Construction and activity evaluation of novel dual-target (SE/CYP51) anti-fungal agents containing amide naphthyl structure.

With the increase of fungal infection and drug resistance, it is becoming an urgent task to discover the highly effective antifungal drugs. In the study, we selected the key ergosterol bio-synthetic enzymes (Squalene epoxidase, SE; 14 α-demethylase, CYP51) as dual-target receptors to guide the construction of novel antifungal compounds, which could achieve the purpose of improving drug efficacy and reducing drug-resistance. Three different series of amide naphthyl compounds were generated through the method of skeleton growth, and their corresponding target products were synthesized. Most of compounds displayed the obvious biological activity against different Candida spp. and Aspergillus fumigatus. Among of them, target compounds 14a-2 and 20b-2 not only possessed the excellent broad-spectrum anti-fungal activity (MIC50, 0.125-2 µg/mL), but also maintained the anti-drug-resistant fungal activity (MIC50, 1-4 µg/mL). Preliminary mechanism study revealed the compounds (14a-2, 20b-2) could block the bio-synthetic pathway of ergosterol by inhibiting the dual-target (SE/CYP51) activity, and this finally caused the cleavage and death of fungal cells. In addition, we also discovered that compounds 14a-2 and 20b-2 with low toxic and side effects could exert the excellent therapeutic effect in mice model of fungal infection, which was worthy for further in-depth study.

MiR-205-driven downregulation of cholesterol biosynthesis through SQLE-inhibition identifies therapeutic vulnerability in aggressive prostate cancer.

Prostate cancer (PCa) shows strong dependence on the androgen receptor (AR) pathway. Here, we show that squalene epoxidase (SQLE), an enzyme of the cholesterol biosynthesis pathway, is overexpressed in advanced PCa and its expression correlates with poor survival. SQLE expression is controlled by micro-RNA 205 (miR-205), which is significantly downregulated in advanced PCa. Restoration of miR-205 expression or competitive inhibition of SQLE led to inhibition of de novo cholesterol biosynthesis. Furthermore, SQLE was essential for proliferation of AR-positive PCa cell lines, including abiraterone or enzalutamide resistant derivatives, and blocked transactivation of the AR pathway. Inhibition of SQLE with the FDA approved antifungal drug terbinafine also efficiently blocked orthotopic tumour growth in mice. Finally, terbinafine reduced levels of prostate specific antigen (PSA) in three out of four late-stage PCa patients. These results highlight SQLE as a therapeutic target for the treatment of advanced PCa.

Design, synthesis and bioactivity evaluation of novel arylalkene-amide derivatives as dual-target antifungal inhibitors.

Ergosterol as the core component of fungal cell membrane plays a key role in maintaining cell morphology and permeability. The squalenee epoxidase (SE) and 14-demethylase (CYP51) are the important rate-limiting enzymes for ergosterol synthesis. In the study, these active fragments, which is derived from the structural groups of the common antifungal agents, were docked into the active sites of dual targets (SE, CYP51), respectively. Some of active fragments with the matching MCSS_Score values were selected and connected to construct three different series of novel arylalkene-amide derivatives as dual-target (SE, CYP51) antifungal inhibitors. Subsequently, these compounds were further synthesized, and their bioactivity was evaluated. Most of compounds showed a certain degree of antifungal activity in vitro. It was worth noting that the target compounds 17a and 25a with excellent antifungal activity (0.125-4 µg/mL) can inhibit the fluconazole-resistant Candida Strain 17#, CaR, 632, and 901 in the range of MIC values (4-8 µg/mL). Furthermore, their molecular mechanism, structural stability and low toxicity were further confirmed. The molecular docking and ADMET properties were predicted to guide the subsequent optimization of target compounds.

Preclinical toxicology profile of squalene epoxidase inhibitors.

