Inhibition Of CYP3A5 Research Articles

Abstract 4369: CYP3A5 promotes aggressive and therapeutically resistant prostate cancer by modulating AR and Wnt signaling: Implications for racially disparate clinical outcomes

Abstract Prostate cancer (PC) is the second leading cause of cancer-related death in American men, which disproportionately affects Black/African American (AA) men. The androgen receptor (AR) signaling plays a pivotal role in PC development and is a primary target for intervention in patients with advanced disease. Aberrant activation of AR is also suggested to play a significant role in castration-resistant prostate cancer (CRPC). Despite advancements in anti-androgen therapies with new targeting agents, such as enzalutamide, metastatic CRPC (mCRPC) remains incurable. CYP3A5, a monooxygenase expressed in the prostate, liver and intestine, is involved in drug metabolism and steroid biosynthesis. We earlier reported that intratumoral CYP3A5 activated AR signaling by facilitating its nuclear translocation of AR. More notably, we found a higher CYP3A5 expression in AAs, attributed to the *1 CYP3A5 variant, as compared to non-Hispanic White Americans (NHWA) possessing the *3 variant. Analysis of RNA-seq data using patient tumor samples revealed that elevated CYP3A5 in AA PC patients was associated with Wnt-β catenin signaling activation via TCF4 overexpression. Here, we investigated the role of CYP3A5 in enzalutamide resistance and Wnt signaling by generating enzalutamide-resistant PC cell lines of NHWA (low CYP3A5-LNCaP) and AA-origin (high CYP3A5-MDAPCa2b). RT-qPCR assay was used to examine the changes in AR and CYP3A5 expression between the parental and the enzalutamide-resistant cell lines. The data show heightened CYP3A5 expression in enzalutamide-resistant cells of AA origin (MDAPCa2b/EnzR) but not of NHWA origin (LNCaP/EnzR). Interestingly, MDAPCa2b/EnzR exhibited no change in AR levels whereas it was increased in LNCaP/EnzR cells relative to parental lines. A loss of function assay was performed for siRNA-mediated silencing of CYP3A5 to study its effect on Wnt signaling. RNA sequencing results show that CYP3A5 inhibition in MDAPCa2b downregulated Wnt pathway genes (Wnt5A, Wnt10B, Wnt11, Fzd2, and Dvl3). These observations align with our previous RNA seq results using patient samples where we observed upregulated Wnt-β catenin signaling in high CYP3A5 expressing patient tumor samples. Western analysis post-CYP3A5 inhibition in LNCaP and MDAPCa2b cells revealed upregulated Axin and downregulated LRP6 and Dvl-3, signifying CYP3A5's regulation of the canonical Wnt-β catenin pathway. Additionally, MDAPCa2b saw downregulated Wnt 5A and Wnt 10B, known to influence the Wnt non-canonical pathway. Altogether, our data suggest that *1 CYP3A5 variant in AAs likely contributes to aggressive behavior and therapeutic resistance of PC and serves as a molecular determinant of disparate clinical outcomes. Citation Format: Jake McLean, Ajay P. Singh, Edwin Oh, Ranjana Mitra. CYP3A5 promotes aggressive and therapeutically resistant prostate cancer by modulating AR and Wnt signaling: Implications for racially disparate clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4369.

Abstract 5414: RNA sequencing reveals that CYP3A5 siRNA treatment induces cell cycle blockade and senescence in MDAPCa2b human prostate cancer cells

