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Two clinically approved anticancer drugs targeting BRAF in melanoma patients - dabrafenib (DAB) and vemurafenib (VEM) - have been successfully encapsulated into nanomicelles formed upon self-assembly of an amphiphilic dendrimer AD based on two C18 aliphatic chains and a G2 PAMAM head. The process resulted in the formation of well-defined (∼10 nm) core-shell nanomicelles (NMs) with excellent encapsulation efficiency (∼70% for DAB and ∼60% for VEM) and good drug loading capacity (∼27% and ∼24% for DAB and VEM, respectively). Dynamic light scattering (DLS), transmission electron microscopy (TEM), small-angle x-ray scattering (SAXS), nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), and molecular simulation (MS) experiments were used, respectively, to determine the size and structure of the empty and drug-loaded nanomicelles (DLNMs), along with the interactions between the NMs and their cargoes. The in vitro release data revealed profiles governed by Fickian diffusion; moreover, for both anticancer molecules, an acidic environment (pH = 5.0) facilitated drug release with respect to physiological pH conditions (pH = 7.4). Finally, both DAB- and VEM-loaded NMs elicited enhanced response with respect to free drug treatments in 4 different melanoma cell lines.

CHO is the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. Manipulation of small non-coding RNAs—miRNAs—is a good alternative to a single gene knockdown approach due to their post-transcriptional regulation of entire cellular pathways without posing translational burden to the production cell. In this study, we performed a high-throughput screening of 2042-human miRNAs and identified several candidates able to increase cell-specific and overall production of Erythropoietin and Etanercept in CHO cells. Some of these human miRNAs have not been found in Chinese hamster cells and yet were still effective in them. We identified miR-574-3p as being able, when overexpressed in CHO cells, to improve overall productivity of Erythropoietin and Etanercept titers from 1.3 to up to 2-fold. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Furthermore, we demonstrated that stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperform the cells with human pri-miRNA sequences. Our findings highlight the importance of flanking genomic sequences, and their secondary structure features, on pri-miRNA processing offering a novel, cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells.

siRNAs are emerging as promising therapeutic agents due to their ability to inhibit specific genes in many diseases. However, these tools require specific vehicles in order to be safely delivered to the targeted site. Among different siRNA delivery systems, self-assembled nanomicelles based on amphiphilic cationic dendrons (ACDs) have recently outperformed nanovectors based on covalent carriers. This chapter describes how isothermal titration calorimetry (ITC) can be exploited as one of the best techniques to investigate the self-assembly process of ACDs. Specifically, ITC can provide, as such or via specific analysis methods, a full thermodynamic characterization of these nanomicelles, including their critical micellar concentration, micelle aggregation number, degree of counterion binding, Gibbs free energy of micellization, and its enthalpic and entropic components.

Repurposing clinically available drugs to treat the new coronavirus disease 2019 (COVID-19) is an urgent need in the course of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) pandemic, as very few treatment options are available. The iminosugar Miglustat is a well-characterized drug for the treatment of rare genetic lysosome storage diseases, such as Gaucher and Niemann-Pick type C, and has also been described to be active against a variety of enveloped viruses. The activity of Miglustat is here demonstrated in the micromolar range for SARS-CoV-2 in vitro. The drug acts at the post-entry level and leads to a marked decrease of viral proteins and release of infectious viruses. The mechanism resides in the inhibitory activity toward α-glucosidases that are involved in the early stages of glycoprotein N-linked oligosaccharide processing in the endoplasmic reticulum, leading to a marked decrease of the viral Spike protein. Indeed, the antiviral potential of protein glycosylation inhibitors against SARS-CoV-2 is further highlighted by the low-micromolar activity of the investigational drug Celgosivir. These data point to a relevant role of this approach for the treatment of COVID-19.