Small cell lung cancer (SCLC) is a particularly aggressive subset of lung cancer, and identification of new therapeutic options is of significant interest. We recently reported that SCLC cell lines display a specific vulnerability to inhibition of squalene epoxidase (SQLE), an enzyme in the cholesterol biosynthetic pathway that catalyzes the conversion of squalene to 2,3-oxidosqualene. Since it has been reported that SQLE inhibition can result in dermatitis in dogs, we conducted a series of experiments to determine if SQLE inhibitors would be tolerated at exposures predicted to drive maximal efficacy in SCLC tumors. Detailed profiling of the SQLE inhibitor NB-598 showed that dogs did not tolerate predicted efficacious exposures, with dose-limiting toxicity due to gastrointestinal clinical observations, although skin toxicities were also observed. To extend these studies, two SQLE inhibitors, NB-598 and Cmpd-4″, and their structurally inactive analogs, NB-598.ia and Cmpd-4″.ia, were profiled in monkeys. While both active SQLE inhibitors resulted in dose-limiting gastrointestinal toxicity, the structurally similar inactive analogs did not. Collectively, our data demonstrate that significant toxicities arise at exposures well below the predicted levels needed for anti-tumor activity. The on-target nature of the toxicities identified is likely to limit the potential therapeutic utility of SQLE inhibition for the treatment of SCLC.

FT-IR investigation of Terbinafine interaction with stratum corneum constituents.

Terbinafine (Tbf) is a well-established anti-fungal agent used for management of a variety of dermal conditions including ringworm and athlete's foot. Both the biochemical mechanism of Tbf fungicidal action (based on squalene epoxidase inhibition) and the target region for Tbf in vivo (the stratum corneum (SC)) are well determined. However, the biochemical and pharmacokinetic approaches used to evaluate Tbf biochemistry provide no biophysical information about molecular level physical changes in the SC upon Tbf binding. Such information is necessary for improved drug and formulation design. IR spectroscopic methods were used to evaluate the effects of Tbf on keratin structure in environments commonly used in pharmaceutics to mimic those in vivo. The Amide I and II spectral regions (1500-1700 cm-1) provided an effective means to monitor keratin secondary structure changes, while a Tbf spectral feature near 775 cm-1 provides a measure of relative Tbf levels in skin. Interaction of Tbf with the SC induced substantial ß-sheet formation in the keratin, an effect which was partially reversed both by ethanol washing and by exposure to high relative humidity. The irreversibility suggests the presence of a Tbf reservoir (consistent with kinetic studies), permitting the drug to be released in a controlled manner into the surrounding tissue.

Squalene epoxidase-induced cholesteryl ester accumulation promotes nasopharyngeal carcinoma development by activating PI3K/AKT signaling.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor and a major cause of mortality and morbidity in southern China. However, the mechanism is still elusive. Here, we focused on studying the role of squalene epoxidase (SQLE), a key enzyme of cholesterol biosynthesis, in the progression of NPC. Clinical study revealed that SQLE expression was significantly upregulated in NPC tissues compared to normal tissues from mRNA level and patients with high expression of SQLE showed a poor prognosis. In vitro experiments showed that SQLE overexpression led to a significant proliferation of cells whereas SQLE knockdown showed an opposite result. In vivo studies also showed that SQLE promoted tumor growth in nude mice. Further study revealed that SQLE promoted NPC proliferation by cholesteryl ester accumulation instead of cholesterol. Mechanism studies indicated that cholesteryl ester promoted NPC cell proliferation by activating the PI3K/AKT pathway and inhibition of this pathway in SQLE-overexpressed or cholesteryl ester-treated cells resulted in a significant reduction of NPC cell proliferation. These results indicate that the oncogenic effect of SQLE in NPC mainly resulted from cholesteryl ester accumulation and PI3K/AKT is a promising target for NPC with SQLE overexpression.

A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate.