Abstract Background: Previously we have demonstrated that CYP3A5 inhibition blocks prostate cancer (PC) cell growth partly by attenuating androgen receptor (AR) activation and downstream signaling. We further investigated whether CYP3A5 inhibition blocks PC cell growth independently of AR signaling. Initially, most PCs respond to androgen deprivation therapy (ADT), but eventually PC becomes castration resistant and does not respond to ADT. With very limited options available for patients with castration resistant prostate cancer (CRPC), there is an urgent need for the development of novel targeted therapies for this subtype of PC. Additionally, understanding the role of CYP3A5 as promoter of PC growth in African Americans (AA) is very relevant as AAs preferentially carry wild type CYP3A5 gene, often present with aggressive disease, and have higher mortality rate versus Caucasian (CA) patients, who typically express the mutant CYP3A5. Method: We used MDAPCa2b PC cell line of AA origin carrying one copy of wild type CYP3A5 for our study. After inhibiting the CYP3A5 using siRNA pool of 3 siRNAs, we performed Illumina RNA sequencing. Differentially expressed genes (DEGs) between non-target and CYP3A5 siRNA triplicate samples were identified using DESeq2, at a false discovery rate of adjusted p < 0.05. Furthermore, KEGG pathway analysis was performed to identify the roles of the differentially expressed genes in regulating prostate cancer growth. Results: We identified 1814 DEGs after inhibiting CYP3A5 using siRNA pool in MDAPCa2b cells, 825 of which increased or decreased expression greater than two-fold. The differentially down-regulated genes associate with several growth regulatory pathways: cell cycle progression (39), cellular senescence (16) DNA replication (18) and base excision repair (9). Additionally, we identified that CYP3A5 siRNA downregulates several genes which are dysregulated (including oncogenes and tumor suppressors) in different cancer type including RAS, c-Met, ERBB2, FGFR2, cyclin E, p53, Myc, PTEN, RARβ and CDK2. The upregulated genes correspond to two pathways, retinol metabolism pathway (arrests proliferation) and steroid hormone biosynthesis. We also identified several unique genes which are down regulated with CYP3A5 siRNA treatment like Glo1, FoxM1 (often upregulated in several cancers) and Tk1 (marker for chemotherapy sensitivity). Conclusion: CYP3A5 siRNA inhibits several genes involved in cell growth and proliferation. Targeting CYP3A5 represents a potential novel therapeutic approach, especially for AAs expressing wild type CYP3A5. Citation Format: Richard L. Tillett, Shirley Shen, Edwin C. Oh, Oscar B. Goodman Jr., Ranjana Mitra. RNA sequencing reveals that CYP3A5 siRNA treatment induces cell cycle blockade and senescence in MDAPCa2b human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5414.

Unraveling the Structural Basis of Selective Inhibition of Human Cytochrome P450 3A5.

The human cytochrome P450 (CYP) CYP3A4 and CYP3A5 enzymes metabolize more than one-half of marketed drugs. They share high structural and substrate similarity and are often studied together as CYP3A4/5. However, CYP3A5 preferentially metabolizes several clinically prescribed drugs, such as tacrolimus. Genetic polymorphism in CYP3A5 makes race-based dosing adjustment of tacrolimus necessary to minimize acute rejection after organ transplantation. Moreover, the differential tissue distribution and expression levels of CYP3A4 and CYP3A5 can aggravate toxicity during treatment. Therefore, selective inhibitors of CYP3A5 are needed to distinguish the role of CYP3A5 from that of CYP3A4 and serve as starting points for potential therapeutic development. To this end, we report the crystal structure of CYP3A5 in complex with a previously reported selective inhibitor, clobetasol propionate (CBZ). This is the first CYP3A5 structure with a type I inhibitor, which along with the previously reported substrate-free and type II inhibitor-bound structures, constitute the main CYP3A5 structural modalities. Supported by structure-guided mutagenesis analyses, the CYP3A5-CBZ structure showed that a unique conformation of the F-F' loop in CYP3A5 enables selective binding of CBZ to CYP3A5. Several polar interactions, including hydrogen bonds, stabilize the position of CBZ to interact with this unique F-F' loop conformation. In addition, functional and biophysical assays using CBZ analogs highlight the importance of heme-adjacent moieties for selective CYP3A5 inhibition. Our findings can be used to guide further development of more potent and selective CYP3A5 inhibitors.

Bergamottin a CYP3A inhibitor found in grapefruit juice inhibits prostate cancer cell growth by downregulating androgen receptor signaling and promoting G0/G1 cell cycle block and apoptosis.

Prostate cancer is the second leading cause of cancer related death in American men. Several therapies have been developed to treat advanced prostate cancer, but these therapies often have severe side effects. To improve the outcome with fewer side effects we focused on the furanocoumarin bergamottin, a natural product found in grapefruit juice and a potent CYP3A inhibitor. Our recent studies have shown that CYP3A5 inhibition can block androgen receptor (AR) signaling, critical for prostate cancer growth. We observed that bergamottin reduces prostate cancer (PC) cell growth by decreasing both total and nuclear AR (AR activation) reducing downstream AR signaling. Bergamottin’s role in reducing AR activation was confirmed by confocal microscopy studies and reduction in prostate specific antigen (PSA) levels, which is a marker for prostate cancer. Further studies revealed that bergamottin promotes cell cycle block and accumulates G0/G1 cells. The cell cycle block was accompanied with reduction in cyclin D, cyclin B, CDK4, P-cdc2 (Y15) and P-wee1 (S642). We also observed that bergamottin triggers apoptosis in prostate cancer cell lines as evident by TUNEL staining and PARP cleavage. Our data suggests that bergamottin may suppress prostate cancer growth, especially in African American (AA) patients carrying wild type CYP3A5 often presenting aggressive disease.

