Listen to what the animals say: a systematic review and meta-analysis of sterol 14-demethylase inhibitor efficacy for in vivo models of Trypanosoma cruzi infection.

Sterol 14-demethylase (CYP51) inhibitors, encompassing new chemical entities and repurposed drugs, have emerged as promising candidates for Chagas disease treatment, based on preclinical studies reporting anti-Trypanosoma cruzi activity. Triazoles like ravuconazole (RAV) and posaconazole (POS) progressed to clinical trials. Unexpectedly, their efficacy was transient in chronic Chagas disease patients, and their activity was not superior to benznidazole (BZ) treatment. This paper aims to summarize evidence on the global activity of CYP51 inhibitors against T. cruzi by applying systematic review strategies, risk of bias assessment, and meta-analysis from in vivo studies. PubMed and Embase databases were searched for original articles, obtaining fifty-six relevant papers meeting inclusion criteria. Characteristics of animal models, parasite strain, treatment schemes, and cure rates were extracted. Primary outcomes such as maximum parasitaemia values, survival, and parasitological cure were recorded for meta-analysis, when possible. The risk of bias was uncertain in most studies. Animals treated with itraconazole, RAV, or POS survived significantly longer than the infected non-treated groups (RR = 4.85 [3.62, 6.49], P < 0.00001), and they showed no differences with animals treated with positive control drugs (RR = 1.01 [0.98, 1.04], P = 0.54). Furthermore, the overall analysis showed that RAV or POS was not likely to achieve parasitological cure when compared with BZ or NFX treatment (OD = 0.49 [0.31, 0.77], P = 0.002). This systematic review contributes to understanding why the azoles had failed in clinical trials and, more importantly, how to improve the animal models of T. cruzi infection by filling the gaps between basic, translational, and clinical research.

Strategies of targeting CYP51 for IFIs therapy: Emerging prospects, opportunities and challenges.

CYP51, a monooxygenase associated with the sterol synthesis pathway, is responsible for the catalysis of the 14-methyl hydroxylation reaction of lanosterol precursors. This enzyme is widely present in microorganisms, plants, and mammals. In mammals, CYP51 plays a role in cholesterol production, oligodendrocyte formation, oocyte maturation, and spermatogenesis. In fungal cells, CYP51 is an enzyme that synthesizes membrane sterols. By inhibiting fungal CYP51, ergosterol synthesis can be inhibited and ergosterol membrane fluidity is altered, resulting in fungal cell apoptosis. Thus, targeting CYP51 is a reliable antifungal strategy with important implications for the treatment of invasive fungal infections (IFIs). Many CYP51 inhibitors have been approved by the FDA for clinical treatment. However, several limitations of CYP51 inhibitors remain to be resolved, including fungal resistance, hepatotoxicity, and drug-drug interactions. New broad-spectrum, anti-resistant, highly selective CYP51 inhibitors are expected to be developed to enhance clinical efficacy and minimize adverse effects. Herein, we summarize the structural features and biological functions of CYP51 and emphatically analyze the structure-activity relationship (SAR) and therapeutic potential of different chemical types of small-molecule CYP51 inhibitors. We also discuss the latest progress of novel strategies, providing insights into new drugs targeting CYP51 for clinical practice.

Design, Synthesis, and Biological Evaluation of Dual-Target COX-2/CYP51 Inhibitors for the Treatment of Fungal Infectious Diseases.

The design of novel dual-target (COX-2/CYP51) inhibitors was proposed in the study, and three series of compounds were constructed though the pathway of skeleton screening and combination; their molecular structures were synthesized and evaluated. Most of the compounds exhibited significant antifungal ability. Among them, potential compounds (10a-2, 16b-3) with excellent antifungal and anti-drug-resistant fungal ability (MIC50, 0.125-2.0 µg/mL) were selected for the subsequent mechanistic study. On the one hand, these compounds could block the ergosterol biosynthesis pathway by inhibiting CYP51 and influence the internal physiological function of fungal cells, which included the increase of the ROS level, the anomaly of ΔΨm, and the emergence of an apoptotic state. On the other hand, these compounds also effectively showed COX-2 inhibition ability, eliminated the inflammatory reaction of the infected region, and activated the body's immune function. In summary, this study not only provided a novel antifungal drug design pathway but also discovered excellent target compounds.