Cholesterol biosynthesis is a high-cost process and, therefore, tightly regulated by both transcriptional and posttranslational negative feedback mechanisms in response to the level of cellular cholesterol. Squalene monooxygenase (SM, also known as squalene epoxidase or SQLE) is a rate-limiting enzyme in the cholesterol biosynthetic pathway and catalyzes epoxidation of squalene. The stability of SM is negatively regulated by cholesterol via its N-terminal regulatory domain (SM-N100). In this study, using a SM-luciferase fusion reporter cell line, we performed a chemical genetics screen that identified inhibitors of SM itself as up-regulators of SM. This effect was mediated through the SM-N100 region, competed with cholesterol-accelerated degradation, and required the E3 ubiquitin ligase MARCH6. However, up-regulation was not observed with statins, well-established cholesterol biosynthesis inhibitors, and this pointed to the presence of another mechanism other than reduced cholesterol synthesis. Further analyses revealed that squalene accumulation upon treatment with the SM inhibitor was responsible for the up-regulatory effect. Using photoaffinity labeling, we demonstrated that squalene directly bound to the N100 region, thereby reducing interaction with and ubiquitination by MARCH6. Our findings suggest that SM senses squalene via its N100 domain to increase its metabolic capacity, highlighting squalene as a feedforward factor for the cholesterol biosynthetic pathway.

The shape of human squalene epoxidase expands the arsenal against cancer.

Squalene epoxidase (also known as squalene monooxygenase, EC 1.14.99.7) is a key rate-limiting enzyme in cholesterol biosynthesis. Anil Padyana and colleagues report the long awaited structure of human squalene epoxidase (SQLE). They solved the crystal structure of the catalytic domain of human SQLE alone and in complex with two similar pharmacological inhibitors and elucidate their mechanism of action. SQLE is the target of fungicides and of increasing interest in human health and disease, particularly as a new anti-cancer target. Indeed, in a companion paper, Christopher Mahoney and colleagues performed an inhibitor screen with cancer cell lines and identified SQLE as an unique vulnerability in a subset of neuroendocrine tumours, where SQLE inhibition caused a toxic accumulation of the substrate squalene. The SQLE structure will facilitate the development of improved inhibitors. Here, we comment on these two studies in the wider context of the field and discuss possible future directions.

Design, synthesis and biological evaluation of amide-pyridine derivatives as novel dual-target (SE, CYP51) antifungal inhibitors.

Based on the analysis of the squalene cyclooxygenase (SE) and 14α-demethylase (CYP51) inhibitors pharmacophore feature and the dual-target active sites, a series of compounds with amide-pyridine scaffolds have been designed and synthesized to treat the increasing incidence of drug-resistant fungal infections. In vitro evaluation showed that these compounds have a certain degree of antifungal activity. The most potent compounds 11a, 11b with MIC values in the range of 0.125-2 µg/ml had a broad-spectrum antifungal activity and exhibited excellent inhibitory activity against drug-resistant pathogenic fungi. Preliminary mechanism studies revealed that the compound 11b might play an antifungal role by inhibiting the activity of SE and CYP51. Notably compounds did not show the genotoxicity through plasmid binding assay. Finally, this study of molecular docking, ADME/T prediction and the construction of 3D QSAR model were performed. These results can point out the direction for further optimization of the lead compound.

Structure and inhibition mechanism of the catalytic domain of human squalene epoxidase.

Squalene epoxidase (SQLE), also known as squalene monooxygenase, catalyzes the stereospecific conversion of squalene to 2,3(S)-oxidosqualene, a key step in cholesterol biosynthesis. SQLE inhibition is targeted for the treatment of hypercholesteremia, cancer, and fungal infections. However, lack of structure-function understanding has hindered further progression of its inhibitors. We have determined the first three-dimensional high-resolution crystal structures of human SQLE catalytic domain with small molecule inhibitors (2.3 Å and 2.5 Å). Comparison with its unliganded state (3.0 Å) reveals conformational rearrangements upon inhibitor binding, thus allowing deeper interpretation of known structure-activity relationships. We use the human SQLE structure to further understand the specificity of terbinafine, an approved agent targeting fungal SQLE, and to provide the structural insights into terbinafine-resistant mutants encountered in the clinic. Collectively, these findings elucidate the structural basis for the specificity of the epoxidation reaction catalyzed by SQLE and enable further rational development of next-generation inhibitors.

A chemical biology screen identifies a vulnerability of neuroendocrine cancer cells to SQLE inhibition.

Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers.

