Tumor-acquired somatic mutation affects conformation to abolish ABCG2-mediated drug resistance.

ABCG2 is an important ATP-binding cassette transporter impacting the absorption and distribution of over 200 chemical toxins and drugs. ABCG2 also reduces the cellular accumulation of diverse chemotherapeutic agents. Acquired somatic mutations in the phylogenetically conserved amino acids of ABCG2 might provide unique insights into its molecular mechanisms of transport. Here, we identify a tumor-derived somatic mutation (Q393K) that occurs in a highly conserved amino acid across mammalian species. This ABCG2 mutant seems incapable of providing ABCG2-mediated drug resistance. This was perplexing because it is localized properly and retained interaction with substrates and nucleotides. Using a conformationally sensitive antibody, we show that this mutant appears "locked" in a non-functional conformation. Structural modeling and molecular dynamics simulations based on ABCG2 cryo-EM structures suggested that the Q393K interacts with the E446 to create a strong salt bridge. The salt bridge is proposed to stabilize the inward-facing conformation, resulting in an impaired transporter that lacks the flexibility to readily change conformation, thereby disrupting the necessary communication between substrate binding and transport.

ABCG2 requires a single aromatic amino acid to "clamp" substrates and inhibitors into the binding pocket.

ATP-binding cassette sub-family G member 2 (ABCG2) is a homodimeric ATP-binding cassette (ABC) transporter that not only has a key role in helping cancer cells to evade the cytotoxic effects of chemotherapy, but also in protecting organisms from multiple xeno- and endobiotics. Structural studies indicate that substrate and inhibitor (ligands) binding to ABCG2 can be differentiated quantitatively by the number of amino acid contacts, with inhibitors displaying more contacts. Although binding is the obligate initial step in the transport cycle, there is no empirical evidence for one amino acid being primarily responsible for ligand binding. By mutagenesis and biochemical studies, we demonstrated that the phylogenetically conserved amino acid residue, F439, was critical for both transport and the binding of multiple substrates and inhibitors. Structural modeling implied that the π-π interactions from each F439 monomer mediated the binding of a surprisingly diverse array of structurally unrelated substrates and inhibitors and that this symmetrical π-π interaction "clamps" the ligand into the binding pocket. Key molecular features of diverse ABCG2 ligands using the π-π clamp along with structural studies created a pharmacophore model. These novel findings have important therapeutic implications because key properties of ligands interacting with ABCG2 have been disovered. Furthermore, mechanistic insights have been revealed by demonstrating that for ABCG2 a single amino acid is essential for engaging and initiating transport of multiple drugs and xenobiotics.

Evolution of efficacious pangenotypic hepatitis C virus therapies.

Hepatitis C compromises the quality of life of more than 350 million individuals worldwide. Over the last decade, therapeutic regimens for treating hepatitis C virus (HCV) infections have undergone rapid advancements. Initially, structure-based drug design was used to develop molecules that inhibit viral enzymes. Subsequently, establishment of cell-based replicon systems enabled investigations into various stages of HCV life cycle including its entry, replication, translation, and assembly, as well as role of host proteins. Collectively, these approaches have facilitated identification of important molecules that are deemed essential for HCV life cycle. The expanded set of putative virus and host-encoded targets has brought us one step closer to developing robust strategies for efficacious, pangenotypic, and well-tolerated medicines against HCV. Herein, we provide an overview of the development of various classes of virus and host-directed therapies that are currently in use along with others that are undergoing clinical evaluation.

Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9 Å resolution.

