Rifabutin

Specificity for Bacterial RNA Polymerase Subunits (e.g., β-subunit, RpoB)

This compound, like other rifamycins, specifically interacts with bacterial RNAP. drugbank.comhres.cawikidoc.orgpfizer.comwho.intasm.orgpharmacy180.com The binding site for rifamycins, including this compound, is located on the β subunit of bacterial RNAP, which is encoded by the rpoB gene. patsnap.comoup.comasm.orgpharmacy180.comwikipedia.orgnih.govasm.orgfishersci.camdpi.com This binding occurs within the DNA/RNA binding channel of the enzyme. asm.orgnih.gov Mutations in the rpoB gene, particularly within specific regions known as the Rifampicin resistance-determining region (RRDR), can alter the drug's binding site and lead to resistance. patsnap.comwikipedia.orgnih.govasm.orgmdpi.comcabidigitallibrary.org

Differential Binding to Bacterial vs. Mammalian Enzymes

A key aspect of this compound's selective toxicity is its differential binding affinity for bacterial versus mammalian RNAP. drugbank.comhres.cawikidoc.orgpfizer.comwho.intasm.orgpharmacy180.com this compound interacts strongly with prokaryotic RNAP but does not inhibit the mammalian enzyme. drugbank.comhres.cawikidoc.orgpfizer.comwho.intpharmacy180.com The binding constant of rifampicin, a related rifamycin, for eukaryotic RNAP is at least 100 times higher than its binding constant for prokaryotic enzymes, indicating a significantly lower affinity for the mammalian enzyme. asm.org This selectivity is attributed to sequence divergence between bacterial and human RNAPs. asm.org

Molecular Docking Studies with SARS-CoV-2 RNA-Dependent RNA Polymerase

Molecular docking studies have explored the potential binding of this compound to the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, the virus responsible for COVID-19. wikipedia.orgijmm.irijmm.irscienceopen.comarxiv.orguni-muenster.dewjgnet.com These in silico studies have suggested that this compound may have a potential inhibitory interaction with SARS-CoV-2 RdRp. ijmm.irijmm.irscienceopen.comarxiv.org One study using molecular docking found that this compound had a favorable binding affinity for SARS-CoV-2 RdRp, identifying a potential new binding site for the drug. ijmm.irijmm.ir Further research on this compound has been suggested as a potential avenue for discovering new drugs for COVID-19 based on these computational findings. ijmm.irijmm.ir

Molecular docking studies comparing this compound and other compounds for binding to SARS-CoV-2 RdRp have indicated that this compound could be an effective drug for COVID-19, showing a favorable binding energy compared to a positive control like remdesivir in one study. wjgnet.com These computational approaches, including drug repurposing and molecular docking, have aimed to predict effective drug candidates targeting SARS-CoV-2 RdRp. scienceopen.comarxiv.org

Role of Siderophore Receptors (e.g., FhuE) in Bacterial Uptake

Research has indicated that siderophore receptors play a role in the cellular uptake of this compound in certain bacteria, particularly in nutrient-limited environments. In Acinetobacter baumannii, a carbapenem-resistant bacterium, this compound has demonstrated potent activity, which is enabled by its cellular uptake through the siderophore receptor FhuE under iron-depleted growth conditions. nih.govresearchgate.netresearchgate.netnih.gov This FhuE-mediated active transport facilitates the accumulation of this compound inside the bacterial cell. nih.govresearchgate.netresearchgate.net Studies have confirmed the involvement of FhuE in the active translocation of this compound at low concentrations, highlighting the importance of this uptake mechanism for its activity against A. baumannii. nih.govresearchgate.net The FhuE receptor appears to be highly selective for this compound transport compared to other rifamycins like rifampicin. semanticscholar.org

Influence of Growth Medium on Uptake

The composition of the growth medium can significantly influence the uptake of this compound by bacteria. Studies have shown a strong impact of the culture medium on the accumulation of this compound in A. baumannii. nih.govresearchgate.net For instance, this compound demonstrated potent activity against A. baumannii in nutrient-limited medium, such as Roswell Park Memorial Institute (RPMI) 1640 supplemented with fetal calf serum (FCS), where the siderophore receptor FhuE is overexpressed. researchgate.netnih.govsemanticscholar.org In contrast, activity was different in standard media like cation-adjusted Mueller-Hinton broth (CA-MHB). semanticscholar.orgasm.org This suggests that conditions that promote the expression or activity of uptake mechanisms, such as siderophore receptors, can enhance this compound's cellular accumulation and, consequently, its activity. Variations in Minimum Inhibitory Concentration (MIC) values depending on the culture medium used for susceptibility testing have also been noted for Mycobacterium abscessus. asm.org

Comparative Analysis of Intracellular/Plasma Concentration Ratios

Studies in both human and animal models have consistently shown that this compound achieves substantially higher intracellular tissue levels compared to plasma concentrations. pfizermedicalinformation.comwho.int In vitro studies using human leukocytes have demonstrated high intracellular-to-extracellular concentration ratios. For instance, in neutrophils, this ratio has been reported to be around 9, while in monocytes, it is approximately 15. nih.govpfizer.comoup.commedsafe.govt.nz This high intracellular accumulation is considered to play a significant role in its efficacy against intracellular bacteria. medsafe.govt.nz Compared to rifampicin, this compound has shown higher intracellular penetration in human polymorphonuclear leukocytes and mouse spleen lymphocytes. oup.com