Determination of parasitic burden in the brain tissue of infected mice in acute toxoplasmosis after treatment by fluconazole combined with sulfadiazine and pyrimethamine.

BACKGROUND/AIMS: One of the opportunistic pathogens which cause serious problems in the human immune system is Toxoplasma gondii, with toxoplasma encephalitis (TE) seen in patients affected by it. The treatment of these patients is limited, and if not treated on time, death will be possible. METHODS: In this study, the effects of the treatment with different doses of fluconazole (FLZ) in combination with the current treatment of acute toxoplasmosis on reducing the mortality rate and the parasitic load in the murine model in vivo were studied. The mice were treated with different doses of fluconazole alone, sulfadiazine, and pyrimethamine plus fluconazole. A day after the end of the treatment and 1 day before death, the mice's brains were collected, and after DNA extraction and molecular tests, the parasite burden was detected. RESULTS: This study showed that a 10-day treatment with 20 mg/kg of fluconazole combined with sulfadiazine and pyrimethamine 1.40 mg/kg per day affected acute toxoplasmosis and reduced the parasitic load significantly in brain tissues and also increased the survival rate of all mice in this group until the last day of the study, in contrast to other treatment groups. These results also indicate the positive effects of combined therapy on Toxoplasma gondii and the prevention of relapse. CONCLUSIONS: Reducing the parasitic burden and increasing the survival rate were more effective against acute toxoplasmosis in the combined treatment of different doses of fluconazole with current treatments than current treatments without fluconazole. In other words, combination therapy with fluconazole plus pyrimethamine reduced the parasitic burden in the brain significantly, so it could be a replacement therapy in patients with intolerance sulfadiazine.

Lead optimization generates selenium-containing miconazole CYP51 inhibitors with improved pharmacological profile for the treatment of fungal infections.

A series of selenium-containing miconazole derivatives were identified as potent antifungal drugs in our previous study. Representative compound A03 (MIC = 0.01 µg/mL against C.alb. 5314) proved efficacious in inhibiting the growth of fungal pathogens. However, further study showed lead compound A03 exhibited potential hemolysis, significant cytotoxic effect and unfavorable metabolic stability and was therefore modified to overcome these drawbacks. In this article, the further optimization of selenium-containing miconazole derivatives resulted in the discovery of similarly potent compound B17 (MIC = 0.02 µg/mL against C.alb. 5314), exhibiting a superior pharmacological profile with decreased rate of metabolism, cytotoxic effect and hemolysis. Furthermore, compound B17 showed fungicidal activity against Candida albicans and significant effects on the treatment of resistant Candida albicans infections. Meanwhile, compound B17 not only could reduce the ergosterol biosynthesis pathway by inhibiting CYP51, but also inhibited biofilm formation. More importantly, compound B17 also shows promising in vivo efficacy after intraperitoneal injection and the PK study of compound B17 was evaluated. In addition, molecular docking studies provide a model for the interaction between the compound B17 and the CYP51 protein. Overall, we believe that these selenium-containing miconazole compounds can be further developed for the potential treatment of fungal infections.

Domain-Swap Dimerization of Acanthamoeba castellanii CYP51 and a Unique Mechanism of Inactivation by Isavuconazole.

