Alessandra Salerno

Proteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that induce ubiquitination and degradation of a target protein via recruitment to an E3 ligase. The linker influences many steps of the PROTAC mode of action, from cellular permeability to ternary complex formation and target degradation. Much interest has therefore been devoted to linker design to fine-tune molecular and mechanistic properties of PROTACs. In this study, we present FerroTACs, a novel PROTAC design strategy incorporating ferrocene as the linker chemotype. We exemplify the approach across three different PROTAC systems: VHL-VHL (homo-PROTACs), VHL-CRBN, and VHL-BETs. We find that ferrocene's unique organometallic structure, featuring freely rotating cyclopentadienyl rings around a central Fe(II) ion, acts as a molecular hinge enabling structural adjustment to the environment that results in properties alteration, i.e., chameleonicity. Conformational analyses via NMR spectroscopy support ferrocene's role in fostering intramolecular interactions that result in a more folded state in an apolar environment. This property promotes compact conformations, improving cellular permeability and reducing efflux liabilities. Cellular assays demonstrate that FerroTACs exhibit robust target degradation and cell permeability profiles, en-par or enhanced compared to benchmark PROTACs CM11, 14a, and MZ1. These findings highlight ferrocene's potential as a new linker design strategy, offering a versatile platform to install and control molecular chameleonicity into next-generation PROTACs.

Dynamic combinatorial chemistry (DCC) leverages a reversible reaction to generate compound libraries from constituting building blocks under thermodynamic control. The position of this equilibrium can be biased by addition of a target macromolecule towards enrichment of bound ligands. While DCC has been applied to select ligands for a single target protein, its application to identifying chimeric molecules inducing proximity between two proteins is unprecedented. In this proof‐of‐concept study, we develop a DCC approach to select bifunctional proteolysis targeting chimeras (PROTACs) based on their ability to stabilize the ternary complex. We focus on VHL‐targeting Homo‐PROTACs as model system, and show that the formation of a VHL₂ : Homo‐PROTAC ternary complex reversibly assembled using thiol‐disulfide exchange chemistry leads to amplification of potent VHL Homo‐PROTACs with degradation activities which correlated well with their biophysical ability to dimerize VHL. Ternary complex templated dynamic combinatorial libraries allowed identification of novel Homo‐PROTAC degraders. We anticipate future applications of ternary‐complex directed DCC to early PROTAC screenings and expansion to other proximity‐inducing modalities beyond PROTACs.

Dynamic combinatorial chemistry (DCC) leverages a reversible reaction to generate compound libraries from constituting building blocks under thermodynamic control. The position of this equilibrium can be biased by addition of a target macromolecule towards enrichment of bound ligands. While DCC has been applied to select ligands for a single target protein, its application to identifying chimeric molecules inducing proximity between two proteins is unprecedented. In this proof‐of‐concept study, we develop a DCC approach to select bifunctional proteolysis targeting chimeras (PROTACs) based on their ability to stabilize the ternary complex. We focus on VHL‐targeting Homo‐PROTACs as model system, and show that the formation of a VHL₂ : Homo‐PROTAC ternary complex reversibly assembled using thiol‐disulfide exchange chemistry leads to amplification of potent VHL Homo‐PROTACs with degradation activities which correlated well with their biophysical ability to dimerize VHL. Ternary complex templated dynamic combinatorial libraries allowed identification of novel Homo‐PROTAC degraders. We anticipate future applications of ternary‐complex directed DCC to early PROTAC screenings and expansion to other proximity‐inducing modalities beyond PROTACs.

Dynamic combinatorial chemistry (DCC) leverages a reversible reaction to generate compound libraries from constituting building blocks under thermodynamic control. The position of this equilibrium can be biased by addition of a target macromolecule towards enrichment of bound ligands. While DCC has been applied to select ligands for a single target protein, its application to identifying chimeric molecules inducing proximity between two proteins is unprecedented. In this proof-of-concept study, we develop a DCC approach to select bifunctional proteolysis targeting chimeras (PROTACs) based on their ability to stabilize the ternary complex. We focus on VHL-targeting Homo-PROTACs as model system, and show that the formation of a VHL2:Homo-PROTAC ternary complex reversibly assembled using thiol-disulfide exchange chemistry leads to amplification of potent VHL Homo-PROTACs with degradation activities which correlated well with their biophysical ability to dimerize VHL. Ternary complex templated dynamic combinatorial libraries allowed identification of novel Homo-PROTAC degraders. We anticipate future applications of ternary-complex directed DCC to early PROTAC screenings and expansion to other proximity-inducing modalities beyond PROTACs.