The following table summarizes representative intracellular/plasma concentration ratios:

In animal studies, radioactivity studies in rats have indicated a significantly higher tissue distribution of this compound compared to rifampicin. oup.com In humans, concentrations of this compound in lung tissue have been measured to be several times higher than plasma levels, with ratios of approximately 6.5 to 10 observed in surgical patients. pfizermedicalinformation.comnih.govpfizer.commedsafe.govt.nz High concentrations have also been reported in urine and bile, exceeding plasma levels by over 100 times, as well as in gastrointestinal tract tissues. nih.gov Muscle tissue also shows penetration, albeit at lower concentrations than in serum. nih.gov

Lipid Solubility and Membrane Partitioning in Distribution

The high lipophilicity of this compound is a key factor contributing to its extensive distribution and intracellular tissue uptake. pfizermedicalinformation.comnih.govoup.comresearchgate.netoup.com this compound is significantly more lipid-soluble than rifampicin, which is reflected in its higher oil/water partition coefficient. oup.comresearchgate.netoup.com This physicochemical property facilitates its ability to readily cross cell membranes and partition into lipid bilayers. nih.govblackwellpublishing.com

Studies investigating the interaction of this compound with lipid membranes have shown that it interacts both electrostatically and via hydrophobic interactions. nih.gov The membrane surface charge is a predominant factor influencing its selective partitioning. nih.gov this compound has demonstrated enhanced partitioning within the inner membrane of mycobacteria, particularly in the lipid head group/interfacial region. nih.govresearchgate.netacs.org While it also interacts with the outer membrane, its partitioning might be more limited in this layer, suggesting the outer membrane can act as a barrier to optimal drug penetration. nih.govacs.org The insertion of this compound into lipid bilayers has been confirmed, with studies suggesting it is fully immersed. researchgate.netresearchgate.net

The following table provides a comparison of lipid solubility and distribution properties between this compound and rifampicin:

The large volume of distribution observed for this compound (8-9 L/kg) further supports its extensive distribution into tissues. researchgate.netoup.comasm.org This widespread distribution contributes to its ability to reach therapeutic concentrations at the site of infection, particularly in tissues affected by diseases like tuberculosis. researchgate.net

Role of Cytochrome P450 (CYP) Enzymes, particularly CYP3A4

This compound is extensively metabolized, primarily in the liver. drugbank.comnih.govlgmpharma.com The metabolism of this compound is significantly influenced by cytochrome P450 (CYP) enzymes. ingentaconnect.comresearchgate.net Specifically, CYP3A is identified as the major isozyme responsible for the oxidative metabolism of this compound in human enterocytes and liver microsomes. researchgate.net This includes demethylation and hydroxylation reactions. researchgate.net

Studies in primary human hepatocytes have shown that this compound significantly induces CYP3A4 activity in a concentration-dependent manner, although it is a less potent inducer than rifampin. nih.govnih.govnih.govasm.orgnih.gov While some evidence strongly indicates a major role for CYP3A4 in this compound metabolism, other evidence has questioned this. asm.org Nevertheless, multiple dosing of this compound has been associated with the induction of hepatic metabolic enzymes of the CYP3A subfamily. fda.govwikidoc.org This induction can lead to decreased circulating levels of co-administered drugs that are also metabolized by the CYP3A pathway. fda.govwikidoc.org The induction of CYP3A4 by this compound is considered moderate. nih.gov

Identification and Antimicrobial Activity of Predominant Metabolites (e.g., 25-O-desacetyl-rifabutin, 31-hydroxy-rifabutin)

This compound undergoes extensive metabolism, resulting in the formation of more than 20 different metabolites. hres.ca Among these, two metabolites are considered predominant: 25-O-desacetyl-rifabutin and 31-hydroxy-rifabutin. drugbank.comlgmpharma.comfda.govhres.ca

The 25-O-desacetyl metabolite (also known as 16-deacetylthis compound or LM-565) has been determined to be as equally active microbiologically as the parent drug, this compound. fda.govhres.ca This metabolite contributes significantly to the total antimicrobial activity, accounting for up to 10%. drugbank.comlgmpharma.comfda.govhres.ca

The 31-hydroxy metabolite also possesses antimicrobial activity, although it is less potent than the parent drug, with approximately 1/16 of the activity of this compound. hres.ca

The formation of the 25-O-desacetyl metabolite is catalyzed by microsomal cholinesterase, in addition to CYP-mediated metabolism. researchgate.net CYP3A also catalyzes the oxidation of 25-O-desacetyl-rifabutin. researchgate.net

Autoinduction of Metabolism

This compound exhibits autoinduction of its own metabolism during multiple dosing. oup.comnih.govhres.caresearchgate.netnih.govasm.org This phenomenon is similar to that observed with rifampin. oup.comnih.govresearchgate.netnih.gov Evidence for autoinduction comes from studies where repeated administration of this compound led to decreased plasma concentrations or area under the curve (AUC) values compared to initial doses or theoretical values. oup.comhres.caresearchgate.netresearchgate.net

