High-grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype. While 60–80% of HGSOC patients initially respond to treatment, the majority of patients will eventually become platinum resistant. Epigenetic modifications are mechanisms that alter the expression of a gene but do not change the DNA sequence itself. Several types of epigenetic modifications, including DNA methylation, histone deacetylation, and microRNA expression, have been implicated in the progression of HGSOC to chemoresistance. These modifications can be targeted by epigenetic modulating therapies to overcome chemoresistance. This review summarises the epigenetic modifications identified in chemoresistant HGSOC and clinical trials utilizing epigenetic therapies in HGSOC.High-grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype, and the overall survival rate has not improved in the last three decades. Currently, most patients develop recurrent disease within 3 years and succumb to the disease within 5 years. This is an important area of research, as the major obstacle to the treatment of HGSOC is the development of resistance to platinum chemotherapy. The cause of chemoresistance is still largely unknown and may be due to epigenetics modifications that are driving HGSOC metastasis and treatment resistance. The identification of epigenetic changes in chemoresistant HGSOC enables the development of epigenetic modulating drugs that may be used to improve outcomes. Several epigenetic modulating drugs have displayed promise as drug targets for HGSOC, such as demethylating agents azacitidine and decitabine. Others, such as histone deacetylase inhibitors and miRNA-targeting therapies, demonstrated promising preclinical results but resulted in off-target side effects in clinical trials. This article reviews the epigenetic modifications identified in chemoresistant HGSOC and clinical trials utilizing epigenetic therapies in HGSOC. Standard-of-care treatment for high-grade serous ovarian cancer (HGSOC) consists of combination carboplatin and paclitaxel chemotherapy. While 60–80% of HGSOC patients initially respond to treatment, the majority of patients will eventually become platinum resistant . AlthougCancer stem cells (CSCs) are a small subgroup of cancer cells which are characterised by their ability to self-renew and give rise to both CSCs and non-CSCs within a heterogenous tumour . CSCs haEpithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their epithelial characteristics and take on properties of mesenchymal cells, including disruption of adhesions to other cells and the cellular basement membrane, as well as increased cell migration and invasiveness ,7. StudiDefective DNA repair pathways such as the homologous recombination (HR) and nucleotide excision repair (NER) pathways are also crucial in the development of chemoresistance in HGSOC. The HR pathway is involved in repairing double-strand breaks that occur at sites of DNA crosslinks caused by platinum chemotherapy during DNA replication ,6. This Epigenetic modifications are mechanisms that alter the expression of a gene but do not change the DNA sequence itself ,16. ThesTP53 mutations and substantial copy-number changes, key characteristics of HGSOC.The modifications that will be discussed in this review are DNA methylation, histone acetylation, and microRNA (miRNA) as epigenetic modulators, as they are most commonly studied in serous ovarian cancer  enzymes catalyse the addition of a methyl group onto the fifth carbon of a cytosine ring to form methyl cytosine ,19. DNA There are several known histone modifications, such as acetylation, phosphorylation, and methylation. The most comprehensively studied modification in ovarian cancer, not specifically HGSOC, is histone acetylation.DNA is coiled around and octomer of four histone proteins  ,23, resuHDACs are divided into four classes: class I  is found in the nucleus and is the most prevalent, whereas class II  and class IV (HDAC11) are found in both the nucleus and cytoplasm, and all are zinc-dependent and considered classical HDACs. Class III  is nicotinamide adenine dinucleotide (NAD)-dependent and found in both the nucleus and cytoplasm . HDACs aMicroRNAs, or miRNAs, are small, highly conserved single-stranded non-coding RNAs (19–25 nucleotides) that are involved in post-translational regulation of gene expression. miRNAs negatively regulate target protein-coding genes through binding with the 3′-UTR (untranslated region), which causes messenger RNA (mRNA) degradation or translational repression ,29. miRNmiRNAs can act as oncogenic miRNAs (oncomiRs) by targeting mRNA that encode for tumour-suppressor proteins, and as tumour-suppressor miRNAs by targeting mRNA that encode for oncogenic proteins. miRNA expression is typically altered in cancers, with tumour-suppressor miRNA subsets typically downregulated and oncogenic miRNAs upregulated ,31. DysrHGSOC is highly controlled by epigenetic modifications. DNA methylation and histone modification have both been extensively studied and targeted with new treatment regimens to attempt to overcome platinum chemoresistance.The potential role of altered DNA methylation patterns in the acquisition of chemoresistance in HGSOC at both the genome-wide and single-gene level has been extensively studied  between chemoresistant and chemosensitive tumour samples, which were associated with 296 genes that were both differentially methylated and differentially expressed in chemoresistant samples . InteresMSX1 were found to have decreased methylation levels in tumour samples from chemoresistant HGSOC patients [MSX1 is crucial in controlling epithelial–mesenchymal interactions during embryogenesis [MSX1 plays a role in the occurrence of EMT through promoting the transition of cancer cells to a mesenchymal phenotype. LAMA3, an integral part of the cell basement membrane, was also found to be hypermethylated in chemoresistant HGSOC tissue samples [LAMA3 expression has previously been linked to both loss of the basement membrane and EMT [LAMA3 hypermethylation may also be involved in the development of EMT. Lum et al. also identified several differentially methylated genes in chemoresistant HGSOC involved in EMT, including SOX9, ZIC1, and TWIST [Investigations into the role of DNA methylation in HGSOC chemoresistance have also focused on differential methylation of single genes, especially in genes associated with EMT. Biological pathway analysis of the 452 hypermethylated genes discovered by Cardenas et al. found that EMT was one of the most highly enriched biological pathways in chemoresistant HGSOC , indicatpatients . MSX1 isogenesis ,38, and ogenesis , suggest samples . Reduced and EMT , indicatnd TWIST , furtherFZD1 [FZD10 [GSK3B, another Wnt-associated gene, also has altered methylation patterns in chemoresistant and chemosensitive HGSOC tissue samples [The wingless/integrated (Wnt) signalling pathway is highly associated with EMT as well as CSCs and has been implicated in HGSOC chemoresistance due to abnormal methylation of genes within the pathway. Activation of the Wnt pathway occurs through interaction of Wnt proteins with the frizzled (FZD) family of transmembrane receptors, triggering phosphorylation of downstream proteins ,43. Two FZD1  and FZD11 [FZD10 , have bo samples .BRCA1 in a relapsed HGSOC tumour sample in comparison to the patient’s primary tumour [BRCA1 in the acquisition of chemoresistance in HGSOC.While there has not been as much evidence on differential methylation of DNA repair genes being involved in HGSOC chemoresistance, there has been one reported case of hypomethylation of y tumour . Global AKAP12 is a scaffolding protein previously found to be a tumour suppressor gene [AKAP12 was increased in paclitaxel resistant HGSOC cell lines, which was associated with low levels of AKAP12 gene methylation [AKAP12 was found to increase in cells after induced EMT [AKAP12 hypomethylation. BLU and ZNF671, two genes which are thought to act as tumour suppressors in other cancer types [DOK2, a known lung cancer tumour suppressor, is also differentially methylated in chemoresistant samples [Altered methylation patterns of tumour suppressor genes have also been implicated in chemoresistance development in HGSOC. sor gene . Proteinhylation . Interesuced EMT , indicater types ,50, haveer types ,52. DOK2 samples , furtherThe role of post-translation histone modifications in HGSOC chemoresistance is still relatively understudied. Histone hypoacetylation by HDACs, and subsequent gene repression, has been associated with tumorigenesis by upregulating genes involved in cell proliferation and migration, and downregulating genes involved in cell differentiation and apoptosis ,56.In general, ovarian cancer chemoresistance is often associated with increased HDAC activity. Class I HDACs 1, 2, and 3 were highly expressed in a large proportion of HGSOC (64%) and were associated with highly proliferating tumours, as determined by Ki-67 labelling . The oveThe overexpression of HDAC1 in the nucleus was significantly associated with decreased progression-free survival (PFS) and overall survival (OS) in serous ovarian cancer . The expIn a recent study by Ali et al. low expression of HDAC6 was shown to be associated with decreased OS in HGSOC patients . HoweverThe role of miRNAs in chemoresistance is the regulation of genes involved in apoptosis, proliferation, regulation of cell cycle, and DNA repair, all of which are pathways targeted or exploited by chemotherapeutic agents. miRNAs have been shown to both increase sensitivity and promote resistance to platinum chemotherapy. However, those that increase platinum chemoresistance have been compiled in this review (summarised in EphA2 (EPH receptor A2). High expression of EphA2 is significantly associated with poor 5-year OS in patients with HGSOC [RB1, a protein that controls cells division and protects cells from genomic instability in fallopian tube cells. Simultaneously, miR-181a also inhibited the expression of STING, allowing the