Trypanothione reductase (TR) is a suitable target for drug discovery approaches against leishmaniasis, although the identification of potent inhibitors is still challenging. Herein, we harnessed a fragment-based drug discovery (FBDD) strategy to develop new TR inhibitors. Previous crystallographic screening identified fragments 1-3, which provided ideal starting points for a medicinal chemistry campaign. In silico investigations revealed critical hotspots in the TR binding site, guiding our structure- and ligand-based structure-actvity relationship (SAR) exploration that yielded fragment-derived compounds 4-14. A trend of improvement in Leishmania infantum TR inhibition was detected along the optimization and confirmed by the crystal structures of 9, 10, and 14 in complex with Trypanosoma brucei TR. Compound 10 showed the best TR inhibitory profile (K_i = 0.2 μM), whereas 9 was the best one in terms of in vitro and ex vivo activity. Although further fine-tuning is needed to improve selectivity, we demonstrated the potentiality of FBDD on a classic but difficult target for leishmaniasis.

Polypharmacology is a new trend in amyotrophic lateral sclerosis (ALS) therapy and an effective way of addressing a multifactorial etiology involving excitotoxicity, mitochondrial dysfunction, oxidative stress, and microglial activation. Inspired by a reported clinical trial, we converted a riluzole (1)-rasagiline (2) combination into single-molecule multi-target-directed ligands. By a ligand-based approach, the highly structurally integrated hybrids 3-8 were designed and synthesized. Through a target- and phenotypic-based screening pipeline, we identified hit compound 6. It showed monoamine oxidase A (MAO-A) inhibitory activity (IC₅₀ = 6.9 μM) rationalized by in silico studies as well as in vitro brain permeability. By using neuronal and non-neuronal cell models, including ALS-patient-derived cells, we disclosed for 6 a neuroprotective/neuroinflammatory profile similar to that of the parent compounds and their combination. Furthermore, the unexpected MAO inhibitory activity of 1 (IC₅₀ = 8.7 μM) might add a piece to the puzzle of its anti-ALS molecular profile.

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.

The multifactorial nature of Alzheimer’s disease (AD) is a reason for the lack of effective drugs as well as a basis for the development of “multi-target-directed ligands” (MTDLs). As cases increase in developing countries, there is a need of new drugs that are not only effective but also accessible. With this motivation, we report the first sustainable MTDLs, derived from cashew nutshell liquid (CNSL), an inexpensive food waste with anti-inflammatory properties. We applied a framework combination of functionalized CNSL components and well-established acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) tacrine templates. MTDLs were selected based on hepatic, neuronal, and microglial cell toxicity. Enzymatic studies disclosed potent and selective AChE/BChE inhibitors (5, 6, and 12), with subnanomolar activities. The X-ray crystal structure of 5 complexed with BChE allowed rationalizing the observed activity (0.0352 nM). Investigation in BV-2 microglial cells revealed antineuroinflammatory and neuroprotective activities for 5 and 6 (already at 0.01 μM), confirming the design rationale.

The continued drug discovery failures in complex neurodegenerative diseases, including Alzheimer's disease (AD), has raised questions about the classical paradigm “one‐drug, one‐target, one‐disease.” In parallel, the ever‐increasing awareness of the multiplicity of the underlying pathways has led to the affirmation of polypharmacological approaches. Polypharmacology, which broadly embodies the use of pharmaceutical agents acting on multiple targets, seems to be the best way to restore the complex diseased network and to provide disease‐modifying effects in AD. In this review, our aim is to provide a roadmap into a world that is still only partly explored and that should be seen as a continuum of pharmacological opportunities, from drug combinations to multitarget‐directed ligands (both codrugs and hybrids). Each modality has unique features that can be effectively exploited by medicinal chemists. We argue that understanding their advantages and drawbacks is very helpful in choosing a proper approach and developing successful AD multitarget drug‐discovery endeavors. We also briefly dwell on (co)target validation, an aspect that is quite often neglected, but critical for an efficient clinical translation. We substantiate our discussion with instructive examples taken from the recent literature. Our wish is that, in spite of the specter of the high attrition rates, best researchers preferring to enter, stay, and progress in the field would help grow the sector and develop AD polypharmacology to full potential.