In one study, healthy volunteers receiving 450 mg of this compound daily for 10 days showed a 38% decrease in AUC and a 45% decrease in expected mean serum concentrations of the 25-O-desacetyl metabolite. oup.com In HIV-infected patients receiving daily doses, the AUC (normalized for dose) decreased by a mean of 42% from day 1 to day 28. oup.com The extent of this autoinduction response did not appear to be dose-dependent in that study. oup.com

The mechanism behind this compound autoinduction is attributed to the induction of CYP450 isoenzymes, particularly CYP3A, and cholinesterase. ingentaconnect.comresearchgate.net While the specific induced metabolic pathways are not fully known, there is convincing evidence that the CYP3A subfamily is induced by this compound. ingentaconnect.comnih.govwikidoc.org The predominant metabolite, 25-desacetyl this compound, may also contribute to this autoinduction effect. fda.govwikidoc.org

Mycobacteria (e.g., Mycobacterium avium Complex, M. tuberculosis, M. leprae)

This compound is considerably more active in vitro against the Mycobacterium avium complex (MAC), Mycobacterium tuberculosis, and Mycobacterium leprae compared to rifampin. nih.govoup.comselleckchem.com It also shows activity against most atypical mycobacteria, such as Mycobacterium kansasii, though Mycobacterium chelonae is relatively resistant. nih.govoup.comselleckchem.com

Studies have shown that this compound is approximately 10 times more active than rifampin against M. leprae in mouse footpad infections. oup.com In mouse peritoneal macrophages, this compound inhibited the growth of M. leprae at a concentration 10 times lower than that of rifampin. oup.com In an in vitro culture system, this compound also demonstrated greater activity against M. leprae. oup.com

For M. tuberculosis, a large study involving 302 strains found that all strains susceptible to 1 µg/mL of rifampin were susceptible to 0.5 µg/mL of this compound. oup.com Notably, 12% of strains resistant to 10 µg/mL of rifampin were susceptible to 0.5 µg/mL of this compound, indicating that cross-resistance between the two drugs is not always complete. oup.com Differences in susceptibility may be attributed to variations in their molecular affinity to the RNA polymerase subunit β. oup.com this compound has shown in vitro activity against many strains of M. tuberculosis. who.int In one study using the radiometric broth method, all rifampin-naive clinical isolates tested from the United States and Taiwan were susceptible to this compound concentrations of ≤0.125 mcg/mL. who.int

This compound has demonstrated in vitro activity against MAC organisms isolated from HIV-positive patients. who.int In a mouse macrophage cell culture model, this compound was also shown to be active against phagocytized MAC. who.int

Gram-Positive and Gram-Negative Bacteria (e.g., Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Klebsiella pneumoniae)

This compound is active against staphylococci, group A streptococci, Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Haemophilus ducreyi, Campylobacter jejuni, and Helicobacter pylori. nih.govoup.comselleckchem.com It also shows activity against Chlamydia trachomatis and Toxoplasma gondii. nih.govoup.comselleckchem.com

This compound generally exhibits poor activity against Enterobacteriaceae and Pseudomonas species. nih.govoup.com However, recent research indicates that Acinetobacter baumannii is significantly more susceptible to this compound when cultured in specific media, such as RPMI, which may better mimic the in vivo environment. nih.govresearchgate.netnih.gov This hypersensitivity appears to be commonly and uniquely observed in A. baumannii compared to other tested non-Acinetobacter bacteria like Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. nih.govresearchgate.netnih.gov

In a study comparing rifampin and this compound activity against antibiotic-resistant A. baumannii, E. coli, S. aureus, K. pneumoniae, and P. aeruginosa clinical isolates, only A. baumannii was hypersusceptible to this compound in vitro and in vivo using a Galleria mellonella infection model. nih.govresearchgate.netnih.gov Small shifts in this compound MIC50 were observed for E. coli, P. aeruginosa, and K. pneumoniae when comparing different media. nih.gov No significant shift in this compound MICs for S. aureus isolates was observed. nih.gov

While Bacillus subtilis produces bacteriocins with inhibitory activity against some Gram-positive and Gram-negative bacteria, including A. baumannii and K. pneumoniae to a lesser extent, this activity is related to the bacteriocins produced by B. subtilis, not the direct activity of this compound against B. subtilis itself. brieflands.com

Table 1: Select In Vitro Activity of this compound Against Various Microorganisms

Methodologies (Broth Microdilution, Agar Dilution, Radiometric Broth Dilution)

Several methods are used for in vitro susceptibility testing of mycobacteria and other bacteria against this compound, including radiometric broth dilution, tube dilution, microdilution methods using various media, and agar dilution methods. oup.commdpi.com

Radiometric broth dilution, often utilizing systems like BACTEC with 14C-labeled palmitic acid as a substrate, is a method employed for susceptibility testing of MAC and M. tuberculosis. oup.comwho.intmdpi.com