A great deal of scientific and economic efforts is currently being focused on nanotechnology, providing ample opportunity for scientific advances and new products for virtually all fields of application. However, substantial hurdles still remain on the road from the conception of a nanomaterial to its everyday use or, in case of nanomedicine, to its adoption in the clinical practice. Some of these include, for instance, (1) the need to bridge existing interdisciplinary gaps in order to exploit all different fields of knowledge in the effective and efficient design, development and testing of new nanomaterials; (2) the necessity to define appropriate standardized methodologies and procedures along the entire nanomaterial production chain (i.e., from cradle to grave), thereby contributing actively in closing the persistent nanomaterial regulatory gap; and (3) the urgency to raise awareness about risks and benefits associated with nanotechnology and nanomaterials both to policy makers and the general public. This chapter will try to briefly review all these points, with a specific eye on nanomaterials for biomedical applications (aka nanomedicines).

siRNAs are emerging as promising therapeutic agents due to their ability to inhibit specific genes in many diseases. However, these tools require specific vehicles in order to be safely delivered to the targeted site. Among different siRNA delivery systems, self-assembled nanomicelles based on amphiphilic cationic dendrons (ACDs) have recently outperformed nanovectors based on covalent carriers. This chapter describes how isothermal titration calorimetry (ITC) can be exploited as one of the best techniques to investigate the self-assembly process of ACDs. Specifically, ITC can provide, as such or via specific analysis methods, a full thermodynamic characterization of these nanomicelles, including their critical micellar concentration, micelle aggregation number, degree of counterion binding, Gibbs free energy of micellization, and its enthalpic and entropic components.

Repurposing clinically available drugs to treat the new coronavirus disease COVID-19 is an urgent need in these early stages of the SARS-CoV-2 pandemic, when very few treatment options are available. The iminosugar Miglustat is a well-characterized drug for the treatment of rare genetic lysosome storage diseases such as Gaucher and Niemann-Pick type C, and has also been described to be active against a variety of enveloped viruses. The activity of Miglustat is here demonstrated for SARS-CoV-2 at concentrations achievable in the plasma by current clinical regimens without cytotoxicity. The drug acts at the post-entry level and leads to a marked decrease of viral proteins and release of infectious virus. The mechanism resides in the inhibitory activity towards α-glucosidases that are involved in early stages of glycoprotein N-linked oligosaccharide processing in the endoplasmic reticulum, leading to a marked decrease of the viral Spike protein. The wealth of available data on the clinical use of Miglustat for the treatment of lysosomal storage disorders and the antiviral properties against SARS-CoV-2 make it an ideal candidate for drug repurposing.

Brain inclusions mainly composed of misfolded and aggregated TAR DNA binding protein 43 (TDP-43), are characteristic hallmarks of amyotrophic lateral sclerosis (ALS). Irrespective of the role played by the inclusions, their reduction represents an important therapeutic pathway that is worth exploring. Their removal can either lead to the recovery of TDP-43 function by removing the self-templating conformers that sequester the protein in the inclusions, and/or eliminate any potential intrinsic toxicity of the aggregates. The search for curative therapies has been hampered by the lack of ALS models for use in high-throughput screening. We adapted, optimised, and extensively characterised our previous ALS cellular model for such use. The model demonstrated efficient aggregation of endogenous TDP-43, and concomitant loss of its splicing regulation function. We provided a proof-of-principle for its eventual use in high-throughput screening using compounds of the tricyclic family and showed that recovery of TDP-43 function can be achieved by the enhanced removal of TDP-43 aggregates by these compounds. We observed that the degradation of the aggregates occurs independent of the autophagy pathway beyond autophagosome-lysosome fusion, but requires a functional proteasome pathway. The in vivo translational effect of the cellular model was tested with two of these compounds in a Drosophila model expressing a construct analogous to the cellular model, where thioridazine significantly improved the locomotive defect. Our findings have important implications as thioridazine cleared TDP-43 aggregates and recovered TDP-43 functionality. This study also highlights the importance of a two-stage, in vitro and in vivo model system to cross-check the search for small molecules that can clear TDP-43 aggregates in TDP-43 proteinopathies.