genomically unstable cells to be protected from being destroyed by interferon-mediated cell death [An analysis of the TCGA dataset  by Nishith HGSOC . The expll death .VEGFB and VEGFR2 in tumours that were responsive to treatment, suggesting that miR-484 exerts its effects through the regulation of angiogenic factors that control the formation of new vasculature [miR-484, miR-642, and miR-217 were downregulated in tumours that were non-responsive (stable or progressive disease) to platinum and taxane combination . Additioculature .The miR-200 family, consisting of miR-200a, miR-200b, miR-200c, miR-141, and miR-429, has been extensively studied in ovarian cancer chemoresistance. Nam et al. showed that the high expression of miR-141 and miR-200c is associated with platinum chemoresistance in ovarian cancer . HoweverThe inhibition of miR-141 and miR-200c is involved in resistance to platinum- and taxane-based chemotherapies by triggering EMT. The re-expression of the miR-200 family reverts the EMT phenotype by inducing mesenchymal-to-epithelial transition (MET), and resensitizes ovarian cancer cells to platinum- and taxane-based chemotherapies .ZEB1 and repression of E-cadherin, but the re-expression of miR-200c restores E-cadherin and reduces cell migration and invasion [TUBB3), which encodes a tubulin isotype known to mediate chemoresistance [TUBB3 and reduces tumour burden by increasing sensitivity to taxanes [Low expression of miR-200c results in the aberrant expression of invasion . miR-200sistance . Restorasistance . Additio taxanes .These studies show that miRNAs can affect the response to standard platinum- and taxane-based chemotherapies used to treat HGSOC, by targeting multiple cellular pathways including the EMT/MET pathways and microtubule assembly. miRNAs can also target the angiogenesis pathway, which may affect sensitivity anti-angiogenic therapies that are used in recurrent HGSOC.BRCA1. In serous ovarian cancer, higher levels of miR-9 were associated with decreased BRCA1 expression and showed increased response to platinum chemotherapy and longer PFS [The inhibition of DNA damage repair proteins by miRNAs also plays a role in resistance to DNA-damaging chemotherapy in HGSOC treatment. miR-9 downregulates BRCA1 expression by directly binding to the 3′-UTR of nger PFS . This suPTEN. The suppression of miR-93 by miR-93 antisense oligonucleotides increased PTEN expression and apoptotic activity in the ovarian cancer cells, suggesting that miR-93 may play a role in regulating platinum sensitivity [miR-93 was upregulated in platinum-resistant HGSOC cells lines. miR-93 was shown to downregulate PTEN expression in HGSOC cells by, similar to the above, directly binding to the 3′-UTR of sitivity .Cx43 [Several studies comparing platinum-sensitive and platinum-resistant HGSOC patient cohorts have also identified miRNAs that are potential biomarkers of platinum chemoresistance. miR-206 was highly expressed in primary platinum-resistant (majority serous) ovarian cancer patients (classified by incomplete response to primary therapy) . This waCx43 , a gap jCx43 . An analCx43  by Qi etCx43 . The tarCx43 . The tarCx43 .The majority of patients with advanced high-grade serous ovarian cancer (HGSOC) develop recurrent disease within 3 years and succumb to the disease within 5 years . AlthougThe reversibility of epigenetic modifications makes them a potential treatment strategy to enhance response to chemotherapy in chemoresistant HGSOC. One promising area of epigenetic treatments is treatments that reduce DNA methylation by inhibiting the ability of DNMT enzymes. These DNMT inhibitors (DNMTi) are cytosine analogues which are incorporated into the DNA strand during replication and covalently bind to DNMTs, making them inactive. As a result of this decrease in DNMT activity, CpG sites which were previously methylated become unmethylated during cell replication, and transcription of genes previously silenced due to promoter hypermethylation is increased ,81. Two While azacitidine and decitabine have not shown any effectiveness in treating solid tumours as single agents, preclinical studies have found that combination therapy of a DNMT inhibitor alongside chemotherapy increased the sensitivity to platinum in platinum-resistant ovarian cancer cell lines ,86. Pre-HOXA11 and BRCA1 reduced after 8 days [MLH1, RASSF1A, HOXA10, and HOXA11 all had reduced methylation after combination treatment [This combination therapy has therefore been the focus of clinical trials investigating DNMT inhibitors in chemoresistant HGSOC . Fang etr 8 days . This cor 8 days . Of the reatment . These rreatment . Severe reatment ,89 and dreatment . Additioreatment ,92, indiCombination therapy of azacitidine and carboplatin has also been assessed in a phase 1b-2a clinical trial by Fu et al. wherein 1 out of 29 patients enrolled achieved a complete response, 3 achieved partial responses, and 10 developed stable disease , with no toxicities observed . PlatinuClinical trials testing carboplatin in combination with the second-generation DNMTi guadecitabine have also recently been conducted. A phase 1 study conducted by Matei et al.  tested gTo date, DNMTis are the epigenetic modulators that have progressed the furthest in clinical trials, with next-generation DNMTis that confer less severe and dose-limiting side effects being investigated .The mechanism of action of HDAC inhibitors (HDACi) is via the alteration of gene transcription, affecting proteins involved in cell growth, the promotion of cell differentiation, and apoptosis . HDACis In vitro studies showed that HDACis (pan and class-specific) sensitize HGSOC cells to DNA-damaging drugs, such as platinum chemotherapy, by increasing apoptosis-mediated cell death caused by platinum chemotherapy treatment ,98. The Panobinostat downregulated genes of the cyclin E and homologous recombination repair pathways, and synergistically with the PARP inhibitor olaparib, reduce the cell viability and growth in homologous recombination-proficient ovarian cancer cells and xenografts . SimilarSeveral treatment combinations that include HDACis have been trialled to determine whether HDACis can resensitize platinum-resistant HGSOC to platinum chemotherapy . HoweverA phase 1 dose-escalating clinical trial of HDACi vorinostat and chemotherapy agents carboplatin and gemcitabine in platinum-sensitive ovarian cancer (including HGSOC) with a first recurrence was terminated early due to haematological toxicity . Of the Belinostat is better tolerated than vorinostat in combination treatments regimens. In a phase 2 clinical trial evaluating the efficacy of combined belinostat and carboplatin, with 27 patients, the majority of which had HGSOC, the ORR was 7.4% . The study was closed due to the lack of drug activity and concluded that the addition of belinostat did not resensitize platinum-resistant ovarian cancer to carboplatin .To date, there have not been any clinical trials utilising panobinostat or romidepsin in combination with standard-of-care therapy for chemoresistant HGSOC. There is evidence that HDACis could potentially play a role in the treatment of ovarian cancer, but they would need to be further improved to increase their efficacy whilst having tolerable side effects.Stone et al. have previously showed that epigenetic modulating drugs, DNMT inhibitors and HDAC6 inhibitors, individually increase immune signalling in HGSOC cell lines . DNMT inTherapeutic approaches to regulate the expression of miRNAs include miRNA replacement by miRNA mimics or miRNA inhibition by antimiRs. As discussed above, each miRNA can potentially target hundreds of mRNAs as they do not require perfect binding complementarity. This can lead to potential off-target effects in miRNA-targeting therapy. Another limitation of miRNA-targeting therapy is the short half-life of miRNA mimics and antimiRs, which are also almost immediately degraded by nucleases and therefore require a safe delivery system . These iMany preclinical studies of miRNA-targeting therapies have not yet developed into clinical trials. As recently reviewed by Zhang et al. the ten miRNA-targeting drugs that have been in clinical trials have hundreds of unapproved targets, compared to approved drugs that have no more than five unapproved targets .PDCD4, and that the suppression of miR-182 increases platinum- and taxane-induced apoptosis [PDCD4 [Previous studies (not in cell lines listed above) showed that miR-182 is involved in chemoresistance of ovarian cancer by downregulating the cell cycle gene poptosis ,110. Trepoptosis . The compoptosis . In mices [PDCD4 .BMI1, a regulator of CSCs [An analysis of the TCGA database by Dwivedi et al. showed that lower expression of miR-15a and miR-16, and subsequent upregulation of their target  of CSCs , is asso of CSCs . The EMT of CSCs . Treatme of CSCs . The com of CSCs .A search of the Australian New Zealand Clinical Trials Registry (ANZCTR) and National Institutes of Health Clinical Trials database  did not show any active or completed clinical trials involving the use of miRNA mimics or anti-miRs in HGSOC chemotherapy regimens.The complexity and constantly evolving nature of the epigenetic features of HGSOC have been a major challenge to developing effective epigenetic therapeutics with tolerable toxicity profiles. The changes in DNA methylation, histone deacetylation, and miRNA expression that occur as HGSOC develops resistance to standard-of-care chemotherapies have been difficult to determine and target for effective therapeutic development. The most promising area of therapeutic potential for treatment-resistant HGSOC is DNMTi in combination with traditional chemotherapy. The results of the early phase 2 clinical trials have provided evidence that the combination should be followed up in larger studies. As the field of HDACi develops further, there is also promise that the combination of HDACi and chemotherapy will provide an alternate treatment option once resistance to standard-of-care chemotherapy occurs.