Abstract 3475: CYP3A5 inhibitor bergamottin present in grape fruit extract blocks prostate cancer cell growth

Abstract Background: Previous research has revealed that the androgen receptor (AR), which upon activation goes to the nucleus, is the driving force of prostate cancer growth. Once activated, AR promotes transcription of genes responsible for promoting cancer growth. Previous studies have shown that inhibition of CYP3A5 blocks the activation process of the AR. CYP3A5 is a liver enzyme and together with CYP3A4 processes 50% of all commonly prescribed drugs. CYP3A5 is the extrahepatic form expressed in both normal and malignant prostate. Bergamottin, a compound found in grapefruit juice, is a known CYP3A4/5 inhibitor. Based on CYP3A5's ability to regulate AR activation, we tested the effects of bergamottin on AR activation to see if the compound can be utilized as a dietary supplement to reduce prostate cancer growth. Methods: Western blotting, cell fractionation, and immunofluorescence were used to observe the effects of bergamottin on AR, amount of AR in nucleus/cytoplasm, and cellular localization of AR, respectively. MTT based cell growth measurement was used to see how prostate cancer cell growth was affected by bergamottin. Results: Cell growth assay revealed that bergamottin reduced prostate cancer cell growth. The IC50 for LNCaP cells was observed to be 2.4 μM. Growth of RWPE1 cells (non transformed prostate epithelium) was not effected at the same concentration of bergamottin. Western blot analysis showed that total AR protein expression was downregulated in the bergamottin treated LNCaP and MDAPCa2b cells. Cell fractionation assay revealed that bergamottin significantly reduced nuclear AR after DHT induction compared to non-treated cells. Immunofluorescence assay confirmed reduced nuclear AR in the bergamottin treated LNCaP and MDAPCa2b cells. Furthermore, bergamottin reduced PSA levels both inside the cells and in the growth media indicating that it inhibits AR downstream signaling. Conclusion: Our results concluded that bergamottin successfully blocked AR activation and downstream signaling, and consequently slowed down prostate cancer cell growth. The implication of this experiment is the potential use of bergamottin as a food supplement alongside other prostate cancer treatments in order to aid in blocking prostate cancer growth. Citation Format: Opalina Vetrichelvan, Priyatham Gorjala, Oscar Goodman Jr, Ranjana Mitra. CYP3A5 inhibitor bergamottin present in grape fruit extract blocks prostate cancer cell growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3475.

Clobetasol Propionate Is a Heme-Mediated Selective Inhibitor of Human Cytochrome P450 3A5.

The human cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A5 metabolize most drugs and have high similarities in their structure and substrate preference. Whereas CYP3A4 is predominantly expressed in the liver, CYP3A5 is upregulated in cancer, contributing to drug resistance. Selective inhibitors of CYP3A5 are, therefore, critical to validating it as a therapeutic target. Here we report clobetasol propionate (clobetasol) as a potent and selective CYP3A5 inhibitor identified by high-throughput screening using enzymatic and cell-based assays. Molecular dynamics simulations suggest a close proximity of clobetasol to the heme in CYP3A5 but not in CYP3A4. UV-visible spectroscopy and electron paramagnetic resonance analyses confirmed the formation of an inhibitory type I heme-clobetasol complex in CYP3A5 but not in CYP3A4, thus explaining the CYP3A5 selectivity of clobetasol. Our results provide a structural basis for selective CYP3A5 inhibition, along with mechanistic insights, and highlight clobetasol as an important chemical tool for target validation.

Abstract 1311A: CYP3A5 regulation of androgen receptor signaling: Relevance in African American prostate cancer patients and its development as novel target