Broth microdilution is another common technique, often performed in 96-well microtiter plates with dehydrated antimicrobial agents. nih.gov This method involves inoculating the plate with a standardized inoculum of the organism and incubating it. nih.gov The MIC is read as the lowest concentration that inhibits visible growth. nih.gov Broth microdilution methods using media like Middlebrook 7H9 and 7H10 have been used for mycobacteria. oup.com For other bacteria, Mueller-Hinton broth (MHB) or cation-adjusted Mueller-Hinton broth (CAMHB) are commonly used. nih.govresearchgate.netnih.govresearchgate.net

Agar dilution methods involve incorporating the antimicrobial agent into agar media, such as Middlebrook 7H10 and 7H11 for mycobacteria, or Mueller-Hinton agar for other bacteria, at various concentrations. oup.comresearchgate.netplos.org Inocula are then spotted onto the agar plates. plos.org The MIC is the lowest concentration of the antibiotic in the agar that inhibits visible growth after incubation. plos.org

Studies have compared different methodologies, noting that MIC values can vary between broth and agar dilution methods. oup.commdpi.com For instance, MICs of this compound are generally lower in broth than in agar for MAC. oup.com

Influence of Medium Composition on MIC Determination

The composition of the culture medium can significantly influence the determined MIC values for this compound. nih.govresearchgate.netfwdamr-reflabcap.eubiorxiv.orgresearchgate.netasm.org This has been observed for both mycobacteria and other bacterial species. nih.govresearchgate.netresearchgate.netasm.org

For Mycobacterium abscessus, MIC values were found to be dependent on the culture medium. asm.org MICs were lower in Middlebrook 7H9 broth than in cation-adjusted Mueller-Hinton broth (CAMHB), but this effect was lost when supplementing the medium with oleic acid-albumin-dextrose-catalase (OADC) enrichment. asm.org Another study also noted that MIC values for this compound against M. abscessus were 2- to 3-fold higher in CAMHB compared to Middlebrook 7H9 broth, clearly implicating an effect of medium on susceptibility testing. asm.org Differences in assay media and conditions, such as Mueller-Hinton versus standard mycobacterial Middlebrook 7H9 broth, have been suggested as reasons for variations in reported MIC values for this compound against M. abscessus.

For other bacteria, such as Acinetobacter baumannii, the antimicrobial activity of this compound is best modeled by the use of RPMI for in vitro susceptibility testing. nih.govresearchgate.netnih.gov A. baumannii is significantly more susceptible to this compound when cultured in RPMI medium compared to standard Mueller-Hinton II (MHII) culture conditions. nih.govresearchgate.net This hypersensitivity in iron-depleted media like RPMI may better mimic an in vivo environment. researchgate.netnih.gov

Small shifts in this compound MIC50 values have also been observed for Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae depending on whether testing was performed in MHII or RPMI broth. nih.gov However, no significant shift in this compound or rifampin MICs for Staphylococcus aureus isolates was observed between MHII and RPMI broth. nih.gov

Table 2: Influence of Medium on this compound MICs for Select Bacteria

Proposed Susceptibility Breakpoints for In Vitro Testing

Establishing standardized susceptibility breakpoints for this compound in vitro testing has been a subject of discussion, particularly for mycobacteria. who.int Breakpoints are typically defined in relation to achievable drug concentrations, such as those in the blood. oup.com However, blood levels may not fully reflect concentrations in infected cells and tissues, or the pharmacokinetic and pharmacodynamic properties of the drug. oup.com

For MAC, a susceptibility breakpoint of approximately 0.125 µg/mL has been proposed for the radiometric (BACTEC) method. oup.com When using the broth dilution method, strains inhibited at ≤0.50 µg/mL are generally considered susceptible. oup.com Higher concentrations, about two times greater, may be needed when testing strains by the agar dilution method. oup.com

For M. tuberculosis, studies have explored breakpoints in relation to rifampin resistance. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) have been recommended to distinguish between susceptible, intermediate, and this compound-resistant strains of M. tuberculosis. Isolates with marginally reduced susceptibility compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml) but lower than the current critical concentration (≤0.5 µg/ml) could be categorized as intermediate.

It is important to note that for MAC and other mycobacterial species, standardized in vitro susceptibility testing methods and breakpoints have not been universally established. who.int Similarly, no this compound breakpoints are defined for many other bacteria, including staphylococci. nih.gov

Table 3: Proposed this compound Susceptibility Breakpoints

Synergistic Interactions with Ethambutol

Additive and frequently synergistic effects have been consistently noted in combinations of this compound and ethambutol. This combination can lead to a reduction in the Minimum Inhibitory Concentrations (MICs) for both drugs. One proposed mechanism for ethambutol's enhancement of this compound activity is an increase in the permeability of the outer cell envelope of MAC. Studies involving three- or four-drug combinations including this compound, ethambutol, and other agents like ciprofloxacin, clarithromycin, and amikacin have demonstrated additive effects, achieving significant bacterial killing at concentrations that are subinhibitory when the drugs are used individually.

Against Mycobacterium paratuberculosis, synergism was observed when this compound was combined with either cefazolin or clofazimine in double combinations. The combination of this compound and cefazolin showed the greatest synergism, including bactericidal synergism against all tested strains. Triple combinations, such as this compound with ethambutol and cefazolin or streptomycin and cefazolin, also exhibited bactericidal synergism against most strains of M. paratuberculosis.

