Studies on Proteolysis Targeting Chimeras: platform technology for targeted protein degradation in drug discovery

Proteolysis Targeting Chimeras (PROTACs) represent an innovative approach to chemical intervention into biology, with attractive therapeutic potential. In contrast to protein inhibition, PROTACs trigger targeted protein degradation inside cells via hijacking the ubiquitinproteasome machinery. PROTACs are bifunctional compounds composed by two small molecules connected by a linker; one molecule binds to a protein of interest and the other one binds to an E3 ubiquitin ligase. The ligases most commonly recruited are the von Hippel- Lindau (VHL) protein complex CRL2VHL and the cereblon (CRBN) complex CRL4CRBN. To date, different classes of target proteins have been successfully degraded, including epigenetic targets such as bromodomain-containing proteins BRD2, BRD3, and BRD4, BRD9, TRIM24, SIRT2, PCAF/GNC5, protein kinases, nuclear receptors and E3 ubiquitin ligases to selfdegrade. The work described in this thesis is divided into two parts: the first part is about the research activity carried out at the University of Dundee, under the supervision of Professor Alessio Ciulli, while a second part involves the work done at the Department of Chemical Core Technologies, within Nerviano Medical Sciences (Milan), under the supervision of Doctor Eduard Felder. The aim of the project carried out in Ciulli Lab was to investigate PROTAC-mediated degradation of BRD7 and BRD9 proteins, subunits of the human SWI/SNF chromatin remodelling complexes. These subunits have gained interest as therapeutic target especially in hematopoietic cancers, for example supporting growth of acute myeloid leukemia (AML) cells. Despite inhibitors able to disrupt the interaction of the BRD7/9 bromodomains are known, their cellular activity has remained limited as other non-bromodomain functions of the target remain unaffected. PROTAC molecules able to induce BRD9 degradation would more profoundly impact on BRD7 and BRD9 function and therefore could be used as chemical tools to better understand its role in BAF complex and in oncogenesis. Following analysis of the available co-crystal structures of the individual target and ligase proteins with their respective ligands, a library of degraders has been designed and synthesised, involving convergent synthetic strategies to conjugate a diverse range of scaffolds and linkers. All compounds were tested in vitro against different cancer cell lines and immunoblotting was carried out to assess the target degradation profile. VZ185 was found as a highly selective, potent and rapid dual BRD7/BRD9 degrader, with slight preference for BRD9 over BRD7. Degradation was demonstrated to be dependent upon proteasomal activity, cullin neddylation and VHL binding. The second section of the thesis reports the design, synthesis and biological characterization of PROTACs targeting protein kinases, carried out at NMS for one-year stage. With the future prospect of extending the development of PROTAC to other targets, it was planned to study a literature example, in order to confirm the proof of concept. Amongst those available, we chose DAS-6-2-2-6-CRBN, based on a potent tyrosine kinase inhibitor and a CRBN ligand that induce degradation of c-ABL and BCR-ABL. Furthermore, we decided to extend the development of degraders focusing on how target ligand and linker composition affect efficacy and selectivity. The biological evaluation of the synthetized compounds was performed by the Department of Biology of NMS throughout in vitro treatments of cancer cell lines and immunoblotting

Toward LOCOSTRA: Blast-resistant Wheels Test

Technical Survey, often an efficient method of achieving land release, can also be prohibitively expensive for certain communities due to the utilization of the same hulking, heavily-armored machines used in clearance operations. If Technical Survey could be achieved through the use of less expensive agricultural equipment that is already present in communities near suspected areas, land release could be achieved at a much lower price. The following study explores this possibility by examining the explosion resilience of four different designs of blast-resistant tractor wheels, each made of commercial off-the-shelf components and designed for easy reproduction in mine-affected communities

Iterative design and optimization of initially inactive Proteolysis Targeting Chimeras (PROTACs) identify VZ185 as a potent, fast and selective von Hippel-Lindau (VHL)-based dual degrader probe of BRD9 and BRD7

Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase-target pair deemed unsuitable: the von Hippel-Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers, and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure-activity relationships guided the discovery of VZ185, a potent, fast and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown, and provide a roadmap for PROTAC development against seemingly incompatible target-ligase combinations

Acute Delta Hepatitis in Italy spanning three decades (1991–2019): Evidence for the effectiveness of the hepatitis B vaccination campaign

Updated incidence data of acute Delta virus hepatitis (HDV) are lacking worldwide. Our aim was to evaluate incidence of and risk factors for acute HDV in Italy after the introduction of the compulsory vaccination against hepatitis B virus (HBV) in 1991. Data were obtained from the National Surveillance System of acute viral hepatitis (SEIEVA). Independent predictors of HDV were assessed by logistic-regression analysis. The incidence of acute HDV per 1-million population declined from 3.2 cases in 1987 to 0.04 in 2019, parallel to that of acute HBV per 100,000 from 10.0 to 0.39 cases during the same period. The median age of cases increased from 27 years in the decade 1991-1999 to 44 years in the decade 2010-2019 (p < .001). Over the same period, the male/female ratio decreased from 3.8 to 2.1, the proportion of coinfections increased from 55% to 75% (p = .003) and that of HBsAg positive acute hepatitis tested for by IgM anti-HDV linearly decreased from 50.1% to 34.1% (p < .001). People born abroad accounted for 24.6% of cases in 2004-2010 and 32.1% in 2011-2019. In the period 2010-2019, risky sexual behaviour (O.R. 4.2; 95%CI: 1.4-12.8) was the sole independent predictor of acute HDV; conversely intravenous drug use was no longer associated (O.R. 1.25; 95%CI: 0.15-10.22) with this. In conclusion, HBV vaccination was an effective measure to control acute HDV. Intravenous drug use is no longer an efficient mode of HDV spread. Testing for IgM-anti HDV is a grey area requiring alert. Acute HDV in foreigners should be monitored in the years to come

Hydrophilic and amphiphilic water-soluble dendrimer prodrugs suitable for parenteral administration of a non-soluble non-nucleoside HIV-1 reverse transcriptase inhibitor thiocarbamate derivative

Drugs delivered by proper carriers enter into the cells much more rapidly and carry out their action much more promptly than in the free forms. A high drug concentration can be sustained for longer periods of time at the target site in the cell. In in vivo conditions, this would translate into a reduction of systemic toxicity, dosage and frequency of dosing. Dendritic polymers significantly affect drug delivery in terms of reaching the target site, modifying the bio-distribution of the drug, and enhancing the efficacy of different drugs including anticancer compounds. 2-([2-([(2-tolyl)amino]carbonothioyloxy)ethyl]aminocarbo-nyl)benzoic acid 1 is a thiocarbamate derivative belonging to an already reported class of non-nucleoside HIV-1 reverse transcriptase inhibitors. In in vitro assay it showed no cytotoxic effects but was endowed with very low solubility and poor activity against wild-type HIV-1 (EC50\u202f=\u202f27\u202f\u3bcM). With the aim at improving its water solubility, 1 has been successfully incorporated inside non-toxic amino acids-modified core-shell hetero dendrimers. IR, NMR, zeta potential, mean size of particles, buffer capacity and in vitro release profile of prepared materials were reported. All dendriplexes were evaluated in cell-based assays to assess their cytotoxic profile. The obtained complexes, which harmonize a peripheral polycationic character and a buffer capacity which presuppose efficient cells penetration and increased residence time with a not PAMAM structured biodegradable scaffold, were well water-soluble and could rationally appear as a promising set of prodrugs for safe in vivo administrations