Abstract Background: Previously we demonstrated that CYP3A5 positively regulates androgen receptor (AR) signaling and growth of prostate cancer (PC) cells. CYP3A5 inhibition decreases nuclear AR localization and downregulates AR downstream signaling (e.g. PSA and TMPRSS2), resulting in decreased PC cell growth both with and without dihydrotestosterone (DHT) induction. Our current work focusses on the effect of CYP3A5 modulation on AR signaling and therapeutic resistance, mostly in African Americans (AA) widely carrying (73%) wild type CYP3A5 (*1/*1). AAs and have higher incidence of PC and double the mortality rate compared to Non-Hispanic White Americans (NHWA) carrying mutated CY3A5 gene (*3/3, 95%) and expressing truncated non-functional CYP3A5 protein. Methods: Immunoprecipitation was used to identify binding partners of AR. q-RT-PCR based profiler assays was used to detect changes in AR downstream signaling and effect on genes responsible for therapeutic resistance after CYP3A5 inhibition. Effect of CYP3A5 on AR nuclear translocation and induction of synthetic lethality in combination with PARP inhibitors were evaluated using cell fractionation and immunocytochemistry studies. Results: Co-immunoprecipitation of flag-tagged CYP3A5 transfected LNCaP cell extracts showed that CYP3A5 was in a complex with HSP90, AR, HSP70 and HSP40, also confirmed with reverse immunoprecipitation using AR/ HSP90 antibodies and agarose A/G beads. AR regulated genes (SCL45A3, FKBP5, MYC, ELL2, and MT2A) were downregulated with CYP3A5 siRNA treatment in MDA PCa 2b cells (*1/*3, AA origin) in a qPCR-based profiler assay with high fold difference and a P value of ≤0.005. PC patients are often prescribed medications for comorbidities, many of which are CYP3A modulators. Our results indicate that CYP inhibitors (amiodarone, ritonavir, fluoxetine etc.) decrease AR nuclear translocation and inducers (phenytoin, rifampicin, pioglitazone and hyperforin) increase AR nuclear translocation and activation. CYP3A5 siRNA treatment downregulates genes involved in cancer drug therapeutic resistance (TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB and HPRT1, P ≤ 0.005 ). Loss of TOP2A, BRCA2 and BRCA1is known to impair homologous recombination (HR). In combination with topoisomerase inhibitor etoposide, CYP3A5 siRNA in PC cells increases number of gamma-H2AX foci indicating synthetic lethality. Conclusion: A-Developing CYP3A5-AR-HSP90 interface as a target may provide a novel strategy to block PC growth. B-CYP3A5 modulation of AR signaling can affect the efficacy of androgen deprivation therapy specifically AA patients carrying wild type CYP3A5. C-Since CYP3A5 inhibition, downregulates DNA damage repair genes and impairs HR, CYP3A5 inhibition in combination with PARP inhibitors can be a novel strategy to induce synthetic lethality in PC patients. Citation Format: Priyatham Gorjala, Oscar B. Goodman, Ranjana Mitra. CYP3A5 regulation of androgen receptor signaling: Relevance in African American prostate cancer patients and its development as novel target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1311A.

Abstract B001: CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance

Abstract Purpose: Androgen receptor (AR) plays a major role in promoting growth and progression of prostate cancer and thus has been successfully targeted therapeutically. Therapeutic resistance to androgen deprivation therapy (ADT) accounts for much of the morbidity and mortality attributable to prostate cancer. Therapeutic resistance is typically accompanied by insufficient blockade of AR activation, whose initial step involves translocation of AR to the nucleus. In recent studies we demonstrated that CYP3A5 facilitates the nuclear translocation of AR, promoting prostate cancer growth. This work focuses on understanding the role of CYP3A5 in regulating AR nuclear translocation and therapeutic resistance. Method: To understand how CYP3A5 effects AR downstream signaling, we used RT2 profiler PCR arrays to help identify set of genes in AR signaling pathway that are affected by CYP3A5 inhibition. Confocal microscopy and cell fractionation experiments were used to further confirm the effect of CYP3A5 modulation of AR signaling. As concomitant CYP3A5 inducer/inhibitor medications can affect intratumoral CYP3A5 activity and thus may modify AR downstream signaling, we utilized cell fractionation and confocal microscopy to analyze the effect of these medications on AR nuclear translocation. Further, to decipher the role of CYP3A5 in the AR translocation process we performed a series of co-immunoprecipitation (co-IP) experiments with CYP3A5 and components of HSP90-AR complex. We used extracts of LNCaP transfected with a CYP3A5-Flag-tagged construct for IP, with either Flag-agarose or HSP90 and AR antibodies with A/G beads. Finally, to decipher CYP3A5’s role in therapeutic resistance we analyzed cDNA after CYP3A5 siRNA treatment using drug resistance RT2 profiler PCR array. Results: The q-PCR based profiler assay revealed a set of nine AR regulated genes (SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1, and SPDEF) downregulated with CYP3A5 siRNA treatment with a p value of ≤0.005. MYC and PIK3R3 are known to be involved in regulating cell growth by either affecting cell cycle progression and/or apoptosis. PSA, known to aid in the cell growth, is regulated by SPDEF, a transcription factor. Downregulation of these genes with CYP3A5 siRNA treatment indicates that CYP3A5 modifier medications can alter AR downstream signaling regulating growth. Concomitantly prescribed CYP3A inhibitors amiodarone and ritonavir blocked AR nuclear migration and decreased PSA expression whereas inducers phenytoin and rifampicin promoted increased AR nuclear migration and increased PSA production, supporting our hypothesis. Co-immunoprecipitation experiments revealed that CYP3A5 is a part of the HSP90-AR complex. Both Flag-immunoprecipitation and indirect immunoprecipitation revealed that CYP3A5 associates with the HSP90-AR complex. The CYP3A5 siRNA also downregulated 11 genes (TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB, and HPRT1) known to confer drug resistance, with a p value of ≤0.005. The role of TOP2A, MYC, and BRCA is well known in prostate cancer drug resistance; thus, their downregulation with CYP3A5 siRNA treatment may predict better outcome. Conclusion: Our results indicate that CYP3A5 is a part of the HSP90-AR complex and positively regulates AR nuclear translocation promoting AR downstream signaling and therapeutic resistance. Therefore, targeting CYP3A5 to block AR nuclear translocation may be a therapeutic adjunctive approach in advanced prostate cancer. As CYP3A5 siRNA also downregulates drug-resistance genes, targeting CYP3A5 may also help overcome therapeutic resistance. As many concomitant medications can alter CYP3A5 activity, thus they can modify AR signaling. We hypothesize that AR directed therapeutic efficacy can be optimized by proper drug selection through minimizing the use of AR inducers while maximizing the use of AR inhibitors. These observations may play a significant role in African Americans who express high level of wild-type CYP3A5 and often have aggressive prostate cancer. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr., Ranjana Mitra. CYP3A5 promotes AR downstream signaling and contributes to therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B001.