Viruses package host RNAs in their virions which are associated with a range of functions in the viral life cycle. Previous transcriptomic profiling of host RNA packaging mostly focused on retroviruses. Which host RNAs are packaged in other viruses at the transcriptome level has not been thoroughly examined. Here we perform proof-of-concept studies using both small RNA and large RNA sequencing of six different SARS-CoV-2 viral isolates grown on VeroE6 cells to profile host RNAs present in cell free viral preparations and to explore SARS-CoV-2 genomic RNA modifications. We find selective enrichment of specific host transfer RNAs (tRNAs), tRNA fragments and signal recognition particle (SRP) RNA in SARS-CoV-2 viral preparations. Different viral preparations contain the same set of host RNAs, suggesting a common mechanism of packaging. We estimate that a single SARS-CoV-2 particle likely contains up to one SRP RNA and four tRNA molecules. We identify tRNA modification differences between the tRNAs present in viral preparations and those in the uninfected VeroE6 host cells. Furthermore, we find uncharacterized candidate modifications in the SARS-CoV-2 genomic RNA. Our results reveal an under-studied aspect of viral-host interactions that may be explored for viral therapeutics. Viral assembly is a critical stage in the viral life cycle that produces mature virus containing the viral genome and proteins needed to infect another host target cell. As early as 1980s it was shown that certain viruses also package host RNAs into their virions. Host transfer RNA (tRNA) is a major cellular RNA family, which is packaged in virions . tRNAs aLys(TTT) from the host cell since it has a fully complementary sequence of ∼20 nucleotides to the primer binding site of the retroviral genome (Lys(TTT), other prominent tRNAs packaged into the virions include tRNALys(CTT), tRNAAsn(GTT) and others  is among the selectively packaged tRNAs, just like HIV-1. We find that a specific tRNA modification may influence tRNA packaging, and some packaged tRNAs are likely tRNA fragments. We also identify a low level of SARS-CoV-2 subgenomic transcripts in the viral preparations, as well as several candidate modification sites in the SARS-CoV-2 genomic RNA.Here, we utilize both small RNA (<200 nt) and large RNA (>200 nt) sequencing to identify host RNAs that are present in cell free viral preparations which consist mostly of the SARS-CoV-2 viruses cultured on VeroE6 cells. We obtain sequencing data for six distinct primary SARS-CoV-2 isolates  and compWe performed Illumina sequencing starting with total RNA extracted from VeroE6 cells and from SARS-CoV-2 viral preparations cultured on VeroE6 cells . To imprGlu(TTC), tRNALys(TTT), tRNALeu(AAG), tRNASer(AGA), tRNASer(GCT), and tRNASer(TGA). These results indicate that SARS-CoV2 virions selectively incorporate tRNA isoacceptors.We next examined the selectivity of packaged tRNA at two levels. Mammalian genomes contain many tRNA isodecoder genes that share the anticodon, but possess different body sequences; all tRNA isodecoders with the same anticodon belong to a single tRNA isoacceptor family . AbundanGlu(TTC) isodecoders, four could be detected in the viral preparations. However, only two isodecoders represent almost all tRNAGlu(TTC) in the viral preparations, even though neither is the most abundant isodecoder in VeroE6 cells (Leu(AAG), tRNALys(TTT), tRNASer(AGA) isodecoders in cells are also the ones in the viral preparations (Ser(GCT) and tRNASer(TGA), two isodecoders each are present at appreciable levels, in each case, the isodecoder at the highest level is also the one in the viral preparations  (Leu(AAG) and tRNALys(TTT) (22G26) in tRNALeu(AAG) which is difficult to remove by the demethylase because it is buried in the tRNA structure (2t6A37) in tRNALys(TTT)  in tRNASer  . The proLys(TTT) . The simtructure  and 2-meLys(TTT)  which dofication . This re tRNASer .Chlorocebus sabaeus kidney, their tRNA modifications have not been reported in the literature. We analyzed the mutation signatures of the cellular tRNAs in the libraries without demethylase treatment and validated the methylations using the results from demethylase treatment  in all tRNAs that are A34 in the genome which include tRNALeu(AAG) and tRNASer(AGA)  ; Table 21A58 (tRNA nomenclature) in tRNALeu(AAG), tRNALys(TTT), and tRNAGlu(TTC), and I34 in tRNALeu(AAG) and tRNASer(AGA). For tRNALeu(AAG) and tRNALys(TTT), the mutation fraction at m1A58 is higher in the tRNA from the viral preparations than the VeroE6 cell tRNA (1A modified tRNA. Among the tRNAGlu(TTC) isodecoders, m1A58 level is variable in cells. Only two of the five abundant tRNAGlu(TTC) isodecoders have high modification levels in VeroE6 cells, but only isodecoders with low modification fractions are present in the viral preparations (Glu(TTC) in the virions are likely tRNA fragments, this result is consistent with low m1A modified tRNAGlu(TTC) being the preferred source of tRNAGlu(TTC) fragments in cells. tRNAs in cells and in the viral preparations are >90% modified with I34 in both tRNALeu(AGA) and tRNASer(AGA)  .We next carried out large RNA-seq (>200 nt) of the viral preparations to characterize the viral RNA and its candidate modifications. We first removed small RNAs by size-selection, followed by chemical fragmentation and library construction. As expected, most of the reads mapped to the SARS-CoV-2 genome (Wuhan reference). We used a mutation threshold of >90% to identify single nucleotide polymorphisms (SNPs) in these samples . These SC. sabaeus SRP from the Rfam database  modification in mRNA (3U) derivatives. 2) A29517 is DM-sensitive without CMC, CMC-sensitive without DM, the mutation signals are reversed in DM with CMC, or in CMC with DM. This site may represent N1-methyl-A (m1A) derivatives. 3) U3877 is DM-insensitive without CMC, CMC-sensitive without DM, DM-sensitive with CMC, not detected in CMC with DM. This site may represent N1-methyl or N3-methyl-pseudouridine  derivatives. 4) A29780 is DM-insensitive without CMC, CMC-sensitive without DM, DM-sensitive with CMC, not detected in CMC with DM. We do not recognize an A modification at this time that would generate such signatures. No stop signature was observed for any of these five sites , and the other with 1-cyclohexyl-(2-morpholinoethyl)carbodiimide (CMC) treatment . Demethy in mRNA . We firs in mRNA . These sve sites .It is intriguing to note that these five candidate modification sites in SARS-CoV-2 do not follow the expected pattern of changes for well-characterized RNA modifications. In all five cases, the mutation levels are at most 20%, which may reflect either low levels of modification at these sites or under-counting the modification levels because of the unknown nature of these modifications. Future studies are needed to pinpoint the exact nature of these modifications.In this work, we identified selective enrichment of host tRNAs and SRP RNA in cell free SARS-CoV-2 viral preparations, differences in tRNA modification between the tRNA in viral preparations and in cells, and candidate modification sites in the packaged SARS-CoV-2 genomic RNA. We estimate that a SARS-CoV-2 virion contains up to one molecule of SRP RNA. Given the roughly equivalent read counts of SRP and tRNA in the viral preparation , and fulLys(TTT) is facilitated by the host lysyl-tRNA synthetase (LysRS) and gag protein interaction (Lys(CTT) is also packaged at similar levels. In our case, however, tRNALys(CTT) was not enriched in the virions. On the other hand, the enrichment of multiple tRNASer isoacceptors may go through this mechanism of facilitating tRNA packaging through host seryl-tRNA synthetase (SerRS) and SARS-CoV-2 protein interactions. Retroviruses also package a large amount of SRP RNA into virions (How and why specific tRNAs and the SRP RNA are enriched in virions remains unclear. Packaging of the HIV primer tRNAeraction . As a co virions . Our resGlu(TTC) fragment, its levels and m1A58 modification profile match in the viral preparations and in cells. However, the full-length tRNALeu(AGA) and tRNALys(TTT) in the viral preparations have higher m1A58 levels than their counterparts in cells. M1A58-modified tRNA can interact differently with cellular components such as eEF1A compared to the hypo-modified tRNA . In future work, we will produce larger amounts of SARS-CoV-2 isolates representative of the early circulating strains as well as the viral variants of concern that have dominated most of the pandemic in 2021. We will culture these isolates on ACE2-expressing human cells rather than on African green monkey VeroE6 cells. These studies will firmly establish the dependency of host RNA packaging on SARS-CoV-2 variants and on host cell source.Residual nasopharyngeal swab specimens were collected after completion of the diagnostic process as part of the Mount Sinai Pathogen Surveillance Program. To culture SARS-CoV-2 isolates, .1 ml of viral transport media was inoculated into one well of a six-well plate seeded with a confluent monolayer of VeroE6 cells. Culture supernatants were harvested when CPE (cytopathic effect) became visible, aliquotted, and stored at −80°C. All work related to SARS-CoV-2 culture was performed in a BSL3 biocontainment facility by trained personnel and in accordance with the research registration approved by the Institutional Biosafety Committee (IBC).We cultured six distinct SARS-CoV-2 isolates representing the early lineages of the pandemic . After iThe viral culture supernatants were spun at 3,000 rpm for 10 min to remove particulates. vRNA from each viral preparation and total RNA from VeroE6 cells was performed using QIAamp Viral RNA Kits (Qiagen) following the manufacturer’s instructions. We did not filter the supernatants as that could result in some viruses being absorbed to the membrane and/or causing a loss of viral infectivity.2B4O7, pH 9.5  and incubated at 37°C for 30 min. To the deacylated samples, 5 µl of a 3′-end clean-up mixture  were added and incubated at 37°C for 20 min, and then heat inactivated at 65°C for 10 min. We used superscript IV RT in this work.The procedure was adapted from DM-tRNA-seq  with the1 µg of total RNA from infected Vero cell culture preparations was diluted to 50 µl in microcentrifuge tubes. Zymo RNA Clean and Concentrator-5 columns  were used to remove small RNAs ≤200 nt by following the manufacturer’s protocol. The large RNA (>200 nt) was eluted in 18 µl RNase free water. PCR machine was preheated at 95°C for RNA fragmentation experiment. Eluted RNA was then transferred to PCR tubes, 2 µl RNA fragmentation buffer  was added to each tube and mixed well. Samples were incubated at 95°C on PCR machine for 6 min (target fragmentation is 200–500 nt) followed by putting on ice immediately. 