-246 is being evaluated as a treatment for pathogenic orthopoxvirus infections in humans. To this end, a phase 2, double-blind, randomized, placebo-controlled, multicenter trial was conducted to assess the safety, tolerability, and pharmacokinetics (PK) of ST-246 when administered as a single daily oral dose (400 mg or 600 mg) for 14 days in fed adult volunteers. ST-246 was safe and well tolerated, with no deaths or serious adverse events reported during the study. There was a low incidence of treatment-emergent adverse events (TEAEs), the most common of which were mild nausea and headache. There were no clinically significant results from laboratory assessments, vital sign measurements, physical examinations, or electrocardiograms. The PK and dose proportionality of ST-246 were determined. The PK analysis showed that steady state was achieved by day 5 for the ST-246 400-mg treatment group and by day 6 for the 600-mg group. The dose proportionality analysis showed that the 400- and 600-mg ratio of dose-normalized peak drug concentration in plasma (C(max)) and relative exposure for each dosing interval (AUC(τ)) ranged from 80% to 85%. However, the 90% confidence intervals did not include 1.0, so dose proportionality could not be concluded. Overall, ST-246 was shown to be safe, and the PK was predictable. These results support further testing of ST-246 in a multicenter pivotal clinical safety study for licensure application.

Pharmacokinetic comparison of a single oral dose of polymorph form i versus form V capsules of the antiorthopoxvirus compound ST-246 in human volunteers.

ST-246, a novel compound that inhibits egress of orthopoxvirus from mammalian cells, is being tested as a treatment for pathogenic orthopoxvirus infections in humans. This phase I, double-blind, randomized, crossover, exploratory study was conducted to compare the pharmacokinetics (PK) of a single daily 400-mg oral dose of ST-246 polymorph form I versus polymorph form V administered to fed, healthy human volunteers. Both forms appeared to be well tolerated, with no serious adverse events. The order of administration of the two forms had no effect on the results of the PK analyses. Form I and form V both exhibited comparable plasma concentration versus time profiles, but complete bioequivalence between the two forms was not found. Maximum drug concentration (C(max)) met the bioequivalence criteria, as the 90% confidence interval (CI) was 80.6 to 96.9%. However, the area under the concentration-time curve from time zero to time t (AUC(0-t)) and AUC(0-∞) did not meet the bioequivalence criteria (CIs of 67.8 to 91.0% and 73.9 to 104.7%, respectively). The extent of absorption of form I, as defined by AUC(0-∞), was 11.7% lower than that of form V. Since ST-246 form I is more thermostable than form V, form I was selected for further development and use in all future studies.

Comparison of the safety and pharmacokinetics of ST-246® after i.v. infusion or oral administration in mice, rabbits and monkeys.

BACKGROUND: ST-246® is an antiviral, orally bioavailable small molecule in clinical development for treatment of orthopoxvirus infections. An intravenous (i.v.) formulation may be required for some hospitalized patients who are unable to take oral medication. An i.v. formulation has been evaluated in three species previously used in evaluation of both efficacy and toxicology of the oral formulation. METHODOLOGY/PRINCIPAL FINDINGS: The pharmacokinetics of ST-246 after i.v. infusions in mice, rabbits and nonhuman primates (NHP) were compared to those obtained after oral administration. Ten minute i.v. infusions of ST-246 at doses of 3, 10, 30, and 75 mg/kg in mice produced peak plasma concentrations ranging from 16.9 to 238 µg/mL. Elimination appeared predominately first-order and exposure dose-proportional up to 30 mg/kg. Short i.v. infusions (5 to 15 minutes) in rabbits resulted in rapid distribution followed by slower elimination. Intravenous infusions in NHP were conducted at doses of 1 to 30 mg/kg. The length of single infusions in NHP ranged from 4 to 6 hours. The pharmacokinetics and tolerability for the two highest doses were evaluated when administered as two equivalent 4 hour infusions initiated 12 hours apart. Terminal elimination half-lives in all species for oral and i.v. infusions were similar. Dose-limiting central nervous system effects were identified in all three species and appeared related to high C(max) plasma concentrations. These effects were eliminated using slower i.v. infusions. CONCLUSIONS/SIGNIFICANCE: Pharmacokinetic profiles after i.v. infusion compared to those observed after oral administration demonstrated the necessity of longer i.v. infusions to (1) mimic the plasma exposure observed after oral administration and (2) avoid C(max) associated toxicity. Shorter infusions at higher doses in NHP resulted in decreased clearance, suggesting saturated distribution or elimination. Elimination half-lives in all species were similar between oral and i.v. administration. The administration of ST-246 was well tolerated as a slow i.v. infusion.