Abstract 5900: Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in prostate cancer cells

Abstract Background and objective: Androgen deprivation therapy (ADT) is among the most effective of all cancer therapies for prostate cancer; however, resistance to therapy leading to castrate resistant prostate cancer (CRPC) is inevitable in most patients. Recently we demonstrated that CYP3A5 expressed in prostate cancer cells facilitates the nuclear translocation of AR, directly impacting the transcriptional regulation of AR-dependent genes promoting prostate cancer cell growth. Our preliminary data show that CYP3A5 is a part of the HSP90-AR complex, known to regulate AR nuclear localization and activation. Additionally, we observed that CYP3A5 inhibition also blocks growth of prostate cancer cells lacking AR ligand binding site (22RV1). This observation suggested presence of an alternative mechanism of regulation of prostate cell growth by CYP3A5, independent of its AR activation. We hypothesize that CYP3A5 inhibition can also sensitize prostate cancer cells to therapy by downregulating expression of drug resistance genes independent of its AR regulation. Our aim is to decipher the role of CYP3A5 in therapeutic resistance, by screening for CYP3A5 regulated cancer drug resistance genes and to further understand the underlying mechanism of this regulation. Method: We used cancer drug resistance PCR array after CYP3A5 siRNA knockdown to identify CYP3A5 regulated genes involved in therapeutic resistance. Further we evaluated the role of CYP3A5 in inducing synthetic lethality in combination with DNA damage repair inhibiting drugs by imagining the γ-H2AX foci. Results: CYP3A5 siRNA knockdown downregulates 11 genes involved in cancer drug resistance: TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB and HPRT1 with a p≤0.005 value. Of the list of downregulated genes, the three genes TOP2A, BRCA2 and BRCA1 have the most fold change and their loss is known to impair homologous recombination (HR), leading to synthetic lethality. HR defects in CRPC, sensitizes these tumors to PARP and topoisomerase inhibitors. We tested the effect of CYP3A5 inhibition in inducing HR defects by analyzing γ-H2AX foci counts in combination with topoisomerase inhibitor etoposide. Increased number of γ-H2AX foci suggest that CYP3A5 siRNA may induce synthetic lethality in prostate cancer cells when used in combination with DNA damage repair inhibiting drugs by impairing HR and downregulating TOP2A and BRCA1/2 expression. Conclusions: CYP3A5 inhibition downregulates DNA damage repair genes (TOP2A and BRCA1/2) and cell cycle progression genes (CCNE1, CDK 2/4) and impairs HR. CYP3A5 inhibition can sensitize CRPC to PARP and topoisomerase inhibitors as it induces synthetic lethality by downregulating TOP2A and BRCA1/2 expression. CYP3A5 inhibition can be potentially exploited therapeutically in combination with PARP inhibitors as it functionally impairs HR and causes synthetic lethality. Citation Format: Priyatham Gorjala, Ranjana Mitra, Oscar Goodman. Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5900.