2 µl RNA fragmentation stop buffer  was added to each tube and mixed well. Samples were transferred to microcentrifuge tubes and diluted to 50 µl. Zymo RNA Clean and Concentrator-5 columns were used to cleanup RNA (>200 nt). RNA was eluted in 8 µl RNase free water. 1 μl T4 PNK buffer and 1 µl 10 U/μl T4 PNK were added to each tube and mixed well. The samples were incubated at 37°C for 30 min to repair RNA fragment 3′ ends. The samples were spun down and incubated at 75°C for 10 min followed by immediately putting on ice to inactivate T4 PNK. All samples (∼10 µl reaction mixture each) were used to build the libraries using bead-based library construction methods developed in our lab. Briefly, the first adaptor ligated RNA fragments were captured on the beads and pooled. The beads were then split into two equal parts for ± demethylase treatment. After demethylase treatment, the beads were split again to two parts for −CMC and +CMC treatment (1:1.5 ratio) . The ± dChlorocebus sabaeus that included non-coding RNA and tRNA were downloaded from Rfam database  to generate a custom reference database.Reference RNA sequences from https://github.com/BioInfoTools/BBMap), which results in merged fastq files. These merged fastq were converted to fasta file format using reformat.sh present within the bbmap package. These fasta files were aligned to our custom reference genome using bowtie2  count feature was utilized to output a wig files using the following parameters: -z 5 -w 1 -e 250 —bases. The resulting wig files were processed using a custom python script to identify nucleotide mutations as well as coverage of aligned reads.Raw reads following sequencing were designated reads 1 and reads 2 and were merged together using bbmerge.sh present within the bbmap package (


Multidrug resistance (MDR) can be a serious complication for the treatment of cancer as well as for microbial and parasitic infections. Dysregulated overexpression of several members of the ATP-binding cassette transporter families have been intimately linked to MDR phenomena. Three paradigm ABC transporter members, ABCB1 (P-gp), ABCC1 (MRP1) and ABCG2 (BCRP) appear to act as brothers in arms in promoting or causing MDR in a variety of therapeutic cancer settings. However, their molecular mechanisms of action, the basis for their broad and overlapping substrate selectivity, remains ill-posed. The rapidly increasing numbers of high-resolution atomic structures from X-ray crystallography or cryo-EM of mammalian ABC multidrug transporters initiated a new era towards a better understanding of structure–function relationships, and for the dynamics and mechanisms driving their transport cycles. In addition, the atomic structures offered new evolutionary perspectives in cases where transport systems have been structurally conserved from bacteria to humans, including the pleiotropic drug resistance (PDR) family in fungal pathogens for which high resolution structures are as yet unavailable. In this review, we will focus the discussion on comparative mechanisms of mammalian ABCG and fungal PDR transporters, owing to their close evolutionary relationships. In fact, the atomic structures of ABCG2 offer excellent models for a better understanding of fungal PDR transporters. Based on comparative structural models of ABCG transporters and fungal PDRs, we propose closely related or even conserved catalytic cycles, thus offering new therapeutic perspectives for preventing MDR in infectious disease settings. The ATP-binding cassette (ABC) transporter family is one of the largest protein superfamilies present in all living organisms, from prokaryotes to eukaryotes ,2,3,4,5.The NBD is a universally conserved domain that consumes ATP and somehow fuels the dynamic switch of the transporter structure from an inward substrate-binding state to an outward substrate-releasing conformation. The catalytic cycle drives the conformational switch at the TMD and enables the substrate translocation through an as yet elusive transport pathway ,23. The The human ABC transporter family of 48 genes served to categorize subfamilies into the ABCA to ABCG nomenclature ,47,48, aMost, if not all, eukaryotic ABC transporters function as unidirectional exporters and use ATP consumption to drive transport. Some have been implicated in uptake processes as well, although this remains controversial ,99. ImpoMDR1 gene) was identified from a multidrug-resistant KB carcinoma cell line [ABCB1 or P-glycoprotein (P-gp) or MDR1  was discovered as the second member of MDR exporters, cloned from a multidrug-resistant P-gp-negative human lung cancer cell line with doxorubicin tolerance [ABCC1 or MRP1 or multidrug resistance-associated protein1  was originally isolated from P-gp-negative multidrug-resistance breast cancer cell lines [ABCG2 or BCRP or Breast Cancer Resistance Protein  [Candida spp. are a major part of the physiological mycobiome species [Candida spp. can cause life-threatening invasive systemic disease in severely immunocompromised individuals [C. auris is a newly emerging pan-resistant fungal pathogen first reported from an ear infection in Japan in 2009 [C. auris appeared in more than 40 countries around the globe, causing hospital outbreaks of invasive candidemia [C. auris is also causing severe superinfections with viruses, as seen in a recent co-infection of COVID-19 patients in a Mexican hospital ICU, leading to a dramatic overall mortality of 83%. In fact, intrinsic MDR in other Candida spp. such as C. glabrata and C. kruzei have also been increasing over the last decade [Invasive fungal diseases account for ~1.5 million deaths per year worldwide ,221. Thes (spp.) . Candida species , along w species ,228,229.ividuals . The smaividuals . For exa in 2009 . Within ndidemia ,239,240.t decade ,241,242.t decade ,244, efft decade ,246,247.C. albicans and C. glabrata harbor 27 and 18 ABC proteins in total, respectively [Cryptococcus neoformans and Aspergillus fumigatus harbor even larger numbers, with 54 and 49 ABC transporters, respectively [C. albicans harbor only Yor1 and Mlt1  [C. auris (Cdr1), in C. tropicalis (Cdr1), in C. dubliniensis (Cdr1 and Cdr2) and in Cryptococcus neoformans (Afr1)  or pleiotropic drug resistance family is the largest subfamily of ABC transporters of the type II exporter class . PDR trand Aus1) , in C. as (Afr1) . All ares (Afr1) . Their oDiversity of fungal PDR transporters and evolutionary relationships to mammalian ABC transporters. Most ABC proteins from non-pathogenic baker’s yeasts  are alsoSeveral high-resolution structures, obtained through X-ray crystallography or cryo-EM approaches in the past five years, provided a better understanding of the structure–function relationships of mammalian type I and type II exporters . HoweverAt present, the available atomic structures of type I exporters are as listed in The mammalian ABCGs are the closest orthologues of yeast PDR transporters ,251,321,Conservation in the NBD. First, the NBDs in fungal PDRs share highly conserved motifs as well as residues with ABCG required for ATP-binding and hydrolysis ,325,326,Conservation in the TMD region. Second, the general architecture and configuration of TMDs are maintained, as each TMD in ABCG/PDR transporters contains six putative membrane-spanning helices, and a rather short first intracellular loop contains the coupling helix. The large ECL is part of the lid architecture in the extracellular region. Interestingly, two putative helices residing at both lipid bilayer leaflets, which are important for function, as they restrict dynamic movements during transport cycle, are only found in the ABCG subfamily ,42. The The elbow helix is a 15-residue amphipathic helix sharing highly conserved residues among all ABCG/PDR members. This elbow helix is an intrinsic part of the triple helical bundle (THB), which also engages the hot spot helix from the NBD and the coupling helix from the first intracellular loop (ICL1) . ImportaThe transmembrane helix 1 (TMH1) is a prototypic 20-residue membrane-spanning helix, following the elbow helix in all ABCG/PDR exporters. The small extracellular loop 1 (ECL1) is a short linker connecting TMH1 and TMH2 at the cell surface. Notably, ECL1 in the first half holds a conserved arginine (R), which is important for salt bridge formation with a conserved glutamate (E) in the re-entry helix of the same molecule . TMH2 isThe intracellular loop 1 (ICL1) spans over 30 residues in the ABCG/PDR subfamily and holds a critical U-turn motif. ICL1 operates as the coupling helix for the NBD–TMD communication by participating in a triple helical bundle (THB). The entire THB functions as a molecular spring that controls the catalytic cycle by regulating the conformational switch. Indeed, two conserved negative residues within ICL1 of human ABCG2, E451 and E585, are thought to control the intracellular gating mechanism and engage in salt bridges with the elbow helix, respectively. Remarkably, ICL1 holds an essential tyrosine residue that is conserved in all ABCG/PDR transporters. This Y464 contributes a salt bridge interaction within the THB complex for stabilization of the transmission interface.TMH3 also has several conserved residues within the ABCG/PDR group such as negative residues (E or D), proline, as well as positive amino acids (K or R). D477 at the start of TMH3 in human ABCG2 is on top of the transmission interface and may contribute to an intracellular gate. ECL2 is again rather short, containing only eight residues with a kink, connecting TMH3 and TMH4 at the cell surface.The hydrophobic valve is a kinked domain just after TMH5, consisting of the conserved “glycine-Φ-Φ” (Φ is hydrophobic residue) motif that subtends the extracellular bilayer leaflet. The valve is pivotal for controlling substrate transport through a putative translocation pathway whose precise nature and dynamics remains obscure. The re-entry helix is the counterpart of the elbow helix and is unique, establishing a kinked hallmark motif in the ABCG/PDR subfamily with several conserved residues. In addition, ECL3 is the only large extracellular loop, and a main part of the extracellular roof architecture. Interestingly, ECL3 in human ABCG2 has three cysteines, which form intra- and inter-molecular disulfide formations, and perhaps facilitate dimerization and drug release ,335. LikC. albicans, using human ABCG2 (PDB ID 6VXF) as a template . The N-terminal and C-terminal NBDs form a head-to-tail dimer upon ATP-binding ,337, witStabilizing salt bridges maintain proper folding and dynamics. At least two salt bridges appear conserved in Cdr1 , connectThe triple helical bundle (THB) is part of the transmission interface and extremely conserved in Cdr1. Remarkably, the THB is present in diverse ABC transporters, including ABCB1 and LPS extractor, as well as in the antibiotic exporter MacB . This maThe mechanism of intracellular gating in PDR/ABCG2 must be crucial for substrate/inhibitor entry into the transport pathway of the exporter, but as yet is little understood. In human ABCG2, two negative residues are conserved in all ABCG/PDR transporters, represented by E451 (between TMH2 and ICL1) and D477 (beginning of TMH3). Interestingly enough, they are pivotal for drug transport, but do not affect ATP hydrolysis  Table 4C. Since The so-called phenylalanine clamp formed by two F residues is located in the substrate-binding zone of ABCG/PDR transporters. Interestingly, the THM2 in the ABCG/PDR subfamily contains at least 4–5 conserved F residues . TMH2 ocExtracellular gating at the membrane interface and subsequent drug release from the outward-facing state is regulated by two conserved motifs, a hydrophobic valve and a flexible lid architecture in the roof . The hydA flexible but compact lid is part of the roof architecture formed by a rather large extracellular domain in ABCG/PDR. The roof is another unique motif in the ABCG/PDR subfamily. In human ABCG2, this roof is maintained by an intramolecular C592–C608 disulfide bond that strengthens the compact lid architecture. This lid may establish the second gating mechanism to regulate drug release from the outer cavity ,335 FigG. NotablMammalian ABCG and fungal PDR share all hallmark domains as well as numerous conserved residues essential for function . Based oThe increase in atomic structures of ABC transporters, and data from extensive biochemical and genetic experiments, have been propelling the field, since they have yielded novel insights and a better understanding of ABC transporter mechanisms. At the same time, while atomic structures have been invaluable, they have to be interpreted with caution, especially when biochemistry or genetics are not in line with structural data  or when Twist and Squeeze may be used by different types of transporters, and these driving mechanisms may appear related or even similar when looking at it from the mechanics, as either Twist or Squeeze or a combination of both can result in peristaltic pressure during the switch. While there are many challenges remaining ahead of us, the reversal of clinical MDR phenomena in fungal pathogens in infectious disease settings have regained attention, especially since the catalytic cycle of human ABCG2 likely reflects the mode of action of fungal PDR transporters implicated in anti-infective drug resistance.The past few years challenge previous notions that a unified transport mechanism exists for MDR exporters from all three major classes, such as ABCB1/MDR1/P-gp ,307,310,


C. elegans UNC-112 (kindlin) is required for muscle sarcomere assembly, and is one component of a conserved four-protein complex that associates with the cytoplasmic tail of integrin at the base of integrin adhesion complexes in muscle. The four-protein complex consists of UNC-112 (kindlin), PAT-4 , PAT-6 , and UNC-97 (PINCH). UNC-112 is comprised of 720 amino acid residues and contains FERM and PH domains. The N-terminal half of UNC-112 (1-396 aa) can bind to the C-terminal half of UNC-112 (397-720 aa), and this interaction is inhibited by the association of PAT-4 (ILK) to the N-terminal half of UNC-112. In support of this model, previously, we reported identification of a D382V mutation that results in lack of binding to PAT-4. However, this residue is not conserved in human Kindlins. Here, we report identification of a novel UNC-112 mutation of a conserved residue that cannot bind to PAT-4. UNC-112 E302G cannot bind to PAT-4 and does not localize to integrin adhesion complexes in muscle. C. elegans UNC-112 (kindlin) is required for muscle sarcomere assembly , and is one component of a conserved four-protein complex that associates with the cytoplasmic tail of integrin at the base of integrin adhesion complexes in muscle . UNC-112 binds directly with the cytoplasmic tail of PAT-3 (beta-integrin). UNC-112 binds to PAT-4 (ILK), and PAT-4 binds to both PAT-6 , and UNC-97 (PINCH). A complex consisting of UNC-112, PAT-4, PAT-6 and UNC-97 has been demonstrated by co-immunoprecipitation . UNC-112 is comprised of 720 amino acid residues and contains FERM and PH domains. The N-terminal half of UNC-112 (1-396 aa) can bind to the C-terminal half of UNC-112 (397-720 aa), and this interaction is inhibited by the association of PAT-4 (ILK) to the N-terminal half of UNC-112 . In support of this model, we identified a D382V mutation that results in lack of binding to PAT-4 . However, this residue is not conserved in human Kindlins . Here, we report identification of a novel UNC-112 mutation of a conserved residue that cannot bind to PAT-4. We found that E302G UNC-112 cannot bind to PAT-4, but still can bind to PAT-3 (beta-integrin) , we generated a homology model of UNC-112 and highlighted the locations of E302 and D382 . In fact, the lack of a sidechain of G, or the smaller sidechain of V, is likely to make these loops even more flexible. These mutations are predicted to not alter the overall structure of UNC-112 but could possibly affect the surface binding of UNC-112 to PAT-4. It should be noted, that in addition to our findings, a L located 6 residues C-terminal of D382, within the same FERM_M domain, and conserved in UNC-112 and all human kindlins . However, the results by Huet-Calderwood et al. were obtained by GST pulldown from lysates of tissue culture cells that overexpress both ILK and alpha-parvin. Our results, using the yeast two hybrid method and localization in worm muscle cells in which only UNC-112 was overexpressed, provide more evidence for an effect on direct binding between UNC-112 (kindlin) and PAT-4 (ILK). Our finding of a conserved residue in UNC-112 in the FERM_N domain that reduces binding to PAT-4, opens the door to testing the comparable residues in human Kindlin-1 and Kindlin-2 for their importance to binding to ILK. We do not know what the phenotype is of a nematode that is homozygous for the UNC-112 E302G mutation. Since UNC-112 function requires PAT-4 , and all known mutations in pat-4 are Pat embryonic lethal, worms homozygous for UNC-112 E302G, might be Pat embryonic lethal. Several mutations in unc-112 are known to result in either the Pat embryonic phenotype or the adult viable Unc phenotype in which adults move slowly and have a disorganized myofilament lattice . The three known Pat alleles are unc-112(st562) and unc-112(st581) that are both nonsense mutations, and unc-112(gk1) that is a deletion. The Unc allele, unc-112(r367), is a missense mutation, T85I, and is also temperature sensitive. The molecular properties of UNC-112 T85I have not been explored, and thus, we do not know if the phenotype is due to reduced binding to PAT-4 or PAT-3, or whether it results in a unstable protein. Finally, there is one unusual allele created by CRISPR/Cas9, unc-112(kq715), L715E, which shows a defect in the migration of the distal tip cell , but whether there was an effect on the myofilament lattice of body wall muscle, was not reported.ntegrin) . When wes muscle , HA taggs muscle . The E30s muscle . Based oand D382 . Both E3kindlins , when muYeast two-hybrid screeninget al. 2006; Qadota et al. 2012). The UNC-112 N-terminal half was cloned into pGAD-C3 (prey plasmid), and the UNC-112 C-terminal half was cloned into pGBDU-C1 (bait plasmid). PAT-4 full length cDNA was cloned into pGBDU-C2 (bait plasmid). The N-terminal UNC-112 fragment was amplified by the error-prone method using the following primers: 5′ primer, AAA AAA GAG ATC GAA TTC CCC GGG GGA TCC; 3′ primer, GGT TTT TCA GTA TCT ACG ATT CAT AGA TCT. These primers were designed to amplify the insert, and each consists of 30 nucleotides of pGAD-C3. The cloning of error-prone PCR-amplified fragments into the acceptor plasmid was accomplished by exploiting yeast recombination in vivo. The mixture of the amplified PCR fragments (∼1 μg) and the acceptor plasmid (1 μg) digested with BamHI and BglII was transformed into PJ69-4A harboring pGBDU-UNC-112C. Transformed yeast cells were spread onto −Leu−Ura−His and 2 mM 3-amino-1,2,4-triazole to screen for His+ colonies. His+ selection ensured that the mutagenized UNC-112N could still interact with UNC-112C. This step was essential for eliminating clones with premature stop mutations or with many other mutations. His+ colonies were streaked onto an −Ade plate and screened for His+Ade+ colonies. After streaking on a 5-fluoroorotic acid plate to eliminate the URA3 marker bait plasmid (pGBDU-UNC-112C), prey clones were isolated from yeast and amplified in E. coli. From His+Ade+ yeast colonies, 64 mutagenized clones were isolated. These prey clones were transformed separately into PJ69-4A carrying either pGBDU-UNC-112C or pGBDU-PAT-4 (full-length) to check for interaction with the C-terminal half of UNC-112 and interaction with full-length PAT-4. Among 64 mutagenized clones of UNC-112N, 8 of these clones could not bind to PAT-4. From DNA sequencing of these 8 clones, we identified one clone with a single amino acid change, E302G. The UNC-112N with E302G was cloned into pACT-Q-UNC-112C , resulting in pACT-Q-UNC-112 E302G (full length), and then used to test for binding to PAT-3.Random mutagenesis using PCR and screening for interactions using the yeast two hybrid method was performed as previously described , resulting in pKS-HA-UNC-112 E302G. The NheI fragment of pKS-HA-UNC-112 E302G was then cloned into NheI-digested pPD49.78 and pPD49.83, resulting in pPD49.78-HA-UNC-112 E302G and pPD49.83-HA-UNC-112 E302G. pPD49.78/83-HA-UNC-112 E302G were mixed with pTG96 (SUR-5::NLS::GFP) as a transformation marker and injected into wild type N2 worms. Transgenic lines with extrachromosomal arrays containing pPD49.78/83-HA-UNC-112 E302G and pTG96  were established by picking GFP-positive worms using a GFP dissection microscope. Generation and characterization of transgenic animals expressing by heat shock the comparable HA-tagged wild type UNC-112 was described previously . Expression of the HA-tagged UNC-112 proteins (E302G and wild type) was induced by incubation of the transgenic worms at 30 °C for 2 h (heat shock).To express HA-UNC-112 E302G in Fluorescence imaginget al. 1993)and stained by anti-GFP , anti-UNC-95 , and anti-HA . Images were captured at room temperature with a Zeiss confocal system (LSM510) equipped with an Axiovert 100M microscope and an Apochromat ×63/1.4 numerical aperture oil objective, in ×2.5 zoom mode. The color balances of the images were adjusted by using Adobe Photoshop version 22.4.3.Heat-shocked transgenic worms were fixed  from transgenic worms with or without heat shock and examined the expression of HA-tagged UNC-112 proteins by Western blot, reacting with anti-HA .We prepared worm lysates  and Phyre2 version 2.0  online tools were used. Human kindlin-3  was used as reference crystal structure. Molecular graphics were generated by using Chimera version 1.15 .For UNC-112 protein structure modeling, CLUSTALW version 1.2.2 , SWISS-MODEL version July 2021 (