Cytochromes P450 (P450, CYP) metabolize a wide variety of endogenous and exogenous lipophilic molecules, including most drugs. Sterol 14α-demethylase (CYP51) is a target for antifungal drugs known as conazoles. Using X-ray crystallography, we have discovered a domain-swap homodimerization mode in CYP51 from a human pathogen, Acanthamoeba castellanii CYP51 (AcCYP51). Recombinant AcCYP51 with a truncated transmembrane helix was purified as a heterogeneous mixture corresponding to the dimer and monomer units. Spectral analyses of these two populations have shown that the CO-bound ferrous form of the dimeric protein absorbed at 448 nm (catalytically competent form), whereas the monomeric form absorbed at 420 nm (catalytically incompetent form). AcCYP51 dimerized head-to-head via N-termini swapping, resulting in formation of a nonplanar protein-protein interface exceeding 2000 Å2 with a total solvation energy gain of -35.4 kcal/mol. In the dimer, the protomers faced each other through the F and G α-helices, thus blocking the substrate access channel. In the presence of the drugs clotrimazole and isavuconazole, the AcCYP51 drug complexes crystallized as monomers. Although clotrimazole-bound AcCYP51 adopted a typical CYP monomer structure, isavuconazole-bound AcCYP51 failed to refold 74 N-terminal residues. The failure of AcCYP51 to fully refold upon inhibitor binding in vivo would cause an irreversible loss of a structurally aberrant enzyme through proteolytic degradation. This assumption explains the superior potency of isavuconazole against A. castellanii The dimerization mode observed in this work is compatible with membrane association and may be relevant to other members of the CYP family of biologic, medical, and pharmacological importance. SIGNIFICANCE STATEMENT: We investigated the mechanism of action of antifungal drugs in the human pathogen Acanthamoeba castellanii. We discovered that the enzyme target [Acanthamoeba castellanii sterol 14α-demethylase (AcCYP51)] formed a dimer via an N-termini swap, whereas drug-bound AcCYP51 was monomeric. In the AcCYP51-isavuconazole complex, the protein target failed to refold 74 N-terminal residues, suggesting a fundamentally different mechanism of AcCYP51 inactivation than only blocking the active site. Proteolytic degradation of a structurally aberrant enzyme would explain the superior potency of isavuconazole against A. castellanii.

Mefenoxam, Itraconazole, and Terbinafine Combination Therapy for Management of Pythiosis in Dogs (Six Cases).

Pythium insidiosum is an oomycete that encysts in the skin or gastrointestinal tract, leading to pythiosis. Pythiosis is reported in tropical and subtropical climates, affecting dogs and rarely cats. Surgical resection is the treatment of choice; however, cases present late in the disease and lesions are often nonresectable. Medical management is typically unsuccessful, with uncommon exceptions; however, mefenoxam, an agricultural fungicide, has in vitro efficacy against P insidiosum. We describe the use of mefenoxam, itraconazole, and terbinafine (MIT) in five dogs with gastrointestinal pythiosis and one dog with cutaneous pythiosis. Two of the gastrointestinal cases had disease extending to surgical margins and received MIT: resolution of clinical signs and seronegativity occurred after 189-193 days. Another case underwent surgical resection and MIT. The dog improved but subsequently developed a rectal mass, which responded to addition of prednisone and immunotherapy. Two cases were treated with MIT alone, and response varied. Efficacy of MIT in cutaneous pythiosis could not be determined. MIT may result in improved survival and seronegativity in dogs with incompletely resected gastrointestinal pythiosis. Mefenoxam is EPA registered, and extralabel use under the Animal Medicinal Drug Use Clarification Act does not apply. Additional research is recommended before use.

Clinical efficacy of combination therapy of itraconazole and prednisolone for treating effusive feline infectious peritonitis.

A 3-month-old male Scottish Fold kitten with pleural fluid and low ratio of albumin to globulin (A/G ratio) was brought to our small animal hospital. Since RNA from the type I feline coronavirus (FCoV) were detected in drained pleural fluid, the cat was tentatively diagnosed with effusive feline infectious peritonitis (FIP). Following the administration of itraconazole and prednisolone, the A/G ratio increased, and the pleural fluid mostly disappeared. The fecal FCoV levels temporarily decreased. However, the cat showed neurological manifestations and was eventually euthanized due to status epilepticus after 38 days of treatment. In conclusion, itraconazole partly exerted a beneficial effect in a cat with FIP. However, further investigation of a possible role of itraconazole in FIP treatment is warranted.