Abstract B48: Effect of concomitant CYP3A5-modulating drugs on androgen-receptor signaling in African American men undergoing androgen deprivation therapy

Abstract Introduction: In a recent study we have established that CYP3A5 augments nuclear translocation of androgen receptor (AR) and promotes prostate cancer (PC) growth. CYP3A isoforms are responsible for metabolizing almost 50% of the commonly prescribed drugs, many of which are CYP3A5 inhibitors and inducers. PC patients are elderly with comorbidities and are often prescribed concomitant medications while undergoing androgen-deprivation therapy (ADT), the most preferred treatment for PC. CYP3A5 is polymorphic, and African Americans (AA, 73%) express higher levels of CYP3A5 due to presence of wild-type CYP3A5 (*1/*1) compared to Caucasians (95%) expressing the mutant (*3/*3) producing only 13% of the active full-length CYP3A5 protein. Due to the expression of wild-type CYP3A5 in AA, concomitant medications modulating CYP3A5 activity can drastically alter AR activity and ADT efficacy in these patients. Our current work is focused on deciphering the mechanism of how commonly used CYP3A5-modulating medications can alter AR signaling in AA carrying the wild-type CYP3A5. Method: We genotyped the androgen-responsive prostate cancer cell lines to determine their CYP3A5 status using TaqMan® Drug Metabolism assay. We used cDNA from cells treated with either nontarget or CYP3A5 siRNA in RT2 profiler PCR arrays to identify set of genes in AR signaling pathway that are affected by CYP3A5 inhibition. Confocal microscopy was used to study AR nuclear localization after exposure to commonly used CYP3A5 inducer/inhibitor drugs. Cell fractionation experiments were used to further confirm the effect of these drugs on AR signaling. Luciferase-based reporter assay was used to test effect of CYP3A5-modulating drugs on AR activity. Results: Our genotyping assay revealed that MDA PCa 2b (African American) carries *1/*3 CYP3A5 gene compared to LNCaP (Caucasian) carrying *3/*3 genotype. To compare the differences due to CYP3A5 genotype on AR signaling, we selected MDAPCa2b and LNCaP cells for further studies. Consistent with earlier observations with LNCaP, MDA PCa 2b cell lines also showed decreased nuclear translocation of AR with CYP3A5 siRNA in both confocal and cell fractionation experiments. We also observed blocking of AR nuclear localization with azamulin, a CYP3A-specific inhibitor in both cell lines. The q-PCR-based profiler assay revealed a set of nine AR-regulated genes (SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1, and SPDEF) downregulated with CYP3A5 siRNA treatment with a p value of ≤0.005 in MDA PCa 2b cells. MYC is known to be involved in cell cycle progression and apoptosis whereas PIK3R3 codes for the regulatory subunit of PI3 kinase regulating cell growth. SPDEF codes for a transcription factor promoting PSA known to aid in the cell growth. Downregulation of these genes with CYP3A5 siRNA treatment indicates that CYP3A5 modifier medications can alter AR downstream signaling regulating growth. We tested some commonly prescribed drugs that are either CYP inhibitors (amiodarone, ritonavir) or inducers (phenytoin, rifampicin) and evaluated their ability to alter AR signaling. The results revealed that the CYP inducers promoted increased AR nuclear migration and increased PSA production whereas CYP3A5 inhibitors blocked AR nuclear migration, decreasing PSA expression in both cell lines. A luminescence-based reporter assay using androgen response elements also supported that these CYP3A inducer and inhibitor drugs alter AR signaling. Conclusion: Our results indicate that concomitant medications that can alter CYP3A5 activity can modify AR signaling and PC growth. These results suggest that these CYP-modulating drugs can alter efficacy of ADT severely in AA men predominantly expressing wild-type CYP3A5 (*1/*1). We hypothesize that AR-directed therapeutic efficacy can be optimized by proper drug selection through minimizing the use of AR inducers while maximizing the use of AR inhibitors in patients undergoing ADT. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr., Ranjana Mitra. Effect of concomitant CYP3A5-modulating drugs on androgen-receptor signaling in African American men undergoing androgen deprivation therapy [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B48.

Difference in Mechanism-Based Inhibition of Cytochrome P450 3A4 and 3A5 by a Series of Fluoroquinolone Antibacterial Agents.

