The central role of gut microbiota in drug metabolism and personalized medicine

The gut microbiota is now considered as a symbiotic organ playing an important role in human health and disease development and has been recently recognized as a modulator of drug metabolism and toxicity. Here, we briefly discuss new findings describing how the gut microbiota is now considered to be a central player in drug metabolism and personalized medicine

Not only P-glycoprotein: amplification of the ABCB1-containing chromosome region 7q21 confers multidrug resistance upon cancer cells by coordinated overexpression of an assortment of resistance-related proteins

The development of drug resistance continues to be a dominant hindrance toward curative cancer treatment. Overexpression of a wide-spectrum of ATP-dependent efflux pumps, and in particular of ABCB1 (P-glycoprotein or MDR1) is a well-known resistance mechanism for a plethora of cancer chemotherapeutics including for example taxenes, anthracyclines, Vinca alkaloids, and epipodopyllotoxins, demonstrated by a large array of published papers, both in tumor cell lines and in a variety of tumors, including various solid tumors and hematological malignancies. Upon repeated or even single dose treatment of cultured tumor cells or tumors in vivo with anti-tumor agents such as paclitaxel and doxorubicin, increased ABCB1 copy number has been demonstrated, resulting from chromosomal amplification events at 7q11.2-21 locus, leading to marked P-glycoprotein overexpression, and multidrug resistance (MDR). Clearly however, additional mechanisms such as single nucleotide polymorphisms (SNPs) and epigenetic modifications have shown a role in the overexpression of ABCB1 and of other MDR efflux pumps. However, notwithstanding the design of 4 generations of ABCB1 inhibitors and the wealth of information on the biochemistry and substrate specificity of ABC transporters, translation of this vast knowledge from the bench to the bedside has proven to be unexpectedly difficult. Many studies show that upon repeated treatment schedules of cell cultures or tumors with taxenes and anthracyclines as well as other chemotherapeutic drugs, amplification, and/or overexpression of a series of genes genomically surrounding the ABCB1 locus, is observed. Consequently, altered levels of other proteins may contribute to the establishment of the MDR phenotype, and lead to poor clinical outcome. Thus, the genes contained in this ABCB1 amplicon including ABCB4, SRI, DBF4, TMEM243, and RUNDC3B are overexpressed in many cancers, and especially in MDR tumors, while TP53TG1 and DMTF1 are bona fide tumor suppressors. This review describes the role of these genes in cancer and especially in the acquisition of MDR, elucidates possible connections in transcriptional regulation (co-amplification/repression) of genes belonging to the same ABCB1 amplicon region, and delineates their novel emerging contributions to tumor biology and possible strategies to overcome cancer MDR

NMR structure of a non-conjugatable, ADP-ribosylation associated, ubiquitin-like domain from Tetrahymena thermophila polyubiquitin locus

Background: Ubiquitin-like domains (UbLs), in addition to being post-translationally conjugated to the target through the E1-E2-E3 enzymatic cascade, can be translated as a part of the protein they ought to regulate. As integral UbLs coexist with the rest of the protein, their structural properties can differ from canonical ubiquitin, depending on the protein context and how they interact with it. In this work, we investigate T.th-ubl5, a UbL present in a polyubiquitin locus of Tetrahymena thermophila, which is integral to an ADP-ribosyl transferase protein. Only one other co-occurrence of these two domains within the same protein has been reported. Methods: NMR, multiple sequence alignment, MD simulations and SPR have been used to characterize the structure of T.th-ubl5, identify putative binders and experimentally test the interaction, respectively. Results: Molecular dynamics simulations showed that T.th-ubl5 is unable to bind the proteasome like ubiquitin due to the lack of the conserved hydrophobic patch. Of other integral UbLs identified by structural and sequence alignment, T.th-ubl5 showed high structural and sequence resemblance with the Ras-binding epitope of FERM UbLs. SPR experiments confirmed that a strong and specific interaction occurs between T.th-ubl5 and T.th-Ras. Conclusion: Data indicate that T. th-ubl5 does not interact with the proteasome like ubiquitin but acts as a decoy for the recruitment of Ras protein by the ADP-ribosyl transferase domain. General significance: Mono-ADP-ribosylation of Ras proteins is known as a prerogative of bacterial toxins. T.th-ubl5 mediated recruitment of Ras highlights the possibility of an unprecedented post-translational modification with interesting implication for signalling pathways.Peer reviewe