Antifungal Agents: Design, Synthesis, Antifungal Activity and Molecular Docking of Phloroglucinol Derivatives.

Pseudoaspidinol is a phloroglucinol derivative with Antifungal activity and is a major active component of Dryopteris fragrans. In our previous work, we studied the total synthesis of pseudoaspidinol belonging to a phloroglucinol derivative and investigated its antifungal activity as well as its intermediates. However, the results showed these compounds have low antifungal activity. In this study, in order to increase antifungal activities of phloroglucinol derivatives, we introduced antifungal pharmacophore allylamine into the methylphloroglucinol. Meanwhile, we remained C1⁻C4 acyl group in C-6 position of methylphloroglucinol using pseudoaspidinol as the lead compound to obtain novel phloroglucinol derivatives, synthesized 17 compounds, and evaluated antifungal activities on Trichophyton rubrum and Trichophyton mentagrophytes in vitro. Molecular docking verified their ability to combine the protein binding site. The results indicated that most of the compounds had strong antifungal activity, in which compound 17 were found to be the most active on Trichophyton rubrum with Minimum Inhibitory Concentration (MIC) of 3.05 µg/mL and of Trichophyton mentagrophytes with MIC of 5.13 µg/mL. Docking results showed that compounds had a nice combination with the protein binding site. These researches could lay the foundation for developing antifungal agents of clinical value.

Studies of 4-arylthiazolylhydrazones derived from 1-indanones as Trypanosoma cruzi squalene epoxidase inhibitors by molecular simulations.

Chagas disease or American trypanosomiasis is a parasitic disease caused by the protozoan Trypanosoma cruzi. Its squalene epoxidase (SE) is a target for drug design and development because it is a key enzyme in the biosynthetic pathway of ergosterol, which is essential for the life cycle of the parasite. Previously, we reported that some 4-arylthiazolylhydrazones derived from 1-indanones (TZHs) active against T. cruzi are able to accumulate squalene probably by SE inhibition. In this work, we performed a series of theoretical studies to verify that TZHs act as inhibitors of this enzyme. Since the crystal structure of SE is unknown for all species, we built a 3D enzyme model of T. cruzi SE by homology modeling. Based on this model, we carried out docking, molecular dynamics, and MM/PBSA calculations and the results were compared with those found for the reference inhibitor compound terbinafine (Tbf). The binding free energy values allowed the discrimination between accumulators and non-accumulators of squalene compounds, in agreement with the experimental findings. Pairwise residue free energy decomposition showed that the key amino acids involved in inhibitor binding for TZHs and Tbf were the same. Also, molecular superposition analysis between these compounds revealed high structural similarity. In addition, we proposed a pharmacophore model for T. cruzi SE inhibitors, which confirmed that TZHs and Tbf share chemical features with respect to their biochemical interaction characteristics at similar positions in 3D space. All theoretical calculations suggest that the experimentally observed squalene accumulation is produced by T. cruzi SE inhibition.

Synthesis, activity, and docking studies of eugenol-based glucosides as new agents against Candida sp.

Seventeen new synthetic derivatives of eugenol (6, 8-15 and 8'-15') were planned following literature reports on antifungal activities of nitroeugenol and eugenol glucoside. The anti-Candida activity of these compounds was investigated by in vitro assay, and the cytotoxicity evaluation was performed with the most active compounds. The peracetylated glucosides presented better biological results than their hydroxylated analogues. The glucoside 11, a 4-nitrobenzamide, showed the best potency (MIC50 range 11.0-151.84 µm), the wider spectrum of action, and overall the best selectivity indexes, especially against C. tropicalis (~30) and C. krusei (~15). To investigate its possible mechanism of action, glucoside 11 was subjected to molecular docking studies with Candida sp. enzymes involved in ergosterol biosynthesis. Results have shown that the peracetyl glucosyl moiety and the 4-nitrobenzamide group in 11 are effectively involved in its high affinity with the active site of squalene epoxidase.

Squalene epoxidase as a promising metabolic target in cancer treatment.