Suppression of Inducible Macrolide Resistance (e.g., whiB7-erm41 system)

Emerging in vitro evidence suggests that this compound can suppress inducible macrolide resistance, a resistance mechanism commonly found in Mycobacterium abscessus (Mab) isolates. Co-treatment of macrolide-resistant Mab with this compound and clarithromycin has shown a synergistic effect. As an inhibitor of transcription, this compound appears to prevent the effective transcriptional induction of the whiB7-erm41 resistance system, potentially restoring macrolide susceptibility. This suggests that this compound may maintain macrolide-resistant Mab in a macrolide-susceptible state. Synergy between this compound and clarithromycin has been observed for erm41-positive Mab strains but not for erm41-negative strains, indicating its potential effectiveness against macrolide-resistant M. abscessus. This compound has been reported to be synergistic with clarithromycin and tigecycline against Mab, reducing the this compound MIC to levels achievable in the lung and producing bactericidal activity. The triple combination of clarithromycin, tigecycline, and this compound was also synergistic and demonstrated a sterilizing effect on Mab cultures at clinically relevant concentrations.

Synergistic Interactions with Ethambutol

Consistent additive and often synergistic effects have been reported when this compound is combined with ethambutol. This interaction can lead to a reduction in the minimum inhibitory concentrations (MICs) for both rifampin and ethambutol when used in combination. It has been hypothesized that ethambutol may enhance the activity of this compound by increasing the permeability of the outer cell envelope of MAC. Furthermore, three- or four-drug regimens incorporating this compound, ethambutol, and other agents such as ciprofloxacin, clarithromycin, and amikacin have demonstrated additive effects, achieving at least a 99% reduction in the initial inoculum after 7 days at concentrations of individual drugs that were subinhibitory on their own.

Against Mycobacterium paratuberculosis, synergistic activity has been noted when this compound is combined with either cefazolin or clofazimine in dual combinations. The combination of this compound and cefazolin demonstrated the most significant synergy, including bactericidal synergy against all tested strains. Triple combinations, such as this compound with ethambutol and cefazolin or streptomycin and cefazolin, also showed bactericidal synergism against the majority of M. paratuberculosis strains.

Suppression of Inducible Macrolide Resistance (e.clinical)

In vitro studies indicate that this compound possesses the ability to suppress inducible macrolide resistance, a mechanism frequently encountered in Mycobacterium abscessus (Mab) isolates. Co-treatment of macrolide-resistant Mab with this compound and clarithromycin has been shown to exert a synergistic effect. As an inhibitor of transcription, this compound appears to prevent the effective transcriptional induction of the resistance-mediating whiB7-erm41 system. This action may effectively maintain genotypically macrolide-resistant Mab in a phenotypically macrolide-susceptible state. Synergy between this compound and clarithromycin has been observed specifically for erm41-positive Mab strains, but not for erm41-negative strains, suggesting its potential utility in treating macrolide-resistant M. abscessus. This compound has also been reported to be synergistic with tigecycline against Mab. These combinations have been shown to reduce the this compound MIC to concentrations achievable in the lung and to produce bactericidal activity. The triple combination of clarithromycin, tigecycline, and this compound was also synergistic and demonstrated a sterilizing effect on Mab cultures at clinically relevant concentrations.

Correlation of Specific Codon Mutations (e.g., S531L, H526Y, H526D, D516V, L533P, L511P, D516G, H526N, S522L) with this compound Susceptibility and Resistance Levels

Drug Inactivation Mechanisms

While mutations in the rpoB gene are the predominant mechanism of resistance to rifamycins like this compound, drug inactivation can also contribute, particularly in certain bacterial species. In Mycobacterium abscessus, for instance, resistance can occur through enzymatic inactivation of rifamycins by an adenosine diphosphate (ADP)-ribosyltransferase enzyme (Arr). researchgate.netnews-medical.net This enzyme catalyzes the ADP-ribosylation of rifamycin, preventing its interaction with bacterial RNA polymerase and thus inactivating the antibiotic. researchgate.net Arr homologues are found in various bacterial species, and their presence can be associated with mobile genetic elements. researchgate.net Research into overcoming this mechanism includes the development of modified rifamycin analogs, such as RFB-5m, which are designed to prevent enzymatic oxidation and ADP-ribosylation by Arr. researchgate.net

Interaction with Bacterial RNA Polymerase

This compound, like other rifamycins, inhibits bacterial RNAP by binding to a specific site on the enzyme. nih.gov This binding event blocks the path of the nascent RNA transcript, preventing elongation beyond a few nucleotides. pnas.orgnih.gov Molecular docking studies have been instrumental in visualizing these interactions and identifying the amino acid residues within the RNAP binding pocket that are crucial for this compound binding. nih.govasm.orguab.edu The binding site for rifamycins, including this compound, is located within the β subunit of bacterial RNAP, deep within the DNA/RNA channel. asm.orgutupub.fi This region, often referred to as the "rif region" or "RIF-binding pocket," is highly conserved among eubacteria, explaining the broad-spectrum activity of rifamycins. nih.govasm.org Mutations in this region are commonly associated with resistance to rifamycins. nih.govasm.org