The novel fungal CYP51 inhibitor VT-1598 displays classic dose-dependent antifungal activity in murine models of invasive aspergillosis.

Aspergillus spp. infections remain a global concern, with ∼30% attributable mortality of invasive aspergillosis (IA). VT-1598 is a novel fungal CYP51 inhibitor designed for exquisite selectivity versus human CYP enzymes to achieve a maximal therapeutic index and therefore maximal antifungal efficacy. Previously, its broad-spectrum in vitro antifungal activity was reported. We report here the pharmacokinetics (PK) and pharmacodynamics (PD) of VT-1598 in neutropenic mouse models of IA. The plasma area-under-the-curve (AUC) of VT-1598 increased nearly linearly between 5 and 40 mg/kg after 5 days of QD administration (155 and 1033 µg*h/ml, respectively), with a further increase with 40 mg/kg BID dosing (1354 µg*h/ml). When A. fumigatus isolates with in vitro susceptibilities of 0.25 and 1.0 µg/ml were used in a disseminated IA model, VT-1598 treatment produced no decrease in kidney fungal burden at QD 10 mg/kg, intermediate decreases at QD 20 mg/kg and maximum or near maximum decreases at 40 mg/kg QD and BID. The PK/PD relationships of AUCfree/MIC for 1-log killing for the two strains were 5.1 and 1.6 h, respectively, similar to values reported for approved CYP51 inhibitors. In a survival study where animals were observed for 12 days after the last treatment, survival was 100% at the doses tested (20 and 40 mg/kg QD), and fungal burden remained suppressed even though drug wash-out was complete. Similar dose-dependent reductions in lung fungal burden were observed in a pulmonary model of IA. These data strongly support further exploration of VT-1598 for the treatment of this lethal mold infection.

ENDOCRINOLOGY IN THE TIME OF COVID-19: Management of Cushing's syndrome.

Clinical evaluation should guide those needing immediate investigation. Strict adherence to COVID-19 protection measures is necessary. Alternative ways of consultations (telephone, video) should be used. Early discussion with regional/national experts about investigation and management of potential and existing patients is strongly encouraged. Patients with moderate or severe clinical features need urgent investigation and management. Patients with active Cushing's syndrome, especially when severe, are immunocompromised and vigorous adherence to the principles of social isolation is recommended. In patients with mild features or in whom a diagnosis is less likely, clinical re-evaluation should be repeated at 3 and 6 months or deferred until the prevalence of SARS-CoV-2 has significantly decreased; however, those individuals should be encouraged to maintain social distancing. Diagnostic pathways may need to be very different from usual recommendations in order to reduce possible exposure to SARS-CoV-2. When extensive differential diagnostic testing and/or surgery is not feasible, it should be deferred and medical treatment should be initiated. Transsphenoidal pituitary surgery should be delayed during high SARS-CoV-2 viral prevalence. Medical management rather than surgery will be the used for most patients, since the short- to mid-term prognosis depends in most cases on hypercortisolism rather than its cause; it should be initiated promptly to minimize the risk of infection in these immunosuppressed patients. The risk/benefit ratio of these recommendations will need re-evaluation every 2-3 months from April 2020 in each country (and possibly local areas) and will depend on the local health care structure and phase of pandemic.

Current and Future Prospective of a Versatile Moiety: Imidazole.

Imidazole containing compounds have been a very much explored field since ancient times. Subsequently, it constitutes a significant moiety for the new drug development. A variety of compounds having imidazole moiety have been synthesized, evaluated and marketed for the treatment of various diseases such as antifungal, antiepileptic, ACE inhibitors and so on, as shown in the figure. The search for imidazole containing compounds with more selective biological potency with low side effects continues to be an active area of research in medicinal chemistry. This review is in an effort to highlight the marketed drugs with imidazole ring. The article also demonstrates the future prospective of marketed imidazoles as antifungal with potential activity targeting 14α-demethylase enzyme.

The Antifungal Drug Isavuconazole Is both Amebicidal and Cysticidal against Acanthamoeba castellanii.