TDP-43 inclusions are an important histopathological feature in various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis and Fronto-Temporal Lobar Degeneration. However, the relation of these inclusions with the pathogenesis of the disease is still unclear. In fact, the inclusions could be toxic themselves, induce loss of function by sequestering TDP-43 or a combination of both. Previously, we have developed a cellular model of aggregation using the TDP-43 Q/N rich amino acid sequence 331–369 repeated 12 times (12xQ/N) and have shown that these cellular inclusions are capable of sequestering the endogenous TDP-43 both in non-neuronal and neuronal cells. We have tested this model in vivo in the Drosophila melanogaster eye. The eye structure develops normally in the absence of dTDP-43, a fact previously seen in knock out fly strains. We show here that expression of EGFP 12xQ/N does not alter the structure of the eye. In contrast, TBPH overexpression is neurotoxic and causes necrosis and loss of function of the eye. More important, the neurotoxicity of TBPH can be abolished by its incorporation to the insoluble aggregates induced by EGFP 12xQ/N. This data indicates that aggregation is not toxic per se and instead has a protective role, modulating the functional TBPH available in the tissue. This is an important indication for the possible pathological mechanism in action on ALS patients.

Dextran sulfate 5,000 Da (DS), a sulfated polysaccharide, has been used in recombinant mammalian cell cultures to prevent cell aggregation, thereby increasing cell viability. Previous studies using Chinese hamster ovary (CHO) suspension cultures had shown that low concentrations of DS are related to an inhibition of apoptosis. In this study, DS was used on anchorage-dependent CHO cells producing erythropoietin (EPO), in order to investigate the effect of this molecule on anti-apoptotic and pro-survival cellular pathways. DS 5,000 Da treatment was shown to prolong the life of cells and increase productivity of EPO by 1.8-fold comparing with controls, in standard batch conditions. At a molecular level, we show that DS inhibits apoptosis by DNA fragmentation delay and decrease of annexin V-labeled cells, causes a G0/G1 cell cycle arrest, decreases p53 expression and increases the pro-survival factor Hsc70 expression. DS treatment also resulted in an enhanced LC3-I to LC3-II conversion and increased autophagosomes formation employing tagged-LC3. Our data show, for the first time, that low doses of DS may promote autophagy in different cell lines. These findings suggest that a better understanding and manipulation of phenomenon of autophagy could be of crucial importance in the bio-pharmaceutical industry, in particular in the field of protein production.

In higher eukaryotes, the 5' splice site (5'ss) is initially recognized through an RNA-RNA interaction by U1 small nuclear ribonucleoprotein (U1 snRNP). This event represents one of the key steps in initial spliceosomal assembly and many disease-associated mutations in humans often disrupt this process. Beside base pair complementarity, 5'ss recognition can also be modified by additional factors such as RNA secondary structures or the specific binding of other nuclear proteins. In this work, we have focused on investigating a few examples of changes detected within the 5'ss in patients, that would not be immediately considered "disease causing mutations". We show that the splicing outcome of very similar mutations can be very different due to variations in trans-acting factor(s) interactions and specific context influences. Using several NF1 donor sites and SELEX approaches as experimental models, we have examined the binding properties of particular sequence motifs such as GGGU found in donor sites, and how the sequence context can change their interaction with hnRNPs such as H/F and A1/A2. Our results clearly show that even minor differences in local nucleotide context can differentially affect the binding ability of these factors to the GGGU core. Finally, using a previously identified mutation in KCNH2 that resulted in intron retention we show how very similar 5'ss mutations found in patients can have a very different splicing outcome due to the neighbouring sequence context, thus highlighting the general need to approach splicing problems with suitable experimental approaches.