P-glycoprotein (P-gp) Binding Sites (Inhibitor vs. Substrate)

P-glycoprotein (P-gp), an efflux transporter, can influence the intracellular concentration of various drugs, including some antibiotics. Molecular docking studies have investigated the interaction of this compound with P-gp to understand if it acts as a substrate, inhibitor, or both. Research indicates that this compound can bind to P-gp. researchgate.netnih.govresearchgate.net Notably, studies comparing this compound and rifampicin binding to P-gp have shown that this compound exhibits a higher binding affinity to the inhibitor binding site (M-site) of P-gp compared to rifampicin. nih.govresearchgate.netnih.gov This suggests that this compound may have a greater potential to inhibit P-gp-mediated efflux. Molecular docking models have illustrated the distinct binding modes of this compound and rifampicin within the P-gp binding sites. researchgate.netresearchgate.net this compound's spiro piperidine moiety has been highlighted as interacting with the inhibitor binding site. researchgate.net

The difference in binding affinities and interaction sites between this compound and rifampicin at P-gp binding sites is summarized in the table below:

Potential Interactions with Viral Enzymes (e.g., SARS-CoV-2 RNA-dependent RNA polymerase)

With the emergence of viral pathogens, computational methods have been employed to explore the potential of existing drugs to inhibit viral enzymes. Molecular docking studies have investigated this compound's potential interactions with viral enzymes, including the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. nih.govwindows.netscienceopen.comarxiv.orgijmm.irresearchgate.netijmm.ir These studies have suggested that this compound may have a potential inhibitory interaction with SARS-CoV-2 RdRp, showing favorable binding energy values in docking simulations. nih.govwindows.netscienceopen.comarxiv.orgijmm.irresearchgate.netijmm.ir Specific amino acid residues within the SARS-CoV-2 RdRp have been identified as being involved in interactions with this compound in these docking models. nih.govwindows.netscienceopen.comarxiv.org

Molecular docking results for this compound binding to SARS-CoV-2 RdRp are presented below:

These findings suggest a potential for this compound to interact with viral RdRp, although further experimental validation is required.

Drug-Membrane Interactions (Lipid Bilayers, Multilamellar Vesicles)

This compound's passage across bacterial cell membranes is crucial for its activity. MD simulations have been employed to investigate the interactions of this compound with lipid bilayers, including those mimicking bacterial cell envelopes. acs.orgnih.govacs.orgnih.govresearchgate.netresearchgate.net These simulations reveal how this compound partitions into and interacts with different membrane environments. Studies on mycobacterial membranes, which have a complex lipid composition, have shown that this compound exhibits differential partitioning and localization within the distinct membrane layers. acs.orgnih.govacs.orgnih.gov For instance, in M. smegmatis membranes, this compound showed higher partitioning in the inner membrane, localizing primarily in the lipid head group/interfacial region. acs.orgnih.govacs.orgnih.gov In the outer membrane (mycomembrane), this compound distributed more uniformly within both the head group/interfacial and hydrophobic acyl chain regions. acs.orgnih.govacs.orgnih.gov

MD simulations can also assess the impact of this compound on membrane properties, such as fluidity and lipid organization. acs.orgresearchgate.netresearchgate.net Studies have indicated that this compound can alter membrane fluidity and lipid clustering distribution, which may influence drug uptake and efficacy. acs.orgresearchgate.netresearchgate.net

Analysis of Drug Partitioning and Interaction Sites within Cell Envelope Layers

MD simulations provide detailed insights into where this compound localizes within the different layers of the bacterial cell envelope and which specific lipids it interacts with. acs.orgnih.govnih.govresearchgate.net In mycobacteria, the distinct lipid compositions of the inner and outer membranes lead to varied interaction profiles with this compound. acs.orgnih.govnih.gov Simulations have shown preferential interactions between this compound and specific lipids within each membrane layer. nih.gov For example, in the mycomembrane, this compound showed preferential interactions with lipids such as LAM, TDM, and SL-1. nih.gov In the inner membrane, it preferentially interacted with cardiolipin and Ac2PIMs. nih.gov

The localization of this compound within the membrane can be visualized through MD simulations, showing its depth of insertion and distribution within the bilayer. researchgate.net This detailed understanding of drug-membrane interactions at a molecular level is crucial for designing more effective antibiotics and strategies to overcome membrane-associated resistance mechanisms. acs.orgnih.govnih.gov

FT-IR, XRD, DSC, SEM for Inclusion Complexes

Fourier Transform Infrared (FT-IR) spectroscopy, X-ray Diffraction (XRD), Differential Scanning Calorimetry (DSC), and Scanning Electron Microscopy (SEM) are commonly used to characterize the formation and properties of this compound inclusion complexes, particularly with cyclodextrins. Studies investigating the complexation of poorly water-soluble drugs like this compound with β-cyclodextrin (β-CD) have utilized these techniques to confirm the formation of solid inclusion complexes researchgate.netnih.govresearchgate.netpreprints.org.