BACKGROUND: P-glycoprotein (ABCB1) is an ATP-binding cassette transporter that plays an important role in the clearance of drugs and xenobiotics and is associated with multi-drug resistance in cancer. Although several P-glycoprotein structures are available, these are either at low resolution, or represent mutated and/or quiescent states of the protein. RESULTS: In the post-hydrolytic state the structure of the wild-type protein has been resolved at about 8 Å resolution. The cytosolic nucleotide-binding domains (NBDs) are separated but ADP remains bound, especially at the first NBD. Gaps in the transmembrane domains (TMDs) that connect to an inner hydrophilic cavity are filled by density emerging from the annular detergent micelle. The NBD-TMD linker is partly resolved, being located between the NBDs and close to the Signature regions involved in cooperative NBD dimerization. This, and the gap-filling detergent suggest steric impediment to NBD dimerization in the post-hydrolytic state. Two central regions of density lie in two predicted drug-binding sites, implying that the protein may adventitiously bind hydrophobic substances even in the post-hydrolytic state. The previously unresolved N-terminal extension was observed, and the data suggests these 30 residues interact with the headgroup region of the lipid bilayer. CONCLUSION: The structural data imply that (i) a low basal ATPase activity is ensured by steric blockers of NBD dimerization and (ii) allocrite access to the central cavity may be structurally linked to NBD dimerization, giving insights into the mechanism of drug-stimulation of P-glycoprotein activity.

Structural conservation of insulin/IGF signalling axis at the insulin receptors level in Drosophila and humans.

The insulin-related hormones regulate key life processes in Metazoa, from metabolism to growth, lifespan and aging, through an evolutionarily conserved insulin signalling axis (IIS). In humans the IIS axis is controlled by insulin, two insulin-like growth factors, two isoforms of the insulin receptor (hIR-A and -B), and its homologous IGF-1R. In Drosophila, this signalling engages seven insulin-like hormones (DILP1-7) and a single receptor (dmIR). This report describes the cryoEM structure of the dmIR ectodomain:DILP5 complex, revealing high structural homology between dmIR and hIR. The excess of DILP5 yields dmIR complex in an asymmetric 'T' conformation, similar to that observed in some complexes of human IRs. However, dmIR binds three DILP5 molecules in a distinct arrangement, showing also dmIR-specific features. This work adds structural support to evolutionary conservation of the IIS axis at the IR level, and also underpins a better understanding of an important model organism.

Kite-Shaped Molecules Block SARS-CoV-2 Cell Entry at a Post-Attachment Step.

Anti-viral small molecules are currently lacking for treating coronavirus infection. The long development timescales for such drugs are a major problem, but could be shortened by repurposing existing drugs. We therefore screened a small library of FDA-approved compounds for potential severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antivirals using a pseudovirus system that allows a sensitive read-out of infectivity. A group of structurally-related compounds, showing moderate inhibitory activity with IC50 values in the 2-5 µM range, were identified. Further studies demonstrated that these "kite-shaped" molecules were surprisingly specific for SARS-CoV-1 and SARS-CoV-2 and that they acted early in the entry steps of the viral infectious cycle, but did not affect virus attachment to the cells. Moreover, the compounds were able to prevent infection in both kidney- and lung-derived human cell lines. The structural homology of the hits allowed the production of a well-defined pharmacophore that was found to be highly accurate in predicting the anti-viral activity of the compounds in the screen. We discuss the prospects of repurposing these existing drugs for treating current and future coronavirus outbreaks.

Structure of ABCB1/P-Glycoprotein in the Presence of the CFTR Potentiator Ivacaftor.

ABCB1/P-glycoprotein is an ATP binding cassette transporter that is involved in the clearance of xenobiotics, and it affects the disposition of many drugs in the body. Conformational flexibility of the protein within the membrane is an intrinsic part of its mechanism of action, but this has made structural studies challenging. Here, we have studied different conformations of P-glycoprotein simultaneously in the presence of ivacaftor, a known competitive inhibitor. In order to conduct this, we used high contrast cryo-electron microscopy imaging with a Volta phase plate. We associate the presence of ivacaftor with the appearance of an additional density in one of the conformational states detected. The additional density is in the central aqueous cavity and is associated with a wider separation of the two halves of the transporter in the inward-facing state. Conformational changes to the nucleotide-binding domains are also observed and may help to explain the stimulation of ATPase activity that occurs when transported substrate is bound in many ATP binding cassette transporters.

Grid-independent Descriptors (GRIND) Analysis and SAR Guided Molecular Docking Studies to Probe Selectivity Profiles of Inhibitors of Multidrug Resistance Transporters ABCB1 and ABCG2.