Echinocystic acid 3,16-O-bisglycosides from Albizia procera.

Three triterpene glycosides and two known ones were isolated from the bark of Albizia procera by using chromatographic techniques. The structures of the compounds were determined to be 3-O-beta-D-xylopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->6)-2-acetamido-2-deoxy-beta-D-glucopyranosyl echinocystic acid 16-O-beta-D-glucopyranoside, 3-O-beta-D-xylopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6)-2-acetamido-2-deoxy-beta-D-glucopyranosyl echinocystic acid 16-O-beta-D-glucopyranoside and 3-O-alpha-L-arabinopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-(1-->6)-2-acetamido-2-deoxy-beta-D-glucopyranosyl echinocystic acid 16-O-beta-D-glucopyranoside. Their structures were determined by NMR techniques including HOHAHA, (1)H-(1)H COSY, ROE, HMQC and HMBC experiments together with FABMS as well as acid hydrolysis. To the best of our knowledge, the new compounds are considered the first examples of echinocystic acid 3,16-O-bisglycosides. In contrast to other cytotoxic echinocystic acid glycosides with N-acetyl glucosamine unit, the new glycosides were found inactive when assayed by MTT method for their cytotoxicities against the human tumor cell lines HEPG2, A549, HT29 and MCF7. The results showed the importance of the free hydroxyl group at the aglycone C-16 for exhibiting cytotoxic properties.

Vaccinia virus p37 interacts with host proteins associated with LE-derived transport vesicle biogenesis.

BACKGROUND: Proteins associated with the late endosome (LE) appear to play a central role in the envelopment of a number of taxonomically diverse viruses. How viral proteins interact with LE-associated proteins to facilitate envelopment is not well understood. LE-derived transport vesicles form through the interaction of Rab9 GTPase with cargo proteins, and TIP47, a Rab9-specific effector protein. Vaccinia virus (VV) induces a wrapping complex derived from intracellular host membranes to envelope intracellular mature virus particles producing egress-competent forms of virus. RESULTS: We show that VV p37 protein associates with TIP47-, Rab9-, and CI-MPR-containing membranes. Mutation of a di-aromatic motif in p37 blocks association with TIP47 and inhibits plaque formation. ST-246, a specific inhibitor of p37 function, inhibits these interactions and also blocks wrapped virus particle formation. Vaccinia virus expressing p37 variants with reduced ST-246 susceptibility associates with Rab9 and co-localizes with CI-MPR in the presence and absence of compound. CONCLUSION: These results suggest that p37 localizes to the LE and interacts with proteins associated with LE-derived transport vesicle biogenesis to facilitate assembly of extracellular forms of virus.

Vaccination of BALB/c mice with Escherichia coli-expressed vaccinia virus proteins A27L, B5R, and D8L protects mice from lethal vaccinia virus challenge.

The potential threat of smallpox use in a bioterrorist attack has heightened the need to develop an effective smallpox vaccine for immunization of the general public. Vaccination with the current smallpox vaccine, Dryvax, produces protective immunity but may result in adverse reactions for some vaccinees. A subunit vaccine composed of protective vaccinia virus proteins should avoid the complications arising from live-virus vaccination and thus provide a safer alternative smallpox vaccine. In this study, we assessed the protective efficacy and immunogenicity of a multisubunit vaccine composed of the A27L and D8L proteins from the intracellular mature virus (IMV) form and the B5R protein from the extracellular enveloped virus (EEV) form of vaccinia virus. BALB/c mice were immunized with Escherichia coli-produced A27L, D8L, and B5R proteins in an adjuvant consisting of monophosphoryl lipid A and trehalose dicorynomycolate or in TiterMax Gold adjuvant. Following immunization, mice were either sacrificed for analysis of immune responses or lethally challenged by intranasal inoculation with vaccinia virus strain Western Reserve. We observed that three immunizations either with A27L, D8L, and B5R or with the A27L and B5R proteins alone induced potent neutralizing antibody responses and provided complete protection against lethal vaccinia virus challenge. Several linear B-cell epitopes within the three proteins were recognized by sera from the immunized mice. In addition, protein-specific cellular responses were detected in spleens of immunized mice by a gamma interferon enzyme-linked immunospot assay using peptides derived from each protein. Our data suggest that a subunit vaccine incorporating bacterially expressed IMV- and EEV-specific proteins can be effective in stimulating anti-vaccinia virus immune responses and providing protection against lethal virus challenge.