Current treatments for Acanthamoeba keratitis rely on a combination of chlorhexidine gluconate, propamidine isethionate, and polyhexamethylene biguanide. These disinfectants are nonspecific and inherently toxic, which limits their effectiveness. Furthermore, in 10% of cases, recurrent infection ensues due to the difficulty in killing both trophozoites and double-walled cysts. Therefore, development of efficient, safe, and target-specific drugs which are capable of preventing recurrent Acanthamoeba infection is a critical unmet need for averting blindness. Since both trophozoites and cysts contain specific sets of membrane sterols, we hypothesized that antifungal drugs targeting sterol 14-demethylase (CYP51), known as conazoles, would have deleterious effects on A. castellanii trophozoites and cysts. To test this hypothesis, we first performed a systematic screen of the FDA-approved conazoles against A. castellanii trophozoites using a bioluminescence-based viability assay adapted and optimized for Acanthamoeba The most potent drugs were then evaluated against cysts. Isavuconazole and posaconazole demonstrated low nanomolar potency against trophozoites of three clinical strains of A. castellanii Furthermore, isavuconazole killed trophozoites within 24 h and suppressed excystment of preformed Acanthamoeba cysts into trophozoites. The rapid action of isavuconazole was also evident from the morphological changes at nanomolar drug concentrations causing rounding of trophozoites within 24 h of exposure. Given that isavuconazole has an excellent safety profile, is well tolerated in humans, and blocks A. castellanii excystation, this opens an opportunity for the cost-effective repurposing of isavuconazole for the treatment of primary and recurring Acanthamoeba keratitis.

Fungal Lanosterol 14α-demethylase: A target for next-generation antifungal design.

The cytochrome P450 enzyme lanosterol 14α-demethylase (LDM) is the target of the azole antifungals used widely in medicine and agriculture as prophylaxis or treatments of infections or diseases caused by fungal pathogens. These drugs and agrochemicals contain an imidazole, triazole or tetrazole substituent, with one of the nitrogens in the azole ring coordinating as the sixth axial ligand to the LDM heme iron. Structural studies show that this membrane bound enzyme contains a relatively rigid ligand binding pocket comprised of a deeply buried heme-containing active site together with a substrate entry channel and putative product exit channel that reach to the membrane. Within the ligand binding pocket the azole antifungals have additional affinity determining interactions with hydrophobic side-chains, the polypeptide backbone and via water-mediated hydrogen bond networks. This review will describe the tools that can be used to identify and characterise the next generation of antifungals targeting LDM, with the goal of obtaining highly potent broad-spectrum fungicides that will be able to avoid target and drug efflux mediated antifungal resistance.

Candida auris and multidrug resistance: Defining the new normal.

Candida auris is an emerging species of yeast characterized by colonization of skin, persistence in the healthcare environment, and antifungal resistance. C. auris was first described in 2009 from a single isolate but has since been reported in more than 25 countries worldwide. Resistance to fluconazole and amphotericin B is common, and resistance to the echinocandins is emerging in some countries. Antifungal resistance has been shown to be acquired rather than intrinsic and the primary mechanisms of resistance to the echinocandins and azoles have been determined. There are a number of new antifungal agents in phase 2 and phase 3 clinical trials and many have activity against C. auris. This review will discuss what is currently known about antifungal resistance in C. auris, limitations to antifungal susceptibility testing, the mechanisms of resistance, and the new antifungals that are on the horizon.

Modern Management of Mild Autonomous Cortisol Secretion.

Incidentally discovered adrenal tumors are reported in ~ 5% of adults undergoing cross-sectional imaging. Mild autonomous cortisol secretion (MACS) from the adrenal mass is demonstrated in 5-48% of patients with adrenal tumors. The diagnosis of MACS represents a challenge due to limitations of the currently used diagnostic tests, differences in the definitions of the clinically relevant MACS, and heterogeneity in an individual's susceptibility to abnormal cortisol secretion from the adrenal mass. Patients with MACS present with increased risk of cardiovascular risk factors, cardiovascular events, metabolic bone disease, and mortality. Adrenalectomy improves or reverses MACS-associated comorbidities in selected patients. The current review will address diagnostic and management challenges in the care of patients with MACS, discuss data on emerging biomarkers, and suggest future directions in the field of MACS.

