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.

Lanosterol 14α-demethylase (CYP51)/histone deacetylase (HDAC) dual inhibitors for treatment of Candida tropicalis and Cryptococcus neoformans infections.

Invasive fungal infections remain a challenge due to lack of effective antifungal agents and serious drug resistance. Discovery of antifungal agents with novel antifungal mechanism is important and urgent. Previously, we designed the first CYP51/HDAC dual inhibitors with potent activity against resistant Candida albicans infections. To better understand the antifungal spectrum and synergistic mechanism, herein new CYP51/HDAC dual inhibitors were designed which showed potent in vitro and in vivo antifungal activity against C. neoformans and C. tropicalis infections. Antifungal mechanism studies revealed that the CYP51/HDAC dual inhibitors acted by inhibiting various virulence factors of C. tropicalis and C. neoformans and down-regulating resistance-associated genes. This study highlights the potential of CYP51/HDAC dual inhibitors as a promising strategy for the discovery of novel broad-spectrum antifungal agents.

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.

Molecular Modeling Studies of Halogenated Imidazoles against 14α- Demethylase from Candida Albicans for Treating Fungal Infections.

BACKGROUND: Imidazole is one of the most explored and marketed azole utilized for the treatment of fungal infections. Lanosterol 14α-demethylase (Cytochrome P450DM) is the active target site for azole antifungals. AIM AND OBJECTIVE: This study emphasized on evaluation of a series of halogenated imidazole analogues using molecular docking studies for anti-Candidal activity. Furthermore, the model was refined by molecular dynamic simulation. METHODS: Halogenated imidazole analogues (PS1-PS30) were obtained from literature for the study. The imidazole analogues were prepared using Chem sketch and molecular docking was performed using Molergo Virtual Docker program and ADMET study was carried out by using Accelry's Accord for Excel programme. RESULTS: The docking study indicated that all the imidazole analogues (PS1-PS30) and standard drugs i.e., Ketoconazole, Miconazole and Clotrimazole possessed interaction with protein residue, heme cofactor and water molecule positioned above Heme cofactor of 14α-demethylase. Further, the ADMET study indicated that most of the halogenated imidazoles possessed good absorption, human intestinal absorption, aqueous solubility and blood brain penetration. CONCLUSION: Halogenated imidazole analogues may be used as potential lead molecules as 14α- demethylase inhibitors.

Identification of Pyrazolo[3,4-e][1,4]thiazepin based CYP51 inhibitors as potential Chagas disease therapeutic alternative: In vitro and in vivo evaluation, binding mode prediction and SAR exploration.

American trypanosomiasis or Chagas disease (CD) is a vector borne pathology caused by the parasite Trypanosoma cruzi (T. cruzi), which remains a serious global health problem. The current available treatment for CD is limited to two nitroderivatives with limited efficacy and several side effects. The rational design of ergosterol synthetic route inhibitors (e.g. CYP51 inhibitors) represents a promising strategy for fungi and trypanosomatids, exhibiting excellent anti-T.cruzi activity in pre-clinical assays. In the present work, we evaluate through different approaches (molecular docking, structure activity relationships, CYP51 inhibitory assay, and phenotypic screenings in vitro and in vivo) the potency and selectivity of a novel CYP51 inhibitor (compound 1) and its analogues against T.cruzi infection. Regarding anti-parasitic effect, compound 1 was active in vitro with EC50 3.86 and 4.00 µM upon intracellular (Tulahuen strain) and bloodstream forms (Y strain), respectively. In vivo assays showed that compound 1 reduced in 43% the parasitemia peak but, unfortunately failed to promote animal survival. In order to promote an enhancement at the potency and pharmacological properties, 17 new analogues were purchased and screened in vitro. Our findings demonstrated that five compounds were active against intracellular forms, highlighting compounds 1e and 1f, with EC50 2.20 and 2.70 µM, respectively, and selectivity indices (SI) = 50 and 36, respectively. Against bloodstream trypomastigotes, compound 1f reached an EC50 value of 20.62 µM, in a similar range to Benznidazole, but with low SI (3). Although improved the solubility of compound 1, the analogue 1f did not enhance the potency in vitro neither promote better in vivo efficacy against mouse model of acute T.cruzi infection arguing for the synthesis of novel pyrazolo[3,4-e][1,4]thiazepin derivatives aiming to contribute for alternative therapies for CD.

