Identification, analysis and inference of point mutations associated to drug resistance in bacteria: a lesson learnt from the resistance of Streptococcus pneumoniae to quinolones

Antibiotic resistance is one of the biggest public health challenges of our time. Bacterial chemoresistance is the phenomenon whereby bacteria develop the ability to survive and multiply in the presence of an antibacterial drug; the expression of a resistant phenotype may be due to three fundamental mechanisms, including the expression of enzymes that inactivate the antibacterial drug, changes in the membrane permeability to antibiotics and the onset of point mutations causing the physical-chemical alteration of the antimicrobial targets. In recent decades, new antibiotic resistance mechanisms have emerged and are spreading globally, threatening human health and the ability to fight the most common infectious diseases. Quinolones, a novel class of antibiotics that bind bacterial topoisomerases and inhibit cell replication, have been important in limiting the spread of penicillin- and macrolides-resistant Streptococcus pneumoniae. However, alarmingly, resistance to quinolones is spreading recently. Resistance is caused by the appearance of point mutations in the bacterial topoisomerase and gyrase. Some mutations are well known, but some are not and the information about known molecular mechanisms causing resistance is sparse and not systematically collected and organised. This means that it cannot be used to infer new mutations in newly sequenced bacterial genes and study how they may affect the drug binding. The lack of structured, organized, and reusable information about point mutations associated with antibiotic resistance represents a critical issue and is a common pattern in the field. Here, we present a structural analysis of point mutations involved in the resistance to quinolones affecting the gyrase and topoisomerase genes in Streptococcus pneumoniae. Results, extended to other bacterial species, have been collected in a database, Quinores3D db, and can now be used – through a web server, Quinores3D finder - to analyze both known and yet unknown mutations occurring in bacterial topoisomerases and gyrases. The development, testing and deployment of Quinores3D db and Quinores3D finder are further results of this PhD thesis. Furthermore, structural data about point mutations associated with antibiotic resistance were used to train, test and validate a machine learning algorithm for the inference of still unknown mutations potentially involved in bacterial resistance to quinolone. As the performance of the algorithm, measured in terms of accuracy, sensitivity and specificity, is very promising, we plan to incorporate it in the web server to allow users to predict new mutations associated with bacterial resistance to quinolones

Analysis of point mutations leading to antibiotic resistance in Streptococcus pneumoniae

Motivation New antimicrobial resistance mechanisms are emerging and spreading globally, threatening the ability to treat common infectious diseases. The long term goal of this project is to develop an automated procedure to identify and structurally characterise point mutations in bacterial genes that can lead to the rising of antibi- otic resistance. Streptococcus pneumoniae, a gram positive bacterium responsible for several community-acquired diseases (infections of the upper respiratory tract, pneumonia, meningitis), was chosen as case study to develop the long term strat- egy. S. pneumoniae has developed resistance to different classes of drugs including penicillin, macrolides and quinolones. Fluoroquinolones are a novel class of com- pound inhibiting the pathogen’s growth by targeting bacterial topoisomerase IV. Methods We collected the complete genome of 46 different strains of S. pneumoniae from a publicly available database (NCBI microbial genome) and developed a pipeline to retrieve the sequences of the subunit A (parC) of topoisomerase IV. These se- quences were translated into the respective amino acidic sequences and a multiple sequence alignment was generated. In order to identify single point mutations the parC gene from the R6 non-resistant strain was used as reference. In order to eval- uate how the identified amino acidic substitutions may affect the binding of the antibiotic to the topoisomerase IV, for each strain, we mapped them on the ParC protein structure and carried out a structural analysis. Results Here, we present the results of the S. pneumoniae case study. Using the parC gene from the R6 non-resistant strain as reference, we identified several single point mu- tations in the parC genes of other strains, some of which were already known from the literature to be related to antibiotic resistance

Bioinformatics analysis of Ras homologue enriched in the striatum, a potential target for Huntington's disease therapy

Huntington's disease (HD) is a lethal neurodegenerative disorder for which no cure is available yet. It is caused by abnormal expansion of a CAG triplet in the gene encoding the huntingtin protein (Htt), with consequent expansion of a polyglutamine repeat in mutated Htt (mHtt). This makes mHtt highly unstable and aggregation prone. Soluble mHtt is linked to cytotoxicity and neurotoxicity, whereas mHtt aggregates are thought to be neuroprotective. While Htt and mHtt are ubiquitously expressed throughout the brain and peripheral tissues, HD is characterized by selective degradation of the corpus striatum, without notable alterations in peripheral tissues. Screening for mRNAs preferentially expressed in rodent striatum led to the discovery of a GTP binding protein homologous to Ras family members. Due to these features, the newly discovered protein was termed Ras Homolog Enriched in Striatum (RHES). The aetiological role of RHES in HD has been ascribed to its small ubiquitin-like modifier (SUMO)-E3 ligase function. RHES sumoylates mHtt with higher efficiency than wild-type Htt, thereby protecting mHtt from degradation and increasing the amounts of the soluble form. Although RHES is an attractive target for HD treatment, essential information about protein structure and function are still missing. With the aim of investigating RHES 3D structure and function, bioinformatic analyses and molecular modelling have been performed in the present study, based on which, RHES regions predicted to be involved in the interaction with mHtt or the SUMO-E2 ligase Ubc9 have been identified. These regions have been used to design peptides aimed at inhibiting RHES interactions and, therefore, mHtt sumoylation; in turn, these peptides will be used to develop small molecule inhibitors by both rational design and virtual screening of large compound libraries. Once identified, RHES sumoylation inhibitors may open the road to the development of therapeutic agents against the severe, and currently untreatable, HD