Eukaryotic gene expression relies on several complex molecular machineries that act in a highly coordinated fashion. These machineries govern all the different steps of mRNA maturation, from gene transcription and pre-mRNA processing in the nucleus to the export of the mRNA to the cytoplasm and its translation. In particular, the pre-mRNA splicing process consists in the joining together of sequences (known as “exons”) that have to be differentiated from their intervening sequences commonly referred to as “introns.” The complex required to perform this process is a very dynamic macromolecular ribonucleoprotein assembly that functions as an enzyme, and is called the “spliceosome.” Because of its flexibility, the splicing process represents one of the main mechanisms of qualitative and quantitative regulation of gene expression in eukaryotic genomes. This flexibility is mainly due to the possibility of alternatively recognizing the various exons that are present in a pre-mRNA molecule and therefore enabling the possibility of obtaining multiple transcripts from the same gene. However, regulation of gene expression by the spliceosome is also achieved through its ability to influence many other gene expression steps that include transcription, mRNA export, mRNA stability, and even protein translation. Therefore, from a biotechnological point of view the splicing process can be exploited to improve production strategies and processes of molecules of interest. In this work, we have aimed to provide an overview on how biotechnology applications may benefit from the introduction of introns within a sequence of interest.

Acquisition of mutations in the BCR-ABL1 kinase domain (KD) is frequently associated with tyrosine kinase inhibitor (TKI) failure in CML. Recently, we revealed a novel mutation “hot- spot” in the BCR-ABL1 KD region (residues 295 - 312), associated with high resistance and poor clinical outcomes [1]. Some of these new BCR-ABL1 variants locate in a protein region, which might not be directly involved in TKI interaction. Therefore, we hypothesized that some of these mutations might be linked to TKI resistance via a direct mechanism while others might exert an indirect effect on drug resistance. The present contribution integrates molecular, computational, and structural biology techniques to understand the eventual role of the newly reported hot-spot mutations of the BCR-ABL1 KD in TKI resistance observed in CML patients using a "two-tier level" investigation: isolated KDs and SH2-linker-SH3-KD BCR- ABL1 constructs. Molecular Modeling will assess in silico whether the clinically observed “hot-spot” BCR-ABL1 mutations might be directly related to a change in the thermodynamic stability and/or protein structure and how these modifications could be involved in the BCR-ABL1 binding profile to specific TKIs (e.g., imatinib, dasatinib, and ponatinib) compared with wild-type (WT) systems and “prototypical” BCR-ABL1 mutations (e.g. T315I). In parallel, in vitro analysis will evaluate the effects exerted by the selected mutations on the intrinsic activity of the kinase and to determine the inhibition activity of the selected drugs on these BCR-ABL1 variants. Specifically, direct drug binding to both KD and SSK constructs will be measured using isothermal titration calorimetry and in vitro kinase assays will monitor the ability of these mutant isoforms to auto-phosphorylate and to phosphorylate a substrate peptide. Finally, structural biology (e.g., SAXS and far-UV CD) evidences of WT and mutant isolated KDs and SH2-linker-SH3-KD (SSK) constructs (per se and in complex with TKIs) will be discussed to support in silico predictions. [1] Gibbons DL, Pricl S et al., Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy. Proc Natl Acad Sci U S A. 2014 Mar 4;111(9):3550-5. doi: 10.1073/pnas.1321173111. Citation Format: Erik Laurini, Suzana Aulic, Natasa Skoko, Sulena Polez, Marco Baralle, Maurizio Romano, Sabrina Pricl. In silico/in vitro studies of novel hot spot mutations in BCR-ABL1: A multidisciplinary approach to shed a new light on TKI resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2382.

N1-[1-[1-aryl-3-[4-(1H-imidazol-1-yl)phenyl]-3-oxo]propyl]-pyridine-2-carboxamidrazone derivatives were design, synthesized and tested for their in vitro antimycobacterial activity. The new compounds showed a moderate antimycobacterial activity against the tested strain of Mycobacterium tuberculosis H37Ra and a significant antimycobacterial activity against several mycobacteria other than tuberculosis strains.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this study, we found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration.

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting.

Background The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries. Results A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae α-factor secretory signal and integrated into the genome of P. pastoris strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth. Conclusions A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture.