These elements contain non‐base‐paired regions that potentially harbor ligand‐binding pockets. Here, we performed an NMR‐based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1H‐based 1D NMR binding assays. The screening identified common as well as RNA‐element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS‐CoV‐2.SARS‐CoV‐2 contains a positive single‐stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS‐CoV and SARS‐CoV‐2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and  The RNA genome of SARS‐CoV‐2 contains 15 independently folded regulatory RNA elements. By NMR‐based fragment screening, the RNA target space for small molecule binding, functional mapping and inhibition is defined. So far, most efforts focus on targeting proteins to inhibit viral propagation, while few attempts have been reported for direct targeting of the large viral RNA genome.Since early 2020, enormous scientific efforts are geared towards antiviral treatment for SARS‐CoV‐2 (SCoV‐2). Impressive advancements are reported in vaccine development. Furthermore, experimental approaches are being actively pursued in drug repurposing and in design and synthesis of new drugs. Such studies are supported by virtual screening and molecular docking campaigns.pyrimidine (18) and the C7‐methoxy‐substituted gramin (37). A last single functional unit‐containing fragment is quinoline, substituted at C2 with a hydroxamic acid functional group (47). Furthermore, we find two methylene‐amino‐substituted benzenes (36 and 49).Hits containing two functional units can be classified into four additional classes. Class 2: five‐membered heterocyclic rings containing a single nitrogen heteroatom, Class 3: five‐membered rings with several heteroatoms, in particular nitrogen, oxygen and sulfur, Class 4: six‐membered heterocyclic rings containing nitrogen atom(s), and Class 5: substituted and unsubstituted benzenes. In class 1, we find three differently 2‐substituted benzo‐thiazoles with either an amino group  , we determined the transverse relaxation time T2 for the binding ligands at 100 μM concentration at an RNA concentration of 35 μM. Within the subset of the fragment hits, the ligands with the highest degree of T2‐reduction also showed the strongest affinity. For ligands with a KD <100 μM, binding affinity correlated with the T2‐reduction (Qbind). No correlation was, however, observed for KD >100 μM. This finding suggests that T2 is a qualitative indicator for binding strength of fragments to one specific target for binding in fast exchange and with a low micromolar KD.For a subset of hits Table  that bouμM Table . In addiKD above 200 μM, it is our experience[KD of 46 μM  in the Supporting Information, Figure S9A. By contrast, for the high‐affinity binder D01, we provide experimental mapping of its binding site to PK  to identify commercially available potential binders and obtained one compound (D01) that bound to both PK and 3_s2m in single‐digit μM affinity Figure . At the We conducted here an NMR‐based fragment screening against all regulatory RNA elements of the viral genome of SCoV‐2. Our results show that the fragments of the DSI‐PL library can cover almost the entire structural space of the viral RNA genome. Thus, the SCoV‐2‐RNA genome can be targeted by ligands of low molecular weight. We detect that several fragments already show specificity at this very first stage of drug development . Assuming that increasing structural complexity enhances the potential to achieve specificity for small molecules,[The current state‐of‐the‐art discovery of small molecules targeting RNAs (RNA drug discovery) and the current status of this field have been described by Juru and Hargrove,Here, we use a fragment library that has previously been used to screen proteins,1 as tertiary amine is strongly basic, 2‐aminopyridine has a pKa of around 3.5. By contrast, 1,2,3 triazole is already not very basic but shows very specific binding . Heteroaromatic rings with two condensed rings including indoles, purines, quinolones  and even the entire 5′‐ and 3′‐untranslated regions of SCoV‐2 comprising more than 337 nts for each of the constructs. The size range of these targets is markedly different. At this point, exact quantification of the NMR screening experiments relies on approximations including effective overall rotational correlation times of the RNA targets, relative population of free vs. bound ligand, and their on‐ and off‐rates. These assumptions are, however, no longer fulfilled when one compares pools of RNAs with significantly different molecular weight and potential anisotropic tumbling. Within a given target, however, the 234 and eWe analyzed the investigated RNA target space towards sequence‐derived properties and derived unique target sites within the SCoV‐2 genome that are conserved among coronaviruses. Exceptionally rare mutations in SCoV‐2 during 2020 have maintained the target space for ligands up to now. By analysis of the experimental hits, we can derive privileged RNA target space from a medicinal chemistry perspective. Further, our approach relies on the hypothesis that the viral genome can be specifically targeted even in the presence of human cellular RNA. The herein provided analysis of secondary structure abundance  is restricted to the viral RNA genome and will have to be extended to the entire cell‐specific human transcriptome. However, given all these theoretical considerations it is striking to recognize that small molecules have been developed that target splicing[The results as well as the methodological approach presented here will impact medicinal chemistry approaches but also cellular targeting of SCoV‐2 RNA. For example, as immediate follow‐up, we currently conduct secondary NMR screens with commercially available compounds using the identified ligands as guide. We consider our broad, systematic and coherent identification of binders to be a significant step forward. The focus of our study is to experimentally validate the druggability of the SCoV‐2 genome. In fact, this potential had so far only been predicted from theoretical studies.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


So far, there has been little comprehension about the effects of m6A RNA methylation regulators on lower-grade gliomas (LGG). Here, we systematically investigated the expression profiles and prognostic significance of 36 m6A RNA methylation regulators in LGG patients from the TCGA and CGGA databases. Most of the m6A RNA methylation regulators are differentially expressed in LGG tissues as compared with normal brain tissues and glioblastoma (GBM) tissues. The consensus clustering for these m6A RNA methylation regulators identified three clusters. Patients in cluster 3 exhibited worse prognosis. In addition, we constructed an m6A-related prognostic signature, which exhibited excellent performance in prognostic stratification of LGG patients according to the results of the Kaplan-Meier curves, ROC curves, and univariate and multivariate Cox regression analyses. In addition, a significant correlation was observed between the m6A-related prognostic signature and the immune landscape of the LGG microenvironment. The high-risk group exhibited higher immune scores, stromal scores, and ESTIMATE scores but lower tumor purity and lower abundance of activated NK cells. Moreover, the expression level of immune checkpoints was positively correlated with the risk score. To conclude, the current research systematically demonstrated the prognostic roles of m6A RNA methylation regulators in LGG.Increasing evidences have revealed that N Gliomas constitute the frequent intracranial malignant tumors, showing significant heterogeneity with respect to tumor biological behavior for different grade gliomas , 2. Desp6-methyladenosine (m6A) constitutes the most frequent type in eukaryotes [6A RNA methylation is regulated by m6A RNA methylation regulators, including methyltransferases, demethylases, and binding proteins, namely, “writers”, “erasers”, and “readers”, respectively. Increasing evidences have suggested that m6A modification contributes to the malignant biological behavior of multiple cancers [6A RNA methylation regulators has also been confirmed in head and neck squamous cell carcinoma [6A RNA methylation regulators in LGG.RNA modification is an emerging field of epigenetics. Among more than 150 RNA modifications, Nkaryotes , 5 and ikaryotes . As a re cancers , 9. The arcinoma , thyroidarcinoma , breast arcinoma , hepatocarcinoma , gastricarcinoma , etc. Ho6A RNA methylation regulators were systematically evaluated in LGG using the data from The Cancer Genome Atlas (TCGA) dataset and Chinese Glioma Genome Atlas (CGGA) dataset. In addition, we constructed an m6A-related prognostic signature with robust ability in predicting survival outcomes of LGG patients.Here, the expression profile and prognostic significance of 36 mTotal 1013 LGG patients with RNA-seq transcriptome data and corresponding clinicopathological features were identified in public databases, including 423 cases from the TCGA dataset and 590 cases from the CGGA dataset. Patients without survival information or OS <30 days, or without definitive histopathological diagnosis had been excluded from further evaluation. These two datasets were used as the training cohort (TCGA) and validation cohort (CGGA). The RNA-seq transcriptome data of 160 GBM tissues and 1152 normal brain tissues were obtained from the TCGA dataset and the Genotype-Tissue Expression (GTEx) database, respectively. All RNA-seq transcriptome data were harmonized using quantile normalization and SVAseq-based batch effect removal [6A RNA methylation regulators with obtainable expression data were identified in the TCGA, CGGA, and GTEx datasets  was performed to confirm the reliability of clustering results [p < 0.05) and functionally annotated by the Gene Ontology (GO) analysis and the Kyoto Encyclopedia of genes and Genomes (KEGG) pathway analysis.Interactions among mterPlus” , distinc results . The dif6A RNA methylation regulators (p < 0.05), which were subsequently incorporated into the least absolute shrinkage and selection operator (LASSO) Cox regression. A total