Enriching Proteolysis Targeting Chimeras with a Second Modality: When Two Are Better Than One

Proteolysis targeting chimera (PROTAC)-mediated protein degradation has prompted a radical rethink and is at a crucial stage in driving a drug discovery transition. To fully harness the potential of this technology, a growing paradigm toward enriching PROTACs with other therapeutic modalities has been proposed. Could researchers successfully combine two modalities to yield multifunctional PROTACs with an expanded profile? In this Perspective, we try to answer this question. We discuss how this possibility encompasses different approaches, leading to multitarget PROTACs, light-controllable PROTACs, PROTAC conjugates, and macrocycle-and oligonucleotide-based PROTACs. This possibility promises to further enhance PROTAC efficacy and selectivity, minimize side effects, and hit undruggable targets. While PROTACs have reached the clinical investigation stage, additional steps must be taken toward the translational development of multifunctional PROTACs. A deeper and detailed understanding of the most critical challenges is required to fully exploit these opportunities and decisively enrich the PROTAC toolbox

Design and synthesis of novel Cystic Fibrosis (CF) modulators - Development of novel inhibitors of the anti-infective target DXS using Dynamic Combinatorial Chemistry (DCC)

Cystic Fibrosis (CF) is a lethal, autosomal recessive genetic disease characterized by an accumulation of viscous mucus at epithelia surface of multiple organs, including lungs, pancreas, gut and testis, which results in obstruction, infection, inflammation and ultimately organ failure. The primary cause of CF is the mutation of a gene, the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), which leads to a decrease in CFTR chloride channel function and ultimately to a reduced ionic and water homeostasis at epithelial surfaces. Historically conventional CF treatments focused on symptomatic therapy, until recently when the growing understanding of the molecular basis of CF pathologies stimulated the development of small-molecule drugs, called CFTR modulators, which address the primary cause of CF with the hope to repair the defects in mutated CFTR. Aiming to expand the portfolio of novel modulators available to CF patients, also considering the relevant but limited pharmacological efficacy elicited by some of the current treatments, there is still the need to develop more CFTR small molecule modulators, primarily correctors, which may address the primary cause of CF by rescuing the activity of defective CFTR (Chapter 1). The present PhD thesis describes the design, synthesis and biological characterization of novel CFTR correctors. Starting from primary hits ARN9364 and ARN5562, selected for their promising initial biological activity after a High-Throughput Screening (HTS) campaign, new analogs were designed and synthesized to elucidate the Structure-Activity Relationship (SAR) patterns around these chemo-types (Chapter 2). The biological test of these novel compounds in a phenotypic cell-based assay (HS-YFP assay) using CFBE41o- cells, allowed to get clear information about the most suited structural modifications needed to improve rescuing activity of defective F508del-CFTR. An iterative process of design, synthesis and biological testing led to the identification of slightly or even more potent CFTR correctors (Chapter 3). In Chapter 4 the development of selective and potent inhibitors of the anti-infective target 1-deoxy-d-xylulose-5-phosphate synthase (DXS), using target-directed Dynamic Combinatorial Chemistry (tdDCC), as hit-identification strategy, was reported. Biochemical evaluation of several hit compounds amplified in the tdDCC experiment against M. tuberculosis DXS and D. radiodurans DXS afforded inhibitors with IC50 in the range of 30 microM \u2013 190 microM

Hit-Optimization Using Target-Directed Dynamic Combinatorial Chemistry: Development of Inhibitors of the Anti-Infective Target 1-Deoxy-D-Xylulose-5-Phosphate Synthase

Target-directed dynamic combinatorial chemistry (tdDCC) enables the identification, as well as optimization of ligands for un(der)explored targets such as the anti-infective target 1‑deoxy‑d‑xylulose-5-phosphate synthase (DXS). We report the unprecedented use of tdDCC to first identify and subsequently optimize inhibitors of the anti-infective target DXS. Using tdDCC, we were able to generate acylhydrazone-based inhibitors for DXS. The tailored tdDCC runs also provided insights into the structure–activity relationship of this novel class of DXS inhibitors. This approach holds the potential to expedite the drug discovery process and could be generally applied to a range of biological targets.</p

Hydroxy-substituted trans-cinnamoyl derivatives as multifunctional tools in the context of Alzheimer's disease

Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. In recent years, a plethora of proteins and biochemical pathways has been proposed as possible targets to counteract neurotoxicity. Although the complex scenario is not completely elucidated, close relationships are emerging among some of these actors. In particular, increasing evidence has shown that aggregation of amyloid beta (A\u3b2), glycogen synthase kinase 3\u3b2 (GSK-3\u3b2) and oxidative stress are strictly interconnected and their concomitant modulation may have a positive and synergic effect in contrasting AD-related impairments. We designed compound 3 which demonstrated the ability to inhibit both GSK-3\u3b2 (IC50 = 24.36 \ub1 0.01 \u3bcM) and A\u3b242 self-aggregation (IC50 = 9.0 \ub1 1.4 \u3bcM), to chelate copper (II) and to act as exceptionally strong radical scavenger (kinh = 6.8 \ub1 0.5 \ub7 105 M 121s 121) even in phosphate buffer at pH 7.4 (kinh = 3.2 \ub1 0.5 \ub7 105 M 121s 121). Importantly, compound 3 showed high-predicted blood-brain barrier permeability, did not exert any significant cytotoxic effects in immature cortical neurons up to 50 \u3bcM and showed neuroprotective properties at micromolar concentration against toxic insult induced by glutamate

Hit-optimization using target-directed dynamic combinatorial chemistry: development of inhibitors of the anti-infective target 1-deoxy-d-xylulose-5-phosphate synthase.

Target-directed dynamic combinatorial chemistry (tdDCC) enables identification, as well as optimization of ligands for un(der)explored targets such as the anti-infective target 1-deoxy-d-xylulose-5-phosphate synthase (DXPS). We report the use of tdDCC to first identify and subsequently optimize binders/inhibitors of the anti-infective target DXPS. The initial hits were also optimized for their antibacterial activity against E. coli and M. tuberculosis during subsequent tdDCC runs. Using tdDCC, we were able to generate acylhydrazone-based inhibitors of DXPS. The tailored tdDCC runs also provided insights into the structure-activity relationship of this novel class of DXPS inhibitors. The competition tdDCC runs provided important information about the mode of inhibition of acylhydrazone-based inhibitors. This approach holds the potential to expedite the drug-discovery process and should be applicable to a range of biological targets

Hit-optimization using target-directed dynamic combinatorial chemistry : development of inhibitors of the anti-infective target 1-deoxy-d-xylulose-5-phosphate synthase

Target-directed dynamic combinatorial chemistry (tdDCC) enables identification, as well as optimization of ligands for un(der)explored targets such as the anti-infective target 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). We report the use of tdDCC to first identify and subsequently optimize binders/inhibitors of the anti-infective target DXPS. The initial hits were also optimized for their antibacterial activity against E. coli and M. tuberculosis during subsequent tdDCC runs. Using tdDCC, we were able to generate acylhydrazone-based inhibitors of DXPS. The tailored tdDCC runs also provided insights into the structure–activity relationship of this novel class of DXPS inhibitors. The competition tdDCC runs provided important information about the mode of inhibition of acylhydrazone-based inhibitors. This approach holds the potential to expedite the drug-discovery process and should be applicable to a range of biological targets