An 19F NMR fragment-based approach for the discovery and development of BRCA2-RAD51 inhibitors to pursuit synthetic lethality in combination with PARP inhibition in pancreatic cancer.

The BRCA2-RAD51 interaction remains an intriguing target for cancer drug discovery due to its vital role in DNA damage repair mechanisms, which cancer cells become particularly reliant on. Moreover, RAD51 has many synthetically lethal partners, including PARP1-2, which can be exploited to induce synthetic lethality in cancer. In this study, we established a 19F-NMR-fragment based approach to identify RAD51 binders, leading to two initial hits. A subsequent SAR program identified 46 as a low micromolar inhibitor of the BRCA2-RAD51 interaction. 46 was tested in different pancreatic cancer cell lines, to evaluate its ability to inhibit the homologous recombination DNA repair pathway, mediated by BRCA2-RAD51 and trigger synthetic lethality in combination with the PARP inhibitor talazoparib, through the induction of apoptosis. Moreover, we further analyzed the 46/talazoparib combination in 3D pancreatic cancer models. Overall, 46 showed its potential as a tool to evaluate the RAD51/PARP1-2 synthetic lethality mechanism, along with providing a prospect for further inhibitors development.

Isolation and Characterization of Monomeric Human RAD51: A Novel Tool for Investigating Homologous Recombination in Cancer.

DNA repair protein RAD51 is a key player in the homologous recombination pathway. Upon DNA damage, RAD51 is transported into the nucleus by BRCA2, where it can repair DNA double-strand breaks. Due to the structural complexity and dynamics, researchers have not yet clarified the mechanistic details of every step of RAD51 recruitment and DNA repair. RAD51 possesses an intrinsic tendency to form oligomeric structures, which make it challenging to conduct biochemical and biophysical investigations. Here, for the first time, we report on the isolation and characterization of a human monomeric RAD51 recombinant form, obtained through a double mutation, which preserves the protein's integrity and functionality. We investigated different buffers to identify the most suitable condition needed to definitively stabilize the monomer. The monomer of human RAD51 provides the community with a unique biological tool for investigating RAD51-mediated homologous recombination, and paves the way for more reliable structural, mechanistic, and drug discovery studies.

Preomic profile of BxPC-3 cells after treatment with BRC4.

BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment. SIGNIFICANCE: RAD51's inhibition has gained increasing attention because of its possible implications in personalized medicine through the SL approach. Chemical disruption of protein-protein interactions (PPIs) between RAD51 and BRCA2, or some of its partner proteins, could potentiate PARPi DNA damage-induced cell death. This could have application for difficult to treat cancers, such as BRCA-competent and olaparib (PARPi) resistant pancreatic adenocarcinoma. Despite RAD51 being a widely studied target, researchers still lack detailed mechanistic information. This has stifled progress in the field with only a few RAD51 inhibitors having been identified, none of which have gained regulatory approval. Nevertheless, the peptide BRC4 is one of the most specific and best characterized RAD51 binder and inhibitor reported to date. Our study is the first to report the proteomic fingerprint consequent to cellular treatment of myr-BRC4, to offer a reference for the discovery of specific protein/pathway alterations within DNA damage repair. Our results suggest that, being a direct consequence of myr-BRC4 treatment, and ultimately ofBRCA2/RAD51 disruption, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring DNA damage repair impairment and therefore be used to potentiate the development of new effective therapeutic strategies.

Innovative Strategy toward Mutant CFTR Rescue in Cystic Fibrosis: Design and Synthesis of Thiadiazole Inhibitors of the E3 Ligase RNF5.

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CF transmembrane conductance regulator (CFTR) is associated to misfolding and defective gating of the mutant channel. One of the most promising CF drug targets is the ubiquitin ligase RNF5, which promotes F508del-CFTR degradation. Recently, the first ever reported inhibitor of RNF5 was discovered, i.e., the 1,2,4-thiadiazol-5-ylidene inh-2. Here, we designed and synthesized a series of new analogues to explore the structure-activity relationships (SAR) of this class of compounds. SAR efforts ultimately led to compound 16, which showed a greater F508del-CFTR corrector activity than inh-2, good tolerability, and no toxic side effects. Analogue 16 increased the basal level of autophagy similar to what has been described with RNF5 silencing. Furthermore, co-treatment with 16 significantly improved the F508del-CFTR rescue induced by the triple combination elexacaftor/tezacaftor/ivacaftor in CFBE41o- cells. These findings validate the 1,2,4-thiadiazolylidene scaffold for the discovery of novel RNF5 inhibitors and provide evidence to pursue this unprecedented strategy for the treatment of CF.

GSK-3ß Allosteric Inhibition: A Dead End or a New Pharmacological Frontier?

Most kinase inhibitors are designed to bind to highly homologous ATP-binding sites, which leads to promiscuity and possible off-target effects. Allostery is an alternative approach to pursuing selectivity. However, allostery is difficult to exploit due to the wide variety of underlying mechanisms and the potential involvement of long-range conformational effects that are difficult to pinpoint. GSK-3ß is involved in several pathologies. This critical target has an ATP-binding site that is highly homologous with the orthosteric sites of other kinases. Unsurprisingly, there is also great similarity between the ATP-binding sites of GSK-3ß and its isomer, which is not redundant and thus would benefit from selective inhibition. Allostery would also allow for a moderate and tunable inhibition, which is ideal for GSK-3ß, because this target is involved in multiple pathways, some of which must be preserved. However, despite considerable research efforts, only one allosteric GSK-3ß inhibitor has reached the clinic. Moreover, unlike other kinases, there are no X-ray structures of GSK-3ß in complex with allosteric inhibitors in the PDB data bank. This review aims to summarize the state of the art in allosteric GSK-3ß inhibitor investigations, highlighting the aspects that make this target challenging for an allosteric approach.

Design, Synthesis, In Vitro and In Vivo Characterization of CDC42 GTPase Interaction Inhibitors for the Treatment of Cancer.

CDC42 GTPases (RHOJ, CDC42, and RHOQ) are overexpressed in multiple tumor types and activate pathways critical for tumor growth, angiogenesis, and metastasis. Recently, we reported the discovery of a novel lead compound, ARN22089, which blocks the interaction of CDC42 GTPases with specific downstream effectors. ARN22089 blocks tumor growth in BRAF mutant mouse melanoma models and patient-derived xenografts (PDXs) in vivo. ARN22089 also inhibits tumor angiogenesis in three-dimensional vascularized microtumor models in vitro. Notably, ARN22089 belongs to a novel class of trisubstituted pyrimidines. Based on these results, we describe an extensive structure-activity relationship of ∼30 compounds centered on ARN22089. We discovered and optimized two novel inhibitors (27, ARN25062, and 28, ARN24928), which are optimal back-up/follow-up leads with favorable drug-like properties and in vivo efficacy in PDX tumors. These findings