We have previously identified an ESE in NF1 exon 37 whose disruption by the pathological mutation c.6792C>G caused aberrant splicing. We now investigate the RNA-protein complexes affected by the c.6792C>G mutation observing that this concurrently decreases the affinity for the positive splicing factor YB-1 and increases the affinity for the negative splicing factors, hnRNPA1, hnRNPA2 and a new player in these type of complexes, DAZAP1. Our findings highlight the complexity of the interplay between positive and negative factors in the exon inclusion/skipping outcome. Furthermore, our observations stress the role of a wide genomic context in NF1 exon 37 definition.

A disease-causing G-to-T transversion at position +6 of BRCA1 exon 18 induces exclusion of the exon from the mRNA and, as has been suggested by in silico analysis, disrupts an ASF/SF2-dependent splicing enhancer. We show here using a pulldown assay with an internal standard that wild-type (WT) and mutant T6 sequences displayed similar ASF/SF2 binding efficiencies, which were significantly lower than that of a typical exonic splicing enhancer derived from the extra domain A exon of fibronectin. Overexpression or small interfering RNA (siRNA)-mediated depletion of ASF/SF2 did not affect the splicing of a WT BRCA1 minigene but resulted in an increase and decrease of T6 exon 18 inclusion, respectively. Furthermore, extensive mutation analysis using hybrid minigenes indicated that the T6 mutant creates a sequence with a prevalently inhibitory function. Indeed, RNA-protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a splicing factor-dependent silencer. This sequence specifically binds to the known repressor protein hnRNPA1/A2 and to DAZAP1, the involvement of which in splicing inhibition we have demonstrated. Our results indicate that the binding of the splicing factors hnRNPA1/A2 and DAZAP1 is the primary determinant of T6 BRCA1 exon 18 exclusion.

The screening of microbial natural products continues to represent an important route to the discovery of novel chemicals for development of new therapeutic agents. The aim of this work was to develop an efficient method for the detection of immunosuppressive compounds produced by soil actinomycetes. Mutant strain of Saccharomyces cerevisiae, named FAV20, sensitive to FK506 was constructed by disrupting VMA22 gene using the selectable marker kanMX4 which allowed detection of integration events. Actinomycetes were isolated from different soil samples and in a newly developed test with S. cerevisiae FAV20, six strains have been identified that produce bioactive compounds with the same mechanism of action as FK506. S. cerevisiae FAV20 can be easily used as a test strain in drug screening programs based on inhibition of the calcineurin phosphatase dependent signaling pathway in the cell.

Penicillin G amidase from Providencia rettgeri is a heterodimer of 92 kDa. We have previously expressed the Pr. rettgeri pac gene coding for this enzyme in Saccharomyces cerevisiae, and now we report the expression and characterization in the methylotrophic yeast Pichia pastoris. The recombinant catalytically active enzyme (rPAC(Pr)) was secreted from shake flask-grown P. pastoris cells into the medium at a level of approximately 0.18 U ml(-1). This yield of rPAC(Pr) was higher, by two orders of magnitude, than that obtained using a single-copy expression plasmid in S. cerevisiae. In addition, the secreted recombinant enzyme was entirely N-glycosylated. The recombinant PAC(Pr) was further characterized in terms of specific activity, kinetic parameters and thermostability. Except the significantly higher thermostability of the glycosylated rPAC(Pr) produced in P. pastoris, the other parameters were very similar to those of the corresponding non-glycosylated enzymes produced in bacteria or in S. cerevisiae. The higher thermostability of this recombinant enzyme has a clear industrial advantage.

We describe the heterologous expression of a human interferon-beta1 in the methylotrophic yeast Pichia pastoris. Biologically active recombinant human interferon-beta1 (rHuIFN-beta1) was secreted from shake-flask-grown P. pastoris cells into the medium using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence at the level of (1-3) x 10(5) i.u. (international units)/ml (6-12 mg/litre). An rHuIFN-beta1 with an N-terminal sequence identical with that of native HuIFN-beta1 was purified and the specific activity was determined (2-3 x 10(7) i.u./mg). It was found that the secreted recombinant protein was partially N-glycosylated.