A series of fluoroquinolone antibacterial compounds were found to be irreversible (compounds 1-5) and quasi-irreversible (compounds 6-9) inhibitors of CYP3A4. The purpose of this study was to evaluate their mechanism-based inhibition (MBI) potency against CYP3A5. Compounds 1-5 were also irreversible inhibitors of CYP3A5, whereas compounds 6-9 showed neither irreversible nor quasi-irreversible inhibition of CYP3A5. Compounds 6 and 8 did not form a metabolite-intermediate complex with the heme of CYP3A5 during incubation. The structural analysis of the metabolites after incubation of compounds 1 and 6 with CYP3A5 revealed that their metabolites were identical to those produced by CYP3A4, including the precursors of which are speculated to account for the MBI of CYP3A4. The homology modeling of CYP3A5 suggests that four residues around the nitroso intermediate of compound 6 in the substrate-binding pocket of CYP3A4 correspond with the bulkier residues in CYP3A5-especially Phe210 in CYP3A5-which might contribute to the steric hindrance with the nitroso intermediate of compound 6. The substrate-binding pocket structure of CYP3A5 might prevent the nitroso intermediate from coordinate binding with the heme, thereby preventing quasi-irreversible inhibition. Our study may provide new insights into the observable differences between the inhibition of CYP3A4 and CYP3A5.

CYP3A5 regulates prostate cancer cell growth by facilitating nuclear translocation of AR.

The central role of androgen receptor (AR) signaling is established in prostate cancer growth and progression. We propose CYP3A5 is part of a feedback loop that modulates the sensitivity of AR to androgen exposure. The purpose of this study is to elucidate the mechanism of regulation of AR expression by CYP3A5. To identify the role of CYP3A5 in regulating AR signaling, CYP3A5 protein expression was inhibited using CYP3A5 siRNA and azamulin. Both cell fractionation and immunocytochemical approaches in combination with dihydrotestosterone (DHT) and R1881 treatment were used to evaluate changes in AR nuclear translocation. CYP3A5 siRNA blocked growth of LNCaP and C4-2 cells by 30-60% (P ≤ 0.005). Azamulin, a CYP3A pharmacologic inhibitor, reduced the growth of LNCaP, C4-2 and 22RV1 lines by ∼ 40% (P ≤ 0.005). CYP3A5 siRNA inhibited growth in response to DHT and R1881 treatment in LNCaP and C4-2 by decreasing nuclear AR localization and resulting in diminished PSA and TMPRSS2 expression. Decreased AR nuclear localization resulting from CYP3A5 inhibition resulted in growth inhibition comparable to IC60 and IC40 of bicalutamide in LNCaP and C4-2 cell lines. Conversely, the CYP3A inducer rifampicin enhanced AR nuclear localization. As CYP3A5 regulates the nuclear translocation of AR; co-targeting CYP3A5 may provide a novel strategy for enhancing the efficacy of androgen deprivation therapy. Consequentially, these data suggest that concomitant medications may impact androgen deprivation therapy's efficacy.

In vitro inhibitory effect of West African medicinal and food plants on human cytochrome P450 3A subfamily

Aim of the study In Africa, medicinal plants are used intensively and concomitantly with allopathic medicines in the treatment of opportunity diseases by many patients or by healthy person to prevent diseases. However, there is little information about the interactions between medicines and botanical products used currently in West Africa area. Therefore, the aim of the present investigation is to study the effect of some plant products on CYP3A4, CYP3A5 and CYP3A7, three individual enzymes of CYP3A subfamily, in vitro. Materials and methods Teas and ethanolic extracts of medicinal, food and co-administered plants were evaluated on CYP3A4, CYP3A5 and CYP3A7 individual enzymes in vitro using fluorometric assays. Results Extracts of adjuvant plants such as Aframomum cuspidatum, and Aframomum melegueta, as well as one medicinal plant ( Harrisonia abyssinica) inhibited CYP3A4, CYP3A5 and CYP3A7 activity more than 90%. Phyllanthus amarus showed high inhibition of CYP3A5 and CYP3A7. Food plants (S olanum macrocarpon and Talinum triangulare) inhibited CYP3A4 and CYP3A5 less than 20%. Conclusion These results indicate that plants tested in this study affect in vitro the activity of the main three CYP3A subfamily enzymes. These active plants could interfere with the metabolism at phase I of conventional drugs in vivo as well act as pharmacoenhancers in herbal mixtures.