FT-IR analysis can reveal changes in the vibrational modes of functional groups of this compound upon complexation, indicating interactions between the drug and the host molecule researchgate.netnih.govresearchgate.net. XRD patterns of inclusion complexes often show a reduction in the crystallinity of the drug, suggesting its encapsulation within the amorphous or less crystalline structure of the cyclodextrin complex researchgate.netnih.govresearchgate.netnih.gov. DSC thermograms can exhibit shifts or disappearance of the characteristic melting peak of this compound, providing evidence of complex formation and altered thermal behavior researchgate.netnih.govresearchgate.netnih.gov. SEM images allow for the visualization of the morphology of the inclusion complexes, often showing a change in particle shape and size compared to the pure drug and host material researchgate.netnih.gov. These techniques collectively provide compelling evidence for the successful formation of this compound inclusion complexes, which can lead to improved solubility and dissolution rates researchgate.netnih.govresearchgate.net.

SAXS and WAXS for Drug-Membrane Interactions

Small-angle X-ray Scattering (SAXS) and Wide-angle X-ray Scattering (WAXS) are powerful techniques for studying the interaction of this compound with model membrane systems. These methods provide insights into the effects of the drug on the structure and organization of lipid bilayers, mimicking human and bacterial cell membranes nih.govresearchgate.netacs.org.

SAXS is used to probe the long-range organization and phase symmetry of the lipid bilayers, while WAXS provides information about the packing of hydrocarbon chains within the membrane researchgate.net. Studies using SAXS and WAXS have shown that this compound can perturb lipid bilayers, particularly in bacterial membrane models, potentially inducing phase separation nih.govresearchgate.net. The extent of this perturbation can depend on factors such as drug concentration, the charge of the phospholipid headgroup, and the membrane organization nih.govresearchgate.net. Research using these techniques with multilamellar vesicles (MLVs) composed of different phospholipids (e.g., DMPC for human membranes, DMPG and DPPE/DPPG mixtures for bacterial membranes) has demonstrated that this compound can affect both human and bacterial membrane models, but shows a stronger interaction with negatively charged bacterial membrane models nih.govresearchgate.netflinders.edu.au. This preferential interaction with bacterial membranes may be related to its antibiotic activity nih.govresearchgate.netflinders.edu.au.

NMR Spectroscopy for Structural Characterization of Derivatives

Nuclear Magnetic Resonance (NMR) spectroscopy is a fundamental technique for the structural characterization of this compound and its derivatives. Both ¹H and ¹³C NMR spectroscopy, often coupled with two-dimensional correlation techniques (e.g., COSY, HSQC, HMBC), are used to assign the complex spectra of these molecules and confirm their structures nih.govgoogle.comresearchgate.netresearchgate.net.

Hepatic Metabolic Enzyme Induction (CYP3A, CYP1A2)

In pre-clinical and in vitro studies, this compound has been shown to induce the expression and activity of several hepatic cytochrome P450 enzymes, with a notable effect on CYP3A and CYP1A2. Studies using primary human hepatocytes have demonstrated that this compound significantly induces CYP3A4 gene expression in a concentration-dependent manner. asm.orgnih.gov For instance, this compound elicited a 20-fold upregulation in CYP3A4 gene expression at a concentration of 5 µM in primary human hepatocytes. asm.org While CYP3A4 is a primary target of induction, this compound has also been reported to slightly increase the activity of CYP1A2. asm.org The induction of these enzymes by this compound contributes to its potential for drug-drug interactions.

Differential Induction Potency Compared to Rifampin

This compound is generally considered a less potent inducer of CYP enzymes compared to rifampin, another widely used rifamycin. nih.govfrontiersin.orgucsf.eduasm.orgoup.comasm.org This difference in induction potential is a key factor when considering drug-drug interactions, particularly in complex treatment regimens such as those for HIV-TB coinfection. asm.org In vitro studies comparing this compound and rifampin have shown quantitative differences in their ability to induce CYP3A4. For example, in primary human hepatocytes, rifampin induced CYP3A4 significantly more strongly (80-fold at 10 µM) than this compound (20-fold at 5 µM). asm.orgnih.gov Animal studies have also indicated that the maximally achievable induction of cytochrome P-450 3A by this compound was approximately two-fold lower than that by rifampin. oup.comuzh.ch Regarding CYP1A2 induction, a study in healthy volunteers showed that equal doses of this compound and rifampin administered for two weeks resulted in significantly less induction of CYP1A2, as measured by theophylline clearance, following this compound treatment compared to rifampin. asm.org

Table 1: Comparison of In Vitro CYP3A4 Induction by this compound and Rifampin in Primary Human Hepatocytes

Note: Data are representative and may vary depending on experimental conditions and cell sources.

Despite accumulating to higher intracellular concentrations in hepatocytes than rifampin, this compound has been shown to be a significantly less potent and less efficient activator of the pregnane X receptor (PXR), a key regulator of CYP3A4 and ABCB1 expression, which may explain its weaker inductive effects. researchgate.net

Inhibition and Induction Effects on P-gp

This compound exhibits a dual effect on P-gp, acting as both an inducer and an inhibitor. Studies have demonstrated that this compound can induce the expression of P-gp. asm.orgnih.govnih.govnih.gov For instance, this compound caused a 4-fold upregulation of ABCB1 (encoding P-gp) at 5 µM in primary human hepatocytes. asm.org This induction can lead to increased efflux of P-gp substrate drugs, potentially reducing their intracellular concentrations.