BACKGROUND: ATP-binding cassette (ABC) transporters, P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP/ABCG2) are major determinants of pharmacokinetic, safety and efficacy profiles of drugs thereby effluxing a broad range of endogenous substances across the plasma membrane. Overexpression of these transporters in various tumors is also implicated in the development of multidrug resistance (MDR) and thus, hampers the success of cancer chemotherapy. Modulators of these efflux transporters in combination with chemotherapeutics could be a promising concept to increase the effective intracellular concentration of anticancer drugs. However, broad and overlapped specificity for substrates and modulators of ABCB1 and ABCG2, merely induce toxicity and unwanted drug-drug interactions and thus, lead to late-stage failure of drugs. OBJECTIVE: In present investigation, we aim to identify specific 3D structural requirements for selective inhibition of ABCB1 and ABCG2 transport function. METHOD: GRID Independent Molecular Descriptor (GRIND) models of selective inhibitors of both transporters have been developed, using their most probable binding conformations obtained from molecular docking protocol. RESULTS: Our results demonstrated a dominant role of molecular shape and different H-bonding patterns in drug-ABCB1/ABCG2 selective interactions. Moreover, distinct distances of different pharmacophoric features from steric hot spots of the molecules provided a strong basis of selectivity for both transporters. Additionally, our results suggested the presence of two H-bond donors at a distance of 8.4-8.8 Å in selective modulators of ABCG2. CONCLUSION: Our findings concluded that molecular shape along with three dimensional pattern of Hbonding in MDR modulators play a critical role in determining the selectivity between the two targets.

Mutations in the STAT1­interacting domain of the hepatitis C virus core protein modulate the response to antiviral therapy.

RNA viruses, such as hepatitis C virus (HCV), have markedly error-prone replication, resulting in high rates of mutagenesis. In addition, the standard treatment includes ribavirin, a base analog that is likely to cause mutations in different regions of the HCV genome, resulting in deleterious effects on HCV itself. The N-terminal region of the core protein is reported to block interferon (IFN) signaling by interaction with the STAT1­SH2 domain, resulting in HCV resistance to IFN therapy. In this study, mutations in the HCV core protein from IFN/ribavirin­treated patients were analyzed, with particular focus on the N­terminal domain of the HCV core which is reported to interact with STAT1. HCV PCR positive patients enrolled in this study were either undergoing pegylated IFN/ribavirin bitherapy and had completed 12 weeks of initial treatment or were treatment­naïve patients. The HCV core protein was cloned and sequenced from these patients and mutations observed in the STAT1­interacting domain of the core protein from treated patients were characterized using in silico interaction to depict the role of these mutations in disease outcomes. Our results suggest that the amino acids at positions 2, 3, 8, 16 and 23 of the HCV core protein are critical for core-STAT1 interaction and ribavirin-induced mutations at these positions interfere with the interaction, resulting in a better response of the treated patients. In conclusion, this study anticipates that HCV core residues 2, 3, 8, 16 and 23 directly interact with STAT1. We propose that IFN/ribavirin bitherapy­induced mutations in the STAT1­interacting domain of the HCV core protein may be responsible for the improved therapeutic response and viral clearance, thus amino acids 1-23 of the N-terminus of the core protein are an ideal antiviral target. However, this treatment may give rise to resistant variants that are able to escape the current therapy. We propose similar studies in responsive and non-responsive genotypes in order to gain a broader picture of this proposed mechanism of viral clearance.

Sequence and structural analysis of 3' untranslated region of hepatitis C virus, genotype 3a, from pakistani isolates.