Cytochrome P450 3A Inhibition by Ketoconazole Affects Prasugrel and Clopidogrel Pharmacokinetics and Pharmacodynamics Differently

Prasugrel and clopidogrel inhibit platelet aggregation through active metabolite formation. Prasugrel's active metabolite (R-138727) is formed primarily by cytochrome P450 (CYP) 3A and CYP2B6, with roles for CYP2C9 and CYP2C19. Clopidogrel's activation involves two sequential steps by CYP3A, CYP1A2, CYP2C9, CYP2C19, and/or CYP2B6. In a randomized crossover study, healthy subjects received a loading dose (LD) of prasugrel (60 mg) or clopidogrel (300 mg), followed by five daily maintenance doses (MDs) (15 and 75 mg, respectively) with or without the potent CYP3A inhibitor ketoconazole (400 mg/day). Subjects had a 2-week washout between periods. Ketoconazole decreased R-138727 and clopidogrel active metabolite Cmax (maximum plasma concentration) 34-61% after prasugrel and clopidogrel dosing. Ketoconazole did not affect R-138727 exposure or prasugrel's inhibition of platelet aggregation (IPA). Ketoconazole decreased clopidogrel's active metabolite AUC0-24 (area under the concentration-time curve to 24 h postdose) 22% (LD) to 29% (MD) and reduced IPA 28% (LD) to 33% (MD). We conclude that CYP3A4 and CYP3A5 inhibition by ketoconazole affects formation of clopidogrel's but not prasugrel's active metabolite. The decreased formation of clopidogrel's active metabolite is associated with reduced IPA.

The Development of a Cocktail CYP2B6, CYP2C8, and CYP3A5 Inhibition Assay and a Preliminary Assessment of Utility in a Drug Discovery Setting

Tools for studying the roles of CYP2B6, CYP2C8, and CYP3A5 in drug metabolism have recently become available. The level of interest in these enzymes has been elevated because investigations have revealed substrate promiscuity and/or polymorphic expression. In this study, we aimed to develop a single cocktail inhibition assay for the three enzymes and assess its utility in drug discovery. Bupropion hydroxylation, amodiaquine N-deethylation, and midazolam 1'-hydroxylation were chosen as probe reactions for CYP2B6, CYP2C8, and CYP3A5 and were analyzed using liquid chromatography-tandem mass spectrometry. Kinetic analyses were performed to establish suitable conditions for inhibition assays, which were subsequently automated. CYP2B6, CYP2C8, and CYP3A5 IC(50) values were determined for marketed drugs and almost 200 AstraZeneca discovery compounds from 16 separate discovery projects. For the marketed drugs, results obtained were comparable with literature values. Data were also compared with IC(50) values determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. In this dataset, the majority of compounds were more potent inhibitors of CYP2C9, CYP2C19, CYP2D6, and CYP3A4 than of CYP2B6, CYP2C8, or CYP3A5. The potential impact of these findings on a cytochrome P450 inhibition strategy is discussed.

DIFFERENTIAL MECHANISM-BASED INHIBITION OF CYP3A4 AND CYP3A5 BY VERAPAMIL

The genetic basis for polymorphic expression of CYP3A5 has been recently identified, but the significance of CYP3A5 expression is unclear. The purpose of this study is to quantify the capability of verapamil, a mechanism-based inhibitor of CYP3A, and its metabolites to inhibit the activities of CYP3A4 and CYP3A5, and to determine whether CYP3A5 expression in human liver microsomes alters the inhibitory potency of verapamil. Testosterone 6beta-hydroxylation or midazolam 1'-hydroxylation was used to quantify CYP3A activity. The possibility that verapamil and its metabolites form metabolic-intermediate complex (MIC) with CYP3A was assessed using dual beam spectrophotometry. Verapamil and N-desalkylverapamil (D617) were found to have little inhibitory effect on cDNA-expressed CYP3A5 activity and did not form a MIC with cDNA-expressed CYP3A5 as indicated by the appearance of the characteristic peak at 455 nm. At 50 microM, norverapamil showed time-dependent inhibition of CYP3A5 (30%), but to a much lesser extent compared with that of CYP3A4 (80%). The estimated values of the inactivation parameters k(inact) and K(I) of norverapamil were 4.53 microM and 0.07 min(-1) for cDNA-expressed CYP3A5, and 10.3 microM and 0.30 min(-1) for cDNA-expressed CYP3A4. Human liver microsomes that expressed CYP3A5 were less inhibited by both verapamil and norverapamil. The inactivation efficiency of verapamil and norverapamil was 30 times and 45 times lower, respectively, for CYP3A5-expressing microsomes compared with CYP3A5-non-expressing microsomes. These findings indicate that the presence of variable CYP3A5/CYP3A4 expression in the liver may contribute to the interindividual variability associated with verapamil-mediated drug interactions.