The free-living ameboflagellate Naegleria fowleri is an opportunistic pathogen causing a fulminating brain infection namely primary amoebic meningoencephalitis (PAM) that can result in death within days, with a worldwide distribution and over 97% fatality rate. Even though PAM is considered rare, with 381 global PAM cases reported by the Center for Disease Control and Prevention, this is likely an underestimate of the true worldwide occurrence of PAM. Currently, there is no standard regimen to treat N. fowleri infections in humans and only seven patients have been successfully treated so far using Amphotericin B (AmpB), either alone or in combination with other drugs. However, clinical use of AmpB is limited due to its toxicity, including acute infusion-related reactions and dose-related nephrotoxicity. 4 For these reasons the development of effective and safe drugs for the PAM treatment represents a real unmet medical need. Over the past few years, we validated several steroidogenic enzymes as drug targets. In particular, disruption of sterol 14-demethylase (CYP51) function by sterol biosynthesis inhibitors, induced massive autophagocytosis leading to N. fowleri cell death after 24 h of drug exposure. Notably, in vitro growth inhibition of N. fowleri by CYP51 inhibitors, including antifungal conazole drugs, has been reported in literature and some of them, even though endowed with poor blood-brain barrier (BBB) permeability, have been used in combination therapies with AmpB for the treatment of PAM patients. In this work, we provide evidence that miconazole-like compounds could be considered as drug candidates for the treatment of PAM. We used a combination of the cheminformatics, target-based and phenotypic drug discovery methods to identify a lead scaffold conducive to BBB permeability capable of targeting N. fowleri CYP51 (NfCYP51). 124 compounds pre-selected in silico were tested against N. fowleri trophozoites, allowing to identify nine hits with EC 50 ≤ 10 µM. The top hit was identified via cross- validation in co-crystallization with the NfCYP51 target that singled out a miconazole-like scaffold having the best drug-target fit. Based on the co-crystal structure, a set of analogs was synthesized and biochemically evaluated, confirming the superiority of the S- over R-configuration and the advantage of ether over ester linkage. The two best acting compounds exhibited improved EC 50 and K D compared to hit, both readily distributed into the brain. Brain-to-plasma distribution coefficient of the best acting compound was 1.02±0.12, holding a promise for further optimization into a drug candidate. Synthetic pathways, in vitro activities, X-ray crystallography data and pharmacokinetic study will be shown and discussed.
Design and synthesis of brain permeable anti-Naegleria fowleri agents targeting CYP51 for primary amoebic meningoencephalitis treatment / Madia, V. N.; Ialongo, D.; Messore, A.; Patacchini, E.; Arpacioglu, M.; Tudino, V.; Sharma, V.; Nguyen, J.; Debnath, A.; Podust, L.; Palenca, I.; Basili Franzin, S.; Seguella, L.; Esposito, G.; Petrucci, R.; Di Matteo, P.; Bortolami, M.; Scipione, L.; Costi, R.; Di Santo, R.. - (2023). (Intervento presentato al convegno IX EFMC International Symposium on Advances in Synthetic and Medicinal Chemistry tenutosi a Zagreb; Croatia.).
Design and synthesis of brain permeable anti-Naegleria fowleri agents targeting CYP51 for primary amoebic meningoencephalitis treatment.
Madia, V. N.;Ialongo, D.;Messore, A.;Patacchini, E.;Arpacioglu, M.;Tudino, V.;Scipione, L.;Costi, R.;Di Santo, R.
2023
Abstract
The free-living ameboflagellate Naegleria fowleri is an opportunistic pathogen causing a fulminating brain infection namely primary amoebic meningoencephalitis (PAM) that can result in death within days, with a worldwide distribution and over 97% fatality rate. Even though PAM is considered rare, with 381 global PAM cases reported by the Center for Disease Control and Prevention, this is likely an underestimate of the true worldwide occurrence of PAM. Currently, there is no standard regimen to treat N. fowleri infections in humans and only seven patients have been successfully treated so far using Amphotericin B (AmpB), either alone or in combination with other drugs. However, clinical use of AmpB is limited due to its toxicity, including acute infusion-related reactions and dose-related nephrotoxicity. 4 For these reasons the development of effective and safe drugs for the PAM treatment represents a real unmet medical need. Over the past few years, we validated several steroidogenic enzymes as drug targets. In particular, disruption of sterol 14-demethylase (CYP51) function by sterol biosynthesis inhibitors, induced massive autophagocytosis leading to N. fowleri cell death after 24 h of drug exposure. Notably, in vitro growth inhibition of N. fowleri by CYP51 inhibitors, including antifungal conazole drugs, has been reported in literature and some of them, even though endowed with poor blood-brain barrier (BBB) permeability, have been used in combination therapies with AmpB for the treatment of PAM patients. In this work, we provide evidence that miconazole-like compounds could be considered as drug candidates for the treatment of PAM. We used a combination of the cheminformatics, target-based and phenotypic drug discovery methods to identify a lead scaffold conducive to BBB permeability capable of targeting N. fowleri CYP51 (NfCYP51). 124 compounds pre-selected in silico were tested against N. fowleri trophozoites, allowing to identify nine hits with EC 50 ≤ 10 µM. The top hit was identified via cross- validation in co-crystallization with the NfCYP51 target that singled out a miconazole-like scaffold having the best drug-target fit. Based on the co-crystal structure, a set of analogs was synthesized and biochemically evaluated, confirming the superiority of the S- over R-configuration and the advantage of ether over ester linkage. The two best acting compounds exhibited improved EC 50 and K D compared to hit, both readily distributed into the brain. Brain-to-plasma distribution coefficient of the best acting compound was 1.02±0.12, holding a promise for further optimization into a drug candidate. Synthetic pathways, in vitro activities, X-ray crystallography data and pharmacokinetic study will be shown and discussed.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