The Fungal Cyp51-Specific Inhibitor VT-1598 Demonstrates In Vitro and In Vivo Activity against Candida auris.

Candida auris is an emerging pathogen associated with significant mortality and often multidrug resistance. VT-1598, a tetrazole-based fungal CYP51-specific inhibitor, was evaluated in vitro and in vivo against C. auris Susceptibility testing was performed against 100 clinical isolates of C. auris by broth microdilution. Neutropenic mice were infected intravenously with C. auris, and treatment began 24 h postinoculation with a vehicle control, oral VT-1598 (5, 15, and 50 mg/kg of body weight once daily), oral fluconazole (20 mg/kg once daily), or intraperitoneal caspofungin (10 mg/kg once daily), which continued for 7 days. Fungal burden was assessed in the kidneys and brains on day 8 in the fungal burden arm and on the days the mice succumbed to infection or on day 21 in the survival arm. VT-1598 plasma trough concentrations were also assessed on day 8. VT-1598 demonstrated in vitro activity against C. auris, with a mode MIC of 0.25 µg/ml and MICs ranging from 0.03 to 8 µg/ml. Treatment with VT-1598 resulted in significant and dose-dependent improvements in survival (median survival, 15 and >21 days for VT-1598 at 15 and 50 mg/kg, respectively) and reductions in kidney and brain fungal burden (reductions of 1.88 to 3.61 log10 CFU/g) compared to the control (5 days). The reductions in fungal burden correlated with plasma trough concentrations. Treatment with caspofungin, but not fluconazole, also resulted in significant improvements in survival and reductions in fungal burden compared to those with the control. These results suggest that VT-1598 may be a future option for the treatment of invasive infections caused by C. auris.

Sterol 14α-Demethylase Structure-Based Optimization of Drug Candidates for Human Infections with the Protozoan Trypanosomatidae.

Sterol 14α-demethylases (CYP51) are cytochrome P450 enzymes essential for sterol biosynthesis in eukaryotes and therapeutic targets for antifungal azoles. Multiple attempts to repurpose antifungals for treatment of human infections with protozoa (Trypanosomatidae) have been undertaken, yet so far none of them have revealed sufficient efficacy. VNI and its derivative VFV are two potent experimental inhibitors of Trypanosomatidae CYP51, effective in vivo against Chagas disease, visceral leishmaniasis, and sleeping sickness and currently under consideration as antiprotozoal drug candidates. However, VNI is less potent against Leishmania and drug-resistant strains of Trypanosoma cruzi and VFV, while displaying a broader spectrum of antiprotozoal activity, and is metabolically less stable. In this work we have designed, synthesized, and characterized a set of close analogues and identified two new compounds (7 and 9) that exceed VNI/VFV in their spectra of antiprotozoal activity, microsomal stability, and pharmacokinetics (tissue distribution in particular) and, like VNI/VFV, reveal no acute toxicity.

Synthesis and Biological Activity of Sterol 14α-Demethylase and Sterol C24-Methyltransferase Inhibitors.

Sterol 14α-demethylase (SDM) is essential for sterol biosynthesis and is the primary molecular target for clinical and agricultural antifungals. SDM has been demonstrated to be a valid drug target for antiprotozoal therapies, and much research has been focused on using SDM inhibitors to treat neglected tropical diseases such as human African trypanosomiasis (HAT), Chagas disease, and leishmaniasis. Sterol C24-methyltransferase (24-SMT) introduces the C24-methyl group of ergosterol and is an enzyme found in pathogenic fungi and protozoa but is absent from animals. This difference in sterol metabolism has the potential to be exploited in the development of selective drugs that specifically target 24-SMT of invasive fungi or protozoa without adversely affecting the human or animal host. The synthesis and biological activity of SDM and 24-SMT inhibitors are reviewed herein.