Importin-β/karyopherin-β1 modulates mitotic microtubule function and taxane sensitivity in cancer cells via its nucleoporin-binding region

The nuclear transport receptor importin-β/karyopherin-β1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-β regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-β's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-β form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-β affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-β interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-β, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-β's nucleoporin-binding residues. Concomitantly, importin-β overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-β function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs

A novel resveratrol derivative induces mitotic arrest, centrosome fragmentation and cancer cell death by inhibiting γ-tubulin

Abstract Background Resveratrol and its natural stilbene-containing derivatives have been extensively investigated as potential chemotherapeutic agents. The synthetic manipulation of the stilbene scaffold has led to the generation of new analogues with improved anticancer activity and better bioavailability. In the present study we investigated the anticancer activity of a novel trimethoxystilbene derivative (3,4,4′-trimethoxylstilbene), where two methoxyl groups are adjacent on the benzene ring (ortho configuration), and compared its activity to 3,5,4′-trimethoxylstilbene, whose methoxyl groups are in meta configuration. Results We provide evidence that the presence of the two methoxyl groups in ortho configuration renders 3,4,4′-trimethoxystilbene more efficient than the meta isomer in inhibiting cell proliferation and producing apoptotic death in colorectal cancer cells. Confocal microscopy of α- and γ-tubulin staining shows that the novel compound strongly depolymerizes the mitotic spindle and produces fragmentation of the pericentrosomal material. Computer assisted docking studies indicate that both molecules potentially interact with γ-tubulin, and that 3,4,4′-trimethoxystilbene is likely to establish stronger interactions with the protein. Conclusions These findings demonstrate the ortho configuration confers higher specificity for γ-tubulin with respect to α-tubulin on 3,4,4′ trimethoxystilbene, allowing it to be defined as a new γ-tubulin inhibitor. A strong interaction with γ-tubulin might be a defining feature of molecules with high anticancer activity, as shown for the 3,4,4′ isomer

Known Drugs Identified by Structure-Based Virtual Screening Are Able to Bind Sigma-1 Receptor and Increase Growth of Huntington Disease Patient-Derived Cells

Huntington disease (HD) is a devastating and presently untreatable neurodegenerative disease characterized by progressively disabling motor and mental manifestations. The sigma-1 receptor (σ1R) is a protein expressed in the central nervous system, whose 3D structure has been recently determined by X-ray crystallography and whose agonists have been shown to have neuroprotective activity in neurodegenerative diseases. To identify therapeutic agents against HD, we have implemented a drug repositioning strategy consisting of: (i) Prediction of the ability of the FDA-approved drugs publicly available through the ZINC database to interact with σ1R by virtual screening, followed by computational docking and visual examination of the 20 highest scoring drugs; and (ii) Assessment of the ability of the six drugs selected by computational analyses to directly bind purified σ1R in vitro by Surface Plasmon Resonance and improve the growth of fibroblasts obtained from HD patients, which is significantly impaired with respect to control cells. All six of the selected drugs proved able to directly bind purified σ1R in vitro and improve the growth of HD cells from both or one HD patient. These results support the validity of the drug repositioning procedure implemented herein for the identification of new therapeutic tools against HD

Known Drugs Identified by Structure-Based Virtual Screening Are Able to Bind Sigma-1 Receptor and Increase Growth of Huntington Disease Patient-Derived Cells

Huntington disease (HD) is a devastating and presently untreatable neurodegenerative disease characterized by progressively disabling motor and mental manifestations. The sigma-1 receptor (σ1R) is a protein expressed in the central nervous system, whose 3D structure has been recently determined by X-ray crystallography and whose agonists have been shown to have neuroprotective activity in neurodegenerative diseases. To identify therapeutic agents against HD, we have implemented a drug repositioning strategy consisting of: (i) Prediction of the ability of the FDA-approved drugs publicly available through the ZINC database to interact with σ1R by virtual screening, followed by computational docking and visual examination of the 20 highest scoring drugs; and (ii) Assessment of the ability of the six drugs selected by computational analyses to directly bind purified σ1R in vitro by Surface Plasmon Resonance and improve the growth of fibroblasts obtained from HD patients, which is significantly impaired with respect to control cells. All six of the selected drugs proved able to directly bind purified σ1R in vitro and improve the growth of HD cells from both or one HD patient. These results support the validity of the drug repositioning procedure implemented herein for the identification of new therapeutic tools against HD