Homology model, molecular dynamics simulation and novel pyrazole analogs design of Candida albicans CYP450 lanosterol 14 α-demethylase, a target enzyme for antifungal therapy.

Candida albicans infections and their resistance to clinically approved azole drugs are major concerns for human. The azole antifungal drugs inhibit the ergosterol synthesis by targeting lanosterol 14α-demethylase of cytochrome P450 family. The lack of high-resolution structural information of fungal pathogens has been a barrier for the design of modified azole drugs. Thus, a preliminary theoretical molecular dynamic study is carried out to develop and validate a simple homologous model using crystallographic structure of the lanosterol 14α-demethylase of Mycobacterium tuberculosis (PDB ID-1EA1) in which the active site residues are substituted with that of C. albicans (taxid 5476). Further, novel designed pyrazole analogs (SGS1-16) docked on chimeric 1EA1 and revealed that SGS-16 show good binding affinity through non-bonding interaction with the heme, which is different from the leading azole antifungals. The ADME-T results showed these analogs can be further explored in design of more safe and effective antifungal agents.

Anti-Candida activity and cytotoxicity of a large library of new N-substituted-1,3-thiazolidin-4-one derivatives.

On the basis of the recent findings about the biological properties of thiazolidinones and taking into account the encouraging results about the antifungal activity of some (thiazol-2-yl)hydrazines, new N-substituted heterocyclic derivatives were designed combining the thiazolidinone nucleus with the hydrazonic portion. In details, 1,3-thiazolidin-4-ones bearing (cyclo)aliphatic or (hetero)aromatic moieties linked to the N1-hydrazine at C2 were synthesized and classified into three series according to the aromatic or bicyclic rings connected to the lactam nitrogen of the thiazolidinone. These molecules were assayed for their anti-Candida effects in reference to the biological activity of the conventional topic (clotrimazole, miconazole, tioconazole) and systemic drugs (fluconazole, ketoconazole, amphotericin B). Finally, we investigated the selectivity against fungal cells by testing the compounds endowed with the best MICs on Hep2 cells in order to assess their cell toxicity (CC50) and we noticed that two derivatives were less cytotoxic than the reference drug clotrimazole. Moreover, a preliminary molecular modelling approach has been performed against lanosterol 14-α demethylase (CYP51A1) to rationalize the activity of the tested compounds and to specify the target protein or enzyme.

[Clinical usefulness of triazole derivatives in the management of fungal infections]. / La utilidad clínica de los derivados triazólicos en el tratamiento de las infecciones fúngicas.

Current therapy for mycoses is limited to the use of a relative reduced number of antifungal drugs. Although amphotericin B still remains considered as the "gold standard" for treatment, acute and chronic toxicity, such as impairment of renal function, limits its use and enhances the investigation and clinical use other chemical families of antifungal drugs. One of these chemical class of active drugs are azole derivatives, discovered in 70s and introduced in clinical practice in 80s. Being the most prolific antifungal class, investigation about more molecules, with a safer and better pharmacological profile, active against a wide spectrum of fungi, with a wide range of administration routes gives us some azole representatives.

Design, synthesis and evaluation of benzotriazole derivatives as novel antifungal agents.

Considering the need for discovery of new antifungal drugs with greater potency and broader spectrum of activity, a new series of 5-substituted benzotriazole derivatives were designed, through structure based design, as inhibitors of fungal cytochrome P450 lanosterol 14-α demethylase. These were further optimized by a combination of iterative medicinal chemistry principles and molecular docking. Based on the best docking scores, some benzotriazole derivatives were synthesized and characterized by IR, (1)H NMR and MS/MS. The molecules were evaluated for their antifungal action against Candida albicans by cup plate method and ergosterol quantification method by UV spectroscopy. Reasonably good correlation between docking scores and antifungal activity were observed. The computational predictions were in consensus with the experimental results.

Design and synthesis of new fluconazole analogues.

We have synthesized new fluconazole analogues containing two different 1,2,3-triazole units in the side chain. The synthesis of new amide analogues using a variety of acids is also described. All the compounds showed very good antifungal activity. A hemolysis study of the most active compounds 6e and 13j showed that both compounds did not cause any hemolysis at the dilutions tested. These compounds did not exhibit any toxicity to L929 cells at MIC and lower concentrations. In the docking study, the overall binding mode of 6e and 13j appeared to be reasonable and provided a good insight into the structural basis of inhibition of Candida albicans Cyp51 by these compounds.