Oncogenic alteration of the cholesterol synthesis pathway is a recognized mechanism of metabolic adaptation. In the present review, we focus on squalene epoxidase (SE), one of the two rate-limiting enzymes in cholesterol synthesis, retracing its history since its discovery as an antimycotic target to its description as an emerging metabolic oncogene by amplification with clinical relevance in cancer. We review the published literature assessing the association between SE over-expression and poor prognosis in this disease. We assess the works demonstrating how SE promotes tumor cell proliferation and migration, and displaying evidence of cancer cell demise in presence of human SE inhibitors in in vitro and in vivo models. Taken together, robust scientific evidence has by now accumulated pointing out SE as a promising novel therapeutic target in cancer treatment.

Comparison and analysis of the structures and binding modes of antifungal SE and CYP51 inhibitors.

With the abuse of clinical broad-spectrum antimicrobial agents, immunosuppressive agents, chemotherapy drugs, the emergence of pathogenic fungi resistance is more and more frequent. However, there is still no effective treatment for the fungal resistance. Squalenee epoxidase (SE) and 14 α-demethylase (CYP51) are important antifungal drug targets. In order to achieve a deeper insight into the structural characteristics and the action modes of SE and CYP51inhibitors, the homology model of SE (Candida albicans) was constructed using monooxygenase of Pseudomonas aeruginosa as template, and the reliability of model was confirmed by Ramachandran plots and Verify 3D. Subsequently, the molecular superimposition and molecular docking were performed, and the pharmacophore model based on the CYP51 receptor structure was constructed. The results indicate that SE and CYP51 inhibitors have common structural feature with two parts of essential fragments, which are mainly composed of aromatic groups. In addition, the fragment structures of inhibitors are combined in the similar hydrophobic pockets through the hydrophobic forces. The present study provides a deeper perspective to understand the characteristics and docking modes of SE and CYP51 inhibitors. It can be used to guide the optimization and design of SE and CYP51 inhibitors. In addition, it also provides the oretical support for the development of dual target antifungal inhibitors (SE and CYP51), which can help us solve the problem of fungi resistance.

Understanding the cholesterol metabolism-perturbing effects of docosahexaenoic acid by gas chromatography-mass spectrometry targeted metabonomic profiling.

PURPOSE: Over the past few decades, docosahexaenoic acid (DHA) has gained special attention for management of cholesterol-associated metabolic disorders and neurodegenerative diseases such as Alzheimer's disease (AD) owing to its neuroprotective, anti-inflammatory and hypolipidemic properties. Several epidemiological studies have reported the effect of DHA in reducing the risk of developing AD by lowering cholesterol. Hypercholesterolemia is a pro-amyloidogenic factor influencing the enzymatic processing of amyloid-ß precursor protein (AßPP) to toxic ß-amyloid. However, the mechanism by which DHA modulates the cholesterol pathway has not been established. Thus, the objective of this study was to investigate the mechanism of regulation of cholesterol metabolism by DHA in an AßPP695 overexpressing AD cell model. METHODS: A gas chromatography/mass spectrometry method was developed and validated for the targeted profiling of 11 cholesterol metabolites in DHA-treated Chinese hamster ovary wild-type (CHO-wt) and AßPP695 overexpressing (CHO-AßPP695) cells. The differential metabolite profiles between DHA- and vehicle-treated groups were further analyzed using fold change values of the ratio of concentration of metabolites in CHO-AßPP695 to CHO-wt cells. Effect of DHA on key rate-limiting enzymatic activities within the cholesterol pathway was established using biochemical assays. RESULTS: Our results showed that DHA reduced the levels of key cholesterol anabolites and catabolites in CHO-AßPP695 cells as compared to CHO-wt cells. Further enzymatic studies revealed that the cholesterol-lowering effect of DHA was mediated by regulating HMG-CoA reductase and squalene epoxidase enzyme activities. CONCLUSION: We demonstrate for the first time the dual effects of DHA in inhibiting HMG-CoA reductase and squalene epoxidase and modulating the sterol biosynthesis axis of the cholesterol pathway in AßPP695 overexpressing AD. Our novel findings underscore the potential of DHA as a multi-target hypocholesterolemic agent for the prophylaxis of AD and other cholesterol-associated diseases.