of nine m6A RNA methylation regulators with LASSO coefficients were identified. The calculation formula of risk score is shown below:xi and Coefi refer to the expression level of selected m6A RNA methylation regulator and corresponding LASSO coefficient, respectively. The median risk score was used for the high-/low-risk grouping of LGG patients. The Kaplan-Meier curve with log-rank test was generated by using the R package “survminer” for the comparison of OS between the high- and low-risk groups. The ROC curve analysis was utilized to evaluate the prediction accuracy of the m6A-related prognostic signature via the R package “timeROC”. All these tests were performed simultaneously in the training and validation cohorts.Univariate Cox regression was performed to identify the overall survival- (OS-) associated mBy employing the R package “rms”, “regplot”, and “Hmisc”, a nomogram was established based on the independent prognostic indicators in the training cohort, which were determined through univariate and multivariate Cox regression analyses. The availability of this nomogram was evaluated by the C-indices  and calip ≥ 0.05 were excluded from the subsequent analysis.The immune scores, stromal scores, ESTIMATE scores, and tumor purity of each LGG patients were calculated using the ESTIMATE algorithm via the R package “estimate” . The abut-test or one-way ANOVA test was utilized to compare the continuous variables with normal distribution between two groups or more than two groups. The Wilcoxon test was performed to determine the differences between subgroups in the expression levels of m6A RNA methylation regulators and the abundance of 22 immune cells. The “Spearman” method was used to calculate the correlations between the expression of m6A RNA methylation regulators and between the expression of immune checkpoints and risk scores. Two-tailed p < 0.05 was considered statistically significant.The preprocessing of RNA-seq transcriptome data was performed using the PERL programming language (version 5.32.0). The R software (version 4.0.2) was applied for all statistical analyses and graph visualization. The student 6A RNA methylation regulators, twenty-two upregulated genes and eleven downregulated genes were identified in LGG tissues compared with normal brain tissues  network and coexpression analyses. Noticeably, “writers” had a wide range of interactions with other m6A RNA methylation regulators, while “erasers” were less connected with others , cluster 2 (n = 200), and cluster 3 (n = 43). PCA for total transcriptomic data revealed prominent differences in distribution among three clusters, which confirmed the validity of consensus clustering  were significantly associated with the OS of LGG patients  stood out as the bases of constructing the m6A-related prognostic signature  curve (AUC). The AUCs of 1-, 3-, and 5-year OS in the TCGA cohort were 0.882, 0.847, and 0.757, respectively , higher WHO grade, IDH wide type, 1p19q noncodel, and MGMT promoter unmethylated showed significantly higher levels of risk score, while no risk score differences were observed between patients satisfied by gender . To detefeatures . The worp < 0.001; Figures To identify the independent prognostic indicators for OS, the OS-related factors identified by univariate Cox regression analyses were subsequently analyzed using multivariate Cox regression. The signature-based risk score was an independent prognostic indicator for OS in both TCGA and CGGA cohorts  and the other two are “writers” (RBM15) and “erasers” (ALKBH3), respectively. Among these critical genes, several have been investigated to be associated with glioma. SETD2, a highly mutated gene, contributes to the tumorigenesis of high-grade glioma [6A RNA methylation regulators contribute to the heterogeneity between LGG and GBM in tumor biological behavior, drug resistance, and prognosis. Therefore, we expect that our findings help to identify the prognostic m6A RNA methylation regulators in LGG and provide insights into their potential roles in LGG tumorigenesis and progression.The me glioma . IGF2BP3e glioma . YTHDF2,e glioma . However6A RNA methylation regulators contributes to the heterogeneity of TME [6A RNA methylation regulators on the immune landscape of the LGG microenvironment remains unclear. Firstly, this study found that LGG patients in the high-risk group had higher immune scores, higher stromal scores, higher ESTIMATE scores, and lower tumor purity than those in the low-risk group. Nowadays, immunotherapy, represented by immune checkpoint blockades (PD-1/L1), has been one of the most promising treatment strategies against various cancers. Interestingly, a positive correlation between the risk score and expression level of immune checkpoints was discovered. Thus, the risk stratification based on the m6A-related prognostic signature might help predict the efficacy of immune checkpoint blockades. As a critical part of the complex TME, immune cells have been identified to be associated with the tumor biological behavior and prognosis [6A RNA methylation regulators partly participate in TME regulation of LGG and might provide new insights into the immunotherapy for LGG.Tumor microenvironment (TME) has been identified as an essential regulatory role in the occurrence and progression of tumors . It has y of TME , 33, 34.rognosis –37. The 6A-related prognostic signature was constructed and validated with retrospective data from public databases. Using prospective data to assess its clinical utility would be more convincing. Secondly, this study only focused on the transcriptome data, other data types like methylation, single nucleotide polymorphism (SNP), copy number variation (CNV), and protein level were not covered. In addition, due to the lack of experimental evidences, this study was not able to assess the role and molecular mechanism of individual regulators in depth. Further in vivo and in vitro experiments are essential to explore the regulatory mechanism of m6A RNA methylation regulators in LGG.Undeniably, some limitations of our study should be pointed out. Firstly, the m6A RNA methylation regulators in LGG. We identified three clusters that stratified the prognosis of LGG patients. An m6A-related risk signature was capable to precisely predict the prognosis of LGG patients and was correlated with the immune landscape of the LGG microenvironment. We hope that our findings provide comprehensive evidence for subsequent research about m6A modification in LGG.All in all, the present study systematically investigated the expression pattern, prognostic value, and effect on the immune landscape of m


Results: Interaction with the major reabsorptive urate transporter URAT1 appears to be dominant over interactions with secretory transporters in determining the net effect of a drug on SU levels. In vitro inhibition interpreted using the recommended cut-offs is useful at predicting the clinical outcome. Conclusions: In vitro safety assessments regarding urate transport should be done early in drug development to identify candidates at risk of causing major imbalances. Attention should be paid both to the inhibition of secretory transporters and inhibition or trans-stimulation of reabsorptive transporters, especially URAT1.Background: Serum urate (SU) levels in primates are extraordinarily high among mammals. Urate is a Janus-faced molecule that acts physiologically as a protective antioxidant but provokes inflammation and gout when it precipitates at high concentrations. Transporters play crucial roles in urate disposition, and drugs that interact with urate transporters either by intention or by accident may modulate SU levels. We examined whether in vitro transporter interaction studies may clarify and predict such effects. Methods: Transporter interaction profiles of clinically proven urate-lowering (uricosuric) and hyperuricemic drugs were compiled from the literature, and the predictive value of in vitro-derived cut-offs like C Urate is the end product of purine catabolism in primates including humans. In other mammals, urate is broken down to allantoin by the enzyme uricase (urate oxidase). This function, however, was lost in the evolutionary lineage of primates due to a mutation . PurinesApproximately two-thirds of urate is excreted renally and one-third with feces . About 1Hyperuricemia is a condition characterized by elevated serum urate (SU) levels, and is defined as SU concentration exceeding 357 μmol/L 6.0 mg/dL) in women and 416 μmol/L (7.0 mg/dL) in men .0 mg/dL . HyperurHyperuricemia may lead to kidney stones, urate nephropathy, or even kidney failure . While hHypouricemia, recently reviewed in , is a coTransporter gene polymorphisms associated with disturbed urate homeostasis have been reported  and are generally thought to be responsible for organic anion uptake; thus, both are important contributors to the tubular secretion of anions . Both huCompared to OAT1/3, OAT2 displays different substrate specificity and a broader tissue expression profile; nevertheless, it also efficiently transports urate . Since iAbcg2/Bcrp1 knockout mice showed increased plasma levels of urate [BCRP is expressed in the apical membrane of all major physiological barriers. It is a low affinity, high-capacity urate transporter  and is tof urate , and mulof urate . A recenof urate .Xenopus oocytes [MRP4 is expressed in the luminal membrane of PTCs  and faci oocytes .