Endometrial volume as predictor of malignancy in women with postmenopausal bleeding.

OBJECTIVE: To assess endometrial volume as a predictor of endometrial malignancy in women with postmenopausal bleeding. METHODS: Endometrial volume was measured by virtual organ computer-aided analysis in 170 women with postmenopausal bleeding, and histopathologic results of endometrial biopsies were obtained for all. A group of 100 women without postmenopausal bleeding was used for control. RESULTS: There were 90 cases of benign disease, 53 cases of atypia, and 27 cases of endometrial cancers in the study group. Whereas endometrial thickness was 9.61+/-5.12 mm (range, 5-20 mm) and endometrial volume was 3+/-1.1 mL (range, 1.8-5.4 mL) in women with atypia or cancer, they were 4.87+/-3.43 mm (range, 2-8 mm) and 1.52+/-0.82 (range, 0.6-2.2 mL), respectively, in women with benign disease. In the control group, endometrial volume was 1.15+/-0.14 mL (range, 0.6-1.3 mL). Volume was more sensitive than thickness for predicting malignancy, and a cutoff value of 1.35 mL was found to provide the best sensitivity. CONCLUSION: An endometrial volume of 1.35 mL or greater may predict malignancy in women with postmenopausal bleeding.

The vaccinia virus F13L YPPL motif is required for efficient release of extracellular enveloped virus.

The Tyr-X-X-Leu (YxxL) motif of the vaccinia virus F13L protein was examined for late (L) domain activity. The ability of an F13L deletion virus to form plaques was restored by PCR products containing single alanine substitutions within the motif and a YAAL construct but not by constructs lacking both the Y and L residues. Recombinant viruses possessing alanine substitutions in place of the tyrosine or the leucine residue in the YxxL motif demonstrated small, asymmetrical plaques. RNA interference-dependent depletion of Alix and TSG101 (host proteins involved in L domain-dependent protein trafficking) diminished extracellular enveloped virion production to various degrees, suggesting that the YxxL motif is a genuine L domain.

Mutational analysis of the potential catalytic residues of the VV G1L metalloproteinase.

The vaccinia virus G1L open-reading frame is predicted to be a metalloproteinase based upon the presence of a conserved zinc-binding motif. Western blot analysis demonstrates G1L undergoes proteolytic processing during the course of infection, although the significance of this event is unknown. In order to determine which amino acid residues are important for G1L activity, a plasmid-borne library of G1L constructs containing mutations in and about the active site was created. Transient expression analysis coupled with a trans complementation assay of a conditionally-lethal mutant virus suggest that, of the mutants, only glutamic acid 120 is non-essential for G1L processing to occur.

Molecular cloning of Schistosoma mansoni calcineurin subunits and immunolocalization to the excretory system.

In order to explain the schistosomicidal effect of cyclosporin A, the hypothesis was advanced that the drug, complexed with cyclophilin, inhibits the phosphatase activity of parasite calcineurin (CN), with mechanisms similar to those operating in its immunosuppressive action. As a preparatory step to the testing of this hypothesis, we report the molecular cloning of both CN subunits in Schistosoma mansoni. The catalytic (A) subunit has a predicted sequence of 607 amino acids and shows substantial similarity to other cloned CNs, except for the carboxy-terminal end that is highly divergent. The regulatory (B) subunit consists of 169 amino acids that are 86% identical to those of the human counterpart and, from its anomalous electrophoretic mobility, it appears to be myristoylated. The results of Southern blotting experiments are compatible with the existence of multiple genes for CNA and a single gene for CNB. Western blots showed that both subunits are present at all stages of the parasite life cycle and can be detected both in the soluble and in the membrane fraction. Immunofluorescence confocal microscopy revealed a striking concentration of the anti-CNA reactivity in 6-8 discrete spots in the schistosomula and in distinct spots along the body of the adult parasite, corresponding to the expected localization of flame cells. Both patterns were confirmed by a perfect co-localization of the anti-CNA signal with that of a previously characterized anti-flame cell monoclonal antibody. The preferential confinement of schistosome CN to the protonephridial system suggests that the enzyme in the parasite may fulfil similar functions to those performed in mammalian kidneys.