Transcriptional Regulation of Cytosolic Sulfotransferase 1C2 by Intermediates of the Cholesterol Biosynthetic Pathway in Primary Cultured Rat Hepatocytes.

Cytosolic sulfotransferase 1C2 (SULT1C2) is expressed in the kidney, stomach, and liver of rats; however, the mechanisms regulating expression of this enzyme are not known. We evaluated transcriptional regulation of SULT1C2 by mevalonate (MVA)-derived intermediates in primary cultured rat hepatocytes using several cholesterol synthesis inhibitors. Blocking production of mevalonate with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin (30 µM), reduced SULT1C2 mRNA content by ∼40% whereas the squalene synthase inhibitor squalestatin (SQ1, 0.1 µM), which causes accumulation of nonsterol isoprenoids, increased mRNA content by 4-fold. Treatment with MVA (10 mM) strongly induced SULT1C2 mRNA by 12-fold, and this effect was blocked by inhibiting squalene epoxidase but not by more distal cholesterol inhibitors, indicating the effects of MVA are mediated by postsqualene metabolites. Using rapid amplification of cDNA ends (RACE), we characterized the 5' end of SULT1C2 mRNA and used this information to generate constructs for promoter analysis. SQ1 and MVA increased reporter activity by ∼1.6- and 3-fold, respectively, from a construct beginning 49 base pairs (bp) upstream from the longest 5'-RACE product (-3140:-49). Sequence deletions from this construct revealed a hepatocyte nuclear factor 1 (HNF1) element (-2558), and mutation of this element reduced basal (75%) and MVA-induced (30%) reporter activity and attenuated promoter activation following overexpression of HNF1α or 1ß. However, the effects of SQ1 were localized to a more proximal promoter region (-281:-49). Collectively, our findings demonstrate that cholesterol biosynthetic intermediates influence SULT1C2 expression in rat primary hepatocytes. Further, HNF1 appears to play an important role in mediating basal and MVA-induced SULT1C2 transcription.

[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.

Production of squalene by lactose-fermenting yeast Kluyveromyces lactis with reduced squalene epoxidase activity.

UNLABELLED: Utilization of yeast as squalene source for commercial use is limited by relatively high production costs. The ability of Kluyveromyces lactis to grow on cheap lactose-containing diary industry wastes could improve the economy of the production process. We therefore tested the potential of this yeast for squalene production. Accumulation of squalene was induced by partial inhibition of squalene epoxidase by a specific inhibitor terbinafine. Kluyveromyces lactis cultivated on glucose and lactose media showed similar growth sensitivity to terbinafine as Saccharomyces cerevisiae. The effect of terbinafine on neutral lipid pattern was tested at concentrations with low, moderate and strong growth inhibition (2·5, 5 and 7·5 µg ml(-1) , respectively). Compared to S. cerevisiae, treatment with subinhibitory terbinafine doses had a weaker effect on steryl ester levels and total ergosterol levels in K. lactis. Quantification of squalene levels in terbinafine-treated K. lactis cells revealed high accumulation of squalene particularly in cells treated with 7·5 µg ml(-1) terbinafine in lactose medium. Terbinafine treatment stimulated the development of lipid droplets as lipid storage organelles and this effect was different in K. lactis grown on glucose or lactose media. Present report is the first attempt to utilize lactose-fermenting yeast K. lactis for production of a high-value lipid and it proves squalene epoxidase as a promising target for squalene overproduction in this yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Squalene is a natural substance with wide applications in food, cosmetic and pharmaceutic industries. The suitability of lactose-fermenting yeast Kluyveromyces lactis for the production of squalene was tested in the study. Partial inhibition of squalene epoxidase by specific inhibitor terbinafine resulted in high accumulation of squalene in K. lactis grown on glucose or lactose comparable to values found in terbinafine-treated Saccharomyces cerevisiae. Our results prove that K. lactis is a promising micro-organism for genetic manipulations aimed at the production of squalene on industrial waste like whey as the growth substrate.