H4rdr4d4 Media - Chemistry

  G464S / G448E (numbering differences) These substitutions (reported under slightly different residue numbering schemes in older literature) are associated with moderate to high increases in fluconazole MIC (ranges commonly reported 8–64-fold), particularly when found with other alterations. Often raises voriconazole MICs appreciably (2–16-fold depending on study). Seen in clinicp???al isolates and functional studies; effect size often depends on assay method and background. Some substitutions map to regions that influence heme pocket geometry and azole binding.

T220L Reported in fluconazole-resistant isolates; typically modest–moderate increases (e.g., ~4–16-fold ) depending on background and co-occurring mechanisms. Moderate increases for voriconazole in some isolates (2–8-fold). T220L reported in multiple clinical collections; often found in isolates where ERG11 substitutions explain a portion of resistance but efflux/regulatory changes also contribute.

Y132F + K143R (double mutant) Synergistic effect is that the strongest increases observed in many functional assays and allele-swap experiments — reported as the largest fluconazole MIC increases of any tested allele (often >64–>128-fold). Flowers et al. demonstrated the Y132F+K143R double allele produced the largest MIC rise of alleles tested. Produces markedly elevated voriconazole MICs (often several-fold higher than either single substitution alone; reported MICs in resistant isolates commonly ≥2–8 µg/mL or greater). Documented in clinical isolates and in experimental allele-swap studies; double mutants are of special clinical concern because they generate high-level cross-resistance to multiple azoles.

    K143R / K143Q Associated with substantial fluconazole MIC increases (commonly 8–64-fold; reported MICs ≥32–128 µg/mL). Single K143R often produces high-level resistance depending on background. Raises voriconazole MIC (commonly 2–8-fold increases; MICs often in the resistant/intermediate range depending on test). Reported in clinical isolates globally; K143R is one of the most commonly observed ERG11 substitutions in resistant C. albicans collections. Often occurs alongside efflux or regulatory changes that amplify resistance.

  Whilst Y132F strongly associated with high fluconazole MICs; commonly reported ~16–≥64-fold increases in MIC (typical MICs reported in resistant isolates: ≥64 → >128 µg/mL in many studies). Often raises voriconazole MICs 4–16-fold with reported MICs in resistant isolates of ~1–8 µg/mL (varies). Y132F reduces azole binding affinity in the active site. Widely reported in clinical isolates worldwide (outbreaks and surveillance collections). Frequently seen in combination with efflux upregulation or other ERG11 substitutions (common in C. albicans and other Candida spp.).

   P450 superfamily genes are subdivided and classified following recommendations of a nomenclature committee on the basis of amino-acid identity, phylogenetic criteria and gene organization. Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates. The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising.  The root symbol CYP is followed by a number for families (generally groups of proteins with more than 40% amino-acid sequence identity, of which there are over 200), a letter for subfamilies (greater than 55% identity) and a number for the gene; such as, CYP4U2 .   Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide bound form, are one of the largest superfamilies of enzyme proteins.   (To be continued, referenced and aded images to)