Virtual screening strategies in medicinal chemistry: the state of the art and current challenges.

Virtual screening (VS) techniques are well-established tools in the modern drug discovery process, mainly used for hit finding in drug discovery. The availability of knowledge of structural information, which includes an increasing number of 3D protein structures and the readiness of free databases of commercially available smallmolecules, provides a broad platform for VS. This review summarizes the current developments in VS regarding chemical databases and highlights the achievements as well as the challenges with an emphasis on a recent example of the successful application for the identification of new hits for sterol 14α-demethylase (CYP51) of Trypanosoma cruzi.

Efinaconazole (Jublia) for the treatment of onychomycosis.

Efinaconazole 10% nail solution (Jublia(®)) is a new topical triazole antifungal designed for the topical treatment of distal and lateral subungual onychomycosis. It inhibits ergosterol biosynthesis enzyme sterol 14α-demethylase. Efinaconazole has lower minimum inhibitory concentrations than terbinafine, ciclopirox, itraconazole and amorolfine in Trichophyton rubrum, Trichophyton mentagrophytes and Candida albicans. The solution based formula has low surface tension and keratin binding properties that increase penetrance through the nail plate. Safety studies have shown that this formulation is not associated with atopic dermatitis or contact sensitivity. Duplicate Phase III clinical trials in adults with mild to moderate distal and lateral subungual onychomycosis indicate that efinaconazole 10% solution is an effective therapy with a pooled complete cure rate of 17% and a pooled mycological cure rate of 54%. Efinaconazole 10% nail solution is a safe and effective new topical therapy for onychomycosis, which will fill a pressing need for more effective topical therapy in this disease.

Nitroheterocyclic compounds are more efficacious than CYP51 inhibitors against Trypanosoma cruzi: implications for Chagas disease drug discovery and development.

Advocacy for better drugs and access to treatment has boosted the interest in drug discovery and development for Chagas disease, a chronic infection caused by the genetically heterogeneous parasite, Trypanosoma cruzi. In this work new in vitro assays were used to gain a better understanding of the antitrypanosomal properties of the most advanced antichagasic lead and clinical compounds, the nitroheterocyclics benznidazole, nifurtimox and fexinidazole sulfone, the oxaborole AN4169, and four ergosterol biosynthesis inhibitors--posaconazole, ravuconazole, EPL-BS967 and EPL-BS1246. Two types of assays were developed: one for evaluation of potency and efficacy in dose-response against a panel of T. cruzi stocks representing all current discrete typing units (DTUs), and a time-kill assay. Although less potent, the nitroheterocyclics and the oxaborole showed broad efficacy against all T. cruzi tested and were rapidly trypanocidal, whilst ergosterol biosynthesis inhibitors showed variable activity that was both compound- and strain-specific, and were unable to eradicate intracellular infection even after 7 days of continuous compound exposure at most efficacious concentrations. These findings contest previous reports of variable responses to nitroderivatives among different T. cruzi strains and further challenge the introduction of ergosterol biosynthesis inhibitors as new single chemotherapeutic agents for the treatment of Chagas disease.

Drug discovery for neglected tropical diseases at the Sandler Center.

The Sandler Center's approach to target-based drug discovery for neglected tropical diseases is to focus on parasite targets that are homologous to human targets being actively investigated in the pharmaceutical industry. In this way we attempt to use both the know-how and actual chemical matter from other drug-development efforts to jump start the discovery process for neglected tropical diseases. Our approach is akin to drug repurposing, except that we seek to repurpose leads rather than drugs. Medicinal chemistry can then be applied to optimize the leads specifically for the desired antiparasitic indication.

Homology modeling and screening of new 14α-demethylase inhibitor (DMI) fungicides based on optimized expression of CYP51 from Ustilago maydis in Escherichia coli.

Ustilago maydis infection is a serious disease affecting corn crops worldwide. Sterol 14α-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis and an effective target of antifungal drugs. To further study the interaction between CYP51 and drugs and exploit more specific 14α-demethylase inhibitor (DMI) fungicides for U. maydis, in this study homology modeling of CYP51 from U. maydis (UmCYP51) templated as the eukaryotic orthologues (the human CYP51) and screening of new DMI fungicides based on optimized expression were carried out for the first time. In addition, XF-113 and ZST-4 were screened by analyzing the spectral characteristics between the purified UmCYP51-35 and fungicides. These results provide a theoretical basis and new ideas for efficient design and development of new antifungal drugs.