−-dependent anion effluxer involved in urate excretion under physiological conditions [NPT1 and NPT4 are expressed in the apical membrane of PTCs . NPT1, onditions . NPT4 isnditions . Variantnditions . Notablynditions  was founnditions .URAT1 is localized in the apical membrane of PTCs . URAT1 i− ions. The OAT4 variant rs17300741 was associated with renal underexcretion type gout [Both OAT4 and OAT10 are localized in the apical membrane of PTCs . OAT4 5 and OAT1ype gout , and carype gout .GLUT9 has two isoforms: GLUT9a and GLUT9b. Both mediate urate transport utilizing glucose and fructose as exchange substrates . GLUT9a The effect of drugs on the transport of urate, whether it is the intended pharmacodynamic action or an undesired liability, can be investigated in in vitro model systems, and results obtained from these models can help to predict clinical response by extrapolation to the in vivo setting.In in vitro transporter assays, transporter-dependent accumulation or vectorial translocation of a probe substrate is quantified using an appropriate method. The mode of detection depends on the probe substrate used and may be spectrophotometry for colored substrates, fluorometry or flow cytometry for fluorescent substrates, liquid scintillation counting for radiolabeled substrates, or quantitative mass spectrometry for unlabeled substrates. In transporter interaction assays, a test drug is added at a single dose or multiple doses, and the effect of the drug on probe substrate transport—which can be inhibitory or stimulatory—is determined. Test drugs can be added in cis, i.e., on the same side, or in trans, i.e., on the opposite side of the membrane relative to the probe substrate. While inhibition studies can be performed in either the cis or the trans setup, they are commonly done in the cis setup, whereas the trans configuration is more accustomed in stimulation studies.Xenopus oocyte system has been used since the early days of transporter studies. Heterologous expression of transporters in the Xenopus oocyte is achieved by microinjection of complementary RNA  . More oA . ImmortA e.g., ,69,70). ,70. Xeno) e.g., ,72,73). ,73. Xenoapp) for each direction is calculated. For apically located efflux transporters like BCRP, the efflux ratio (ER) is defined as the ratio of Papp, B-to-A to Papp, A-to-B, where A-to-B flux represents passive transport minus oppositely directed active transport, while B-to-A flux represents passive plus active transport. Interaction of a drug with the transporter is assessed by its effect on the ER. In substrate efflux assays, transporter activity is quantified based on the exclusion of a probe substrate from cells. For example, the extrusion of pheophorbide A from MDCKII-hBCRP cells was measured by flow cytometry [14C-urate from HEK293-MRP4 cells was quantified by liquid scintillation [ATP hydrolysis-driven export pumps of the ATP-binding cassette (ABC) family and facilitative or secondarily active transporters of the solute carrier (SLC) family are assayed by different methodologies. Current methods in widespread use for interaction studies with ABC transporters include the vesicular transport assay, the vectorial transport or monolayer assay, and the substrate efflux assay, while traditional ATPase assays are becoming obsolete. Vesicular transport assays utilize inside-out membrane vesicles prepared from cells overexpressing the transporter of interest. Such inverted membrane vesicles contain the transporter in a reverse orientation and therefore pump substrates in an inward direction. The assay endpoint is ATP-dependent accumulation of the probe substrate. In vectorial transport or monolayer assays, cells forming a tight monolayer like MDCKII  are groytometry , and effillation . In thesXenopus oocytes are typically exposed to the probe substrate and the test drug in cis, and the effect of the drug on cellular uptake of the substrate is assessed. Alternatively, in the case of bidirectional (exchange) transporters such as URAT1 or OATs, the substrate may be pre-loaded into the cells by microinjection or pre-incubation, and the test drug is added externally  while the pre-loaded substrate is being effluxed. For example, non-steroidal anti-inflammatory drugs (NSAIDs) were found to trans-stimulate the efflux of pre-loaded 14C-labeled urate from human OAT1-expressing oocytes [In drug interaction studies with SLC transporters, transfected/transduced cell lines or cRNA-injected  oocytes .It is important to note that probe substrates other than urate were used extensively in the studies referenced in this review. Although all transporters discussed herein contribute significantly to urate disposition, many of them—especially OATs, BCRP, and MRP4—have other important physiological and pharmacological substrates which are used preferentially as probes in in vitro drug interaction assays see . In factTreatment of hyperuricemia may target the metabolism of urate via inhibition of urate production or facilitation of its breakdown. This approach is briefly discussed in 5.1, but otherwise not covered herein. Instead, the present manuscript focuses on another therapeutic strategy, the inhibition of transporters involved in urate reabsorption . Both apUrate-lowering therapies must be administered with caution as accidentally overtreated hyperuricemia may give way to the opposite condition, hypouricemia . The emeCurrent therapeutic options to reduce serum urate levels include inhibition of urate production by xanthine oxidase and administration of recombinant uricase . The lacThe first uricase to enter clinical practice, uricozyme, has been indicated for tumor lysis syndrome-associated hyperuricemia , but it Allopurinol, the prototype inhibitor of xanthine oxidase, has been used for decades in the treatment of hyperuricemia. Newer members of the xanthine oxidase inhibitor family include febuxostat and the most recent topiroxostat. In a meta-analysis of randomized controlled trials, 120 mg/day febuxostat showed superior efficacy compared to 200 mg/day allopurinol or 120/160 mg/day topiroxostat, while topiroxostat and allopurinol had fewer associated adverse events .Mechanistically, urate production can be reduced at an even earlier step by inhibiting purine nucleosidase phosphorylase (PNP), the enzyme that catalyzes conversion of inosine to hypoxanthine. Hypoxanthine is the precursor of xanthine and a substrate of xanthine oxidase . UlodesiUricosuric agents are defined as drugs that shift the balance of urate disposition towards net secretion, thereby decreasing serum urate levels. Pharmacokinetic properties of uricosurics discussed in this review are listed in gut/(IC50 or Ki) ≥ 11; (ii) renal transporters: Cmax,u/(IC50 or Ki) ≥ 0.02 or ≥ 0.1 (both cut-offs have been used by major regulatory agencies)  . Table 4Benzbromarone has been used to treat gout for decades, but it was withdrawn from some markets in 2003 due to hepatotoxicity . It is amax,u of 46.9 µM it likely fully inhibits reabsorption of urate via URAT1 and OAT4.Interestingly, probenecid inhibits OAT1 and OAT3 more potently than URAT1 or OAT4 . HoweverSulfinpyrazone is one of the oldest drugs used to treat hyperuricemia . It mostbona fide uricosuric agent. However, it inhibits URAT1 about 100- to 780-fold more potently than the secretory urate transporters MRP4, OAT1 and OAT3 (Losartan is an angiotensin II receptor blocker (ARB) and thus not a and OAT3  and dispSalicylic acid has a biphasic effect as it increases SU levels at low doses but decreases SU levels at high doses . A mechamax,u /IC50 ≥ 0.1 cut-off [max,u /IC50 ≥ 0.02) cut-off value for apical compared to basolateral transporters of PTC in their respective guidance document. Lesinurad also inhibits intestinal BCRP to a clinically significant extent . This, again, confirms that inhibition of renal urate reabsorption dominates in the antihyperuricemic effect of uricosuric drugs.Dotinurad was the second drug to be approved for targeting URAT1, and termed a selective urate reabsorption inhibitor for its ability to potently inhibit URAT1 without markedly affecting urate secretion transporters . Indeed,max,u/IC50 value of 0.37 would suggest.Verinurad is a drug in clinical development also specific for URAT1. Verinurad inhibited URAT1 about 200-fold more potently than OAT1 or OAT4 . Inhibit50 or Ki data are available for that interaction. Tranilast is highly protein-bound and at the calculated value of Cmax,u = 0.57 µM the Cmax,u/IC50 ≥ 0.1 cut-off did not predict clinically relevant interactions for basolateral OAT3. The Cmax,u/IC50 ≥ 0.02 cut-off did predict clinically relevant interactions for both OAT3 and apical transporters NPT1, URAT1, OAT4, GLUT9. Based on formally calculated cut-offs, the clinical significance of these interactions was borderline. However, tranilast as a carboxylic acid may be an OAT1 and/or OAT3 substrate and thus may reach higher intracellular concentrations, leading to more significant inhibition of apical transporters, including reabsorptive ones.Tranilast was originally identified as an anti-allergic agent, but its additional benefits were later discovered in a variety of diseases . More remax,u/IC50 value of 1.3. Cmax,u/IC50 values for inhibition of URAT1 and OAT10  can only be considered clinically significant if the Cmax,u/IC50 ≥ 0.02 cut-off is used. No data are available in public databases on the effect of arhalofenate on secretory urate transporters.Arhalofenate inhibits multiple urate reuptake transporters, most pronouncedly OAT4 with a Cmax,u/(IC50 or Ki) > 0.02 criteria is used, both inhibitions are predicted to be clinically significant  displayed clinically significant inhibition of BCRP in the gut  ≥ 0.1 or ≥0.02, are useful for the prediction of a clinically relevant antihyperuricemic effect based on in vitro transporter inhibition data  ≥ 0.02 was a somewhat better indication of uricosuric efficacy than Cmax,u/(IC50 or Ki) ≥ 0.1, as 10 of 10 uricosuric agents showed a clinically significant inhibition of URAT1 when the cut-off ≥ 0.02 was used, compared to 8 of 10 with the cut-off of ≥0.1  ≥ 0.02 cut-off seems to provide greater accuracy.Due to their dominant role in determining SU levels, renal reabsorption transporters were chosen to evaluate whether the cut-offs recommended by regulatory guidelines, i.e., Cion data . Known u of ≥0.1 . For OAT of ≥0.1 . TherefoDevelopment of dual inhibitors  is an alternative to combination treatment with XO inhibitors and URAT1 inhibitors. Several inhibitors targeting both proteins are in development . TranspoMost drugs well known to cause hyperuricemia such as bumetanide , furosemkidney, BCRPgut, NPT4). Data on URAT1 inhibition are only available for furosemide. As the Cmax,u of furosemide is 0.697 µM  ≥ 0.02 provided only a slight edge over the Cmax,u/(IC50 or Ki) ≥ 0.1 cut-off as it predicted clinically significant inhibition of OAT3 by furosemide, inhibition of BCRP by cyclosporine A and inhibition of NPT4 by chlorothiazide. No IC50 or Ki data were publicly available for inhibition of URAT1 by these compounds.In summary, all drugs known to cause hyperuricemia showed a clinically significant inhibition of at least one of the transporters participating in urate efflux . The morAbcg2 knockout mice would suggest [The inhibition profiles of renal transporters as well as of intestinal BCRP have been instrumental in understanding the effect of drugs on serum urate levels. Due to the relatively low protein binding of urate, the majority of urate undergoes glomerular filtration and reabsorption. It appears that URAT1 plays a key role in urate reabsorption and in the maintenance of serum urate levels. A recent study demonstrated hypouricemia in a patient homozygous for the Trp258* (rs121907892) mutation in URAT1 despite being heterozygous for a variant of BCRP (ABCG2 Gln141Lys rs2231142)) as well as homozygous for a mutation in NPT1 (SLC17A1 Thr269Ile (rs1165196)), both known to cause hyperuricemia . In addi1142) as  suggest . Most ur