Roles of Sorcin in Drug Resistance in Cancer: One Protein, Many Mechanisms, for a Novel Potential Anticancer Drug Target

The development of drug resistance is one of the main causes of failure in anti-cancer treatments. Tumor cells adopt many strategies to counteract the action of chemotherapeutic agents, e.g., enhanced DNA damage repair, inactivation of apoptotic pathways, alteration of drug targets, drug inactivation, and overexpression of ABC (Adenosine triphosphate-binding cassette, or ATP-binding cassette) transporters. These are broad substrate-specificity ATP-dependent efflux pumps able to export toxins or drugs out of cells; for instance, ABCB1 (MDR1, or P-glycoprotein 1), overexpressed in most cancer cells, confers them multidrug resistance (MDR). The gene coding for sorcin (SOluble Resistance-related Calcium-binding proteIN) is highly conserved among mammals and is located in the same chromosomal locus and amplicon as the ABC transporters ABCB1 and ABCB4, both in human and rodent genomes (two variants of ABCB1, i.e., ABCB1a and ABCB1b, are in rodent amplicon). Sorcin was initially characterized as a soluble protein overexpressed in multidrug (MD) resistant cells and named “resistance-related” because of its co-amplification with ABCB1. Although for years sorcin overexpression was thought to be only a by-product of the co-amplification with ABC transporter genes, many papers have recently demonstrated that sorcin plays an important part in MDR, indicating a possible role of sorcin as an oncoprotein. The present review illustrates sorcin roles in the generation of MDR via many mechanisms and points to sorcin as a novel potential target of different anticancer molecules

Information Transfer in the Penta-EF-hand Protein Sorcin Does Not Operate via the Canonical Structural/Functional Pairing A STUDY WITH SITE-SPECIFIC MUTANTS

Sorcin is a typical penta-EF-hand protein that participates in Ca2+-regulated processes by translocating reversibly from cytosol to membranes, where it interacts with different target proteins in different tissues. Binding of two Ca2+/monomer triggers translocation, although EF1, EF2, and EF3 are potentially able to bind calcium at micromolar concentrations. To identify the functional pair, the conserved bidentate -Z glutamate in these EF-hands was mutated to yield E53Q-, E94A-, and E124A-sorcin, respectively. Limited structural perturbations occur only in E124A-sorcin due to involvement of Glu-124 in a network of interactions that comprise the long D helix connecting EF3 to EF2. The overall affinity for Ca2+ and for two sorcin targets, annexin VII and the ryanodine receptor, follows the order wild-type > E53Q- > E94A- > E124A-sorcin, indicating that disruption of EF3 has the largest functional impact and that disruption of EF2 and EF1 has progressively smaller effects. Based on this experimental evidence, EF3 and EF2, which are not paired in the canonical manner, are the functional EF-hands. Sorcin is proposed to be activated upon Ca2+ binding to EF3 and transmission of the conformational change at Glu-124 via the D helix to EF2 and from there to EF1 via the canonical structural/functional pairing. This mechanism may be applicable to all penta-EF-hand proteins

Cooperative Oxygen Binding to Scapharca inaequivalvis Hemoglobin in the Crystal

Oxygen binding to homodimeric Scapharca inaequivalvis hemoglobin (HbI) crystals has been investigated by single-crystal polarized absorption microspectrophotometry. The saturation curve, characterized by a Hill coefficient n(H) = 1.45 and an oxygen pressure at half saturation p(50) = 4.8 torr, at 15 degrees C, shows that HbI in the crystalline state retains positive cooperativity in ligand binding. This finding will permit the correlation of the oxygen-linked conformational changes in the crystal with the expression of cooperativity. Polarized absorption spectra of deoxy-HbI, oxy-HbI, and oxidized HbI crystals indicate that oxygenation does not induce heme reorientation, whereas oxidation does. Lattice interactions prevent the dissociation of oxidized dimers that occurs in solution and stabilize an equilibrium distribution of pentacoordinate and hexacoordinate high spin species

A failure mode and effect analysis (FMEA)-based approach for risk assessment of scientific processes in non-regulated research laboratories