BACKGROUND: Hepatitis C virus (HCV) is the cause of high morbidity and mortality worldwide, inflicting around one million people in Pakistan alone. The HCV genomic RNA harbors conserved structural elements that are indispensable for its replication. The 3' untranslated region (UTR) contains several of these elements essentially involved in regulating the major steps of the viral life cycle. OBJECTIVES: Differences in regulatory elements of HCV may contribute towards differential infectivity of local isolates. The present study explicates sequence analysis and secondary structure prediction of HCV 3'UTR region of subtype 3a from Pakistan to characterize this particular region. PATIENTS AND METHODS: HCV 3'UTR region was amplified, cloned and sequenced from five different patients. Sequence and structural analysis was performed and phylogenetic analysis was carried out using the 3'UTR sequence reported in NCBI nucleotide data base (http://www.ncbi.nlm.nih.gov/nuccore) by other studies. RESULTS: Sequence analysis of the amplified fragment from five patients indicated that the 3'UTR is composed of 214-235 nts. Its sequence contains a type-specific variable region followed by a poly U/UC region and a highly conserved X-tail of 98 nts. The variable region reported here has 26 nts and one stem loop at the secondary structure that differentiate it from HCV genotype 1a ( GT1a) 3'UTR which contains additional 14 nts and two stem loops. The poly U/UC region varied in length (100-79 nts) and nucleotide sequence within the Pakistani isolates, and among different genotypes. Some substitutions found in the X-tail do not affect secondary structure of this element suggesting that this region might play an important role in replication, stabilization and packaging of HCV genome. Additionally, U residues are not present at the end of the X-tail in Pakistani 3a isolates as otherwise reported for the variants of genotype 1b. CONCLUSIONS: Sequence and structural diversity of the 3'UTR variable region and Poly U/UC region found in the local isolates indicate specificity in the regulating elements of 3'UTR that might be associated with differential replication efficacy of the HCV Pakistani isolates. The study necessitates functional characterization of these regulating elements to elucidate variable viral efficiency and pathogenicity associated with inter-geographical isolates.

Analysis of interleukin-10 gene polymorphisms and hepatitis C susceptibility in Pakistan.

INTRODUCTION: Hepatitis C virus (HCV) commonly causes a chronic infection but few of patients are able to clear the virus naturally. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that can suppress the immune response against HCV. Interindividual variations in IL-10 production are genetically contributed by polymorphisms within the IL-10 promoter region. This study aimed to investigate the association of the IL-10 gene promoter -1082 G/A, -819 C/T, and -592 C/A polymorphisms with HCV infection susceptibility in Pakistani individuals. METHODOLOGY: Eighty-nine chronically infected patients and 99 controls were enrolled in the study. IL-10 (-1,082 G/A, -819 C/T, -592 C/A) genotyping was performed by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). RESULTS: A suggestive evidence of association with hepatitis C was obtained for the IL-10 -819 C/T (-592 C/A) (p: 0.03) promoter polymorphism at the allele level  but not in genotype distribution. The IL-10 -1082 allele showed no association while positive association of GG (p: 0.001) gene and negative association for GA (0.001) gene were observed.  Higher frequencies were observed for GTA (p: 0.02), ACC (p: 0.01) haplotype and GCC/GTA (p: 0.005) diplotype in HCV patients than controls while diplotype GCC/ATA showed protective effect against HCV. CONCLUSIONS: Our findings suggest that different IL-10 gene polymorphisms may lead to an imbalance between the pro-inflammatory and anti-inflammatory cytokine responses which may in turn influence the susceptibility to HCV infection.

Hepatitis C virus in Pakistan: a systematic review of prevalence, genotypes and risk factors.

In Pakistan more than 10 million people are living with Hepatitis C virus (HCV), with high morbidity and mortality. This article reviews the prevalence, genotypes and factors associated with HCV infection in the Pakistani population. A literature search was performed by using the keywords; HCV prevalence, genotypes and risk factors in a Pakistani population, in Pubmed, PakMediNet and Google scholar. Ninety-one different studies dating from 1994 to May 2009 were included in this study, and weighted mean and standard error of each population group was calculated. Percentage prevalence of HCV was 4.95% +/- 0.53% in the general adult population, 1.72% +/- 0.24% in the pediatric population and 3.64% +/- 0.31% in a young population applying for recruitment, whereas a very high 57% +/- 17.7% prevalence was observed in injecting drug users and 48.67% +/- 1.75% in a multi-transfused population. Most prevalent genotype of HCV was 3a. HCV prevalence was moderate in the general population but very high in injecting drug users and multi-transfused populations. This data suggests that the major contributing factors towards increased HCV prevalence include unchecked blood transfusions and reuse of injection syringes. Awareness programs are required to decrease the future burden of HCV in the Pakistani population.