Conversely, this compound has also been identified as a potent inhibitor of P-gp activity. researchgate.netnih.govnih.govmdpi.com In vitro studies have shown that this compound is a considerably more potent inhibitor of P-gp than rifampin. researchgate.netnih.govnih.gov Molecular docking analysis suggests that this compound has a higher binding affinity to the inhibitor binding site of P-gp compared to rifampin. nih.govnih.govresearchgate.net This inhibitory activity can potentially counteract the induction effect, particularly in the intestine, and may enhance the absorption of P-gp substrate drugs. nih.govnih.gov The net effect of this compound on the pharmacokinetics of co-administered P-gp substrates depends on the balance between its induction and inhibition capabilities.

Table 2: In Vitro Effects of this compound and Rifampin on P-glycoprotein

Note: Induction refers to increased expression, while inhibition refers to decreased transport activity.

Membrane Biophysics and Interactions with Mycobacterial Cell Envelope Layers

The mycobacterial cell envelope is a complex, lipid-rich structure that serves as a crucial barrier and mediates interactions with the host environment. It consists of multiple layers, including the inner membrane (plasma membrane) and the outer membrane (mycomembrane), each with distinct lipid compositions and biophysical properties. acs.orgnih.govacs.orgrsc.orgpnas.orgnih.gov Understanding the biophysics of these membranes is essential for comprehending drug uptake and efficacy. rsc.orgpnas.org

This compound interacts with these distinct membrane layers in a varied manner. Studies using membrane biophysics and molecular simulations have revealed that this compound partitions differently within the inner and outer membranes. acs.orgnih.govacs.orgnih.gov The mycobacterial inner membrane has shown the highest partitioning of this compound, with the drug primarily located in the lipid head group/interfacial region. acs.orgnih.govacs.orgnih.gov In contrast, this compound exhibits specific interaction sites in both the head group/interfacial and hydrophobic acyl regions within the outer membrane. acs.orgnih.govacs.orgnih.gov

Table 3: this compound Interaction with Mycobacterial Membrane Layers (In Vitro/Simulation)

These differential interactions and partitioning behaviors within the distinct membrane layers of the mycobacterial cell envelope can influence the intracellular accumulation of this compound and potentially contribute to drug tolerance. acs.orgnih.gov The structural and chemical makeup of the lipids in each layer underlies these varied interaction profiles. acs.orgnih.govacs.org

Specific Interactions with Mycobacterial Lipids (e.g., LAM, TDM, SL-1)

This compound, a rifamycin antibiotic, interacts with the complex lipid-rich cell envelope of mycobacteria. Research utilizing molecular simulations and membrane biophysics has shed light on specific interactions between this compound and various mycobacterial lipids, particularly within the mycomembrane (outer membrane) and inner membrane.

Studies have revealed that this compound interacts preferentially with certain lipids in the mycomembrane, including Lipoarabinomannan (LAM), Trehalose Dimycolate (TDM), and Sulfolipid-1 (SL-1). In contrast, this compound shows no significant interactions with Mycolic Acids (MA) in this layer acs.orgnih.gov. Within the inner membrane, this compound exhibits preferential interactions with cardiolipin and Ac2PIMs, but not with phosphatidylglycerol (PG) or diacylglycerols (DAGs) acs.orgnih.gov. This indicates a lipid-composition specific interaction profile for this compound within the different layers of the mycobacterial cell envelope nih.govresearchgate.net.

Molecular simulations have indicated a high localization of this compound within the inner membrane, primarily in the interfacial lipid head group region. In the mycomembrane, this compound distribution is lower and more uniform, found in both the interfacial and hydrophobic acyl chain regions acs.org. Specific interaction sites have been identified in both the head group/interfacial and hydrophobic acyl regions within the outer membrane researchgate.net.

The interactions with these lipids can influence the physical properties of the mycobacterial membranes. This compound has been shown to alter the fluidity of mycobacterial membranes, including the mycomembrane. This effect is potentially mediated through interactions with lipids in both the head group/interfacial and hydrophobic acyl chain regions acs.orgnih.gov. At higher temperatures (above 50 °C), this compound caused a decrease in the fluidity of the mycomembrane in both the head group/interfacial and hydrophobic acyl chain regions acs.orgnih.gov. The bulky polar head group of LAM may play a role in mitigating electrostatic interactions of this compound with lipid head groups by providing a shielding effect nih.gov.

Studies using model membranes containing TDM have demonstrated enhanced partitioning of this compound into these systems, accompanied by specific changes in membrane structure, order, and fluidity, further supporting the significance of lipid-drug interactions nih.gov.

The following table summarizes the preferential interactions of this compound with key mycobacterial lipids based on research findings:

This table is based on detailed research findings indicating the specific lipids with which this compound shows preferential interaction within the different layers of the mycobacterial cell envelope acs.orgnih.gov.