AbstractNowadays, Quality Management tools such as failure mode and effect analysis (FMEA) are widely used throughout the aeronautical, automotive, software, food services, health care and many other industries to sustain and improve quality and safety. The increasing complexity of scientific research makes it more difficult to maintain all activities under control, in order to guarantee validity and reproducibility of results. Even in non-regulated research, scientists need to be supported with management tools that maximize study performance and outcomes, while facilitating the research process. Frequently, steps that involve human intervention are the weak links in the process. Risk analysis therefore gives considerable benefit to analytical validation, assessing and avoiding failures due to human error, potential imprecision in applying protocols, uncertainty in equipment function and imperfect control of materials. This paper describes in detail how FMEA methodology can be applied as a performance improvement tool in the field of non-regulated research, specifically on a basic Life Sciences research process. We chose as "pilot process" the selection of oligonucleotide aptamers for therapeutic purposes, as an example of a complex and multi-step process, suitable for technology transfer. We applied FMEA methodology, seeking every opportunity for error and its impact on process output, and then, a set of improvement actions was generated covering most aspects of laboratory practice, such as equipment management and staff training. We also propose a useful tool supporting the risk assessment of research processes and its outputs and that we named "FMEA strip worksheet." These tools can help scientists working in non-regulated research to approach Quality Management and to perform risk evaluation of key scientific procedures and processes with the final aim to increase and better control efficiency and efficacy of their research

ERp57 chaperon protein protects neuronal cells from Aβ-induced toxicity

Alzheimer's disease (AD) is a neurodegenerative disorder whose main pathological hallmark is the accumulation of Amyloid-β peptide (Aβ) in the form of senile plaques. Aβ can cause neurodegeneration and disrupt cognitive functions by several mechanisms, including oxidative stress. ERp57 is a protein disulfide isomerase involved in the cellular stress response and known to be present in the cerebrospinal fluid of normal individuals as a complex with Aβ peptides, suggesting that it may be a carrier protein which prevents aggregation of Aβ. Although several studies show ERp57 involvement in neurodegenerative diseases, no clear mechanism of action has been identified thus far. In this work, we gain insights into the interaction of Aβ with ERp57, with a special focus on the contribution of ERp57 to the defense system of the cell. Here, we show that recombinant ERp57 directly interacts with the Aβ25-35 fragment in vitro with high affinity via two in silico-predicted main sites of interaction. Furthermore, we used human neuroblastoma cells to show that short-term Aβ25-35 treatment induces ERp57 decrease in intracellular protein levels, different intracellular localization, and ERp57 secretion in the cultured medium. Finally, we demonstrate that recombinant ERp57 counteracts the toxic effects of Aβ25-35 and restores cellular viability, by preventing Aβ25-35 aggregation. Overall, the present study shows that extracellular ERp57 can exert a protective effect from Aβ toxicity and highlights it as a possible therapeutic tool in the treatment of AD

Widespread in vivo efficacy of The-0504: a conditionally-activatable nanoferritin for tumor-agnostic targeting of CD71-expressing cancers

Background: Cancer is still among the leading causes of death all over the world. Improving chemotherapy and minimizing associated toxicities are major unmet medical needs. Recently, we provided a preliminary preclinical evaluation of a human ferritin (HFt)-based drug carrier (The -0504) that selectively delivers the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors. The-0504 has so far been evaluated on four different human tumor xenotransplant models (breast, colorectal, pancreatic and liver cancers).Methods: Herein, we extend our studies, by: (a) testing DNA damage in vitro, (b) treating eight additional tumor xenograft models in vivo with The-0504; (c) performing pharmacokinetic (PK) studies in rats; and (d) evaluating The-0504 anti-tumor xenotransplant efficacy by optimizing its administration schedule based on PK considerations.Results: Immunofluorescence demonstrated that The-0504 induces foci expressing the DNA double-strand break marker gamma H2AX. Expression increases up to 4-fold and is more persistent ascompared to free Genz-644282. In vivo studies confirmed a remarkable anti-tumor activity of The 0504, resulting in tumor eradication in most murine xenograft models, regardless of embryological origin (e.g. epithelial, mesenchymal or neuroendocrine), and molecular subtypes. PK studies demonstrated a long persistence of The-0504 in rat serum (half-life of about 40 h as compared to 15 h of the free drug), with a 400-fold increase in peak concentrations as compared to the free drug. On this basis, we reduced The-0504 administration frequency from twice to once per week, with no appreciable loss in therapeutic efficacy in mice. Conclusion: The results presented here confirm that The-0504 is highly active against several human tumor xenotransplants, even when administered less frequently than previously reported. The-0504 may be a good candidate for further clinical development in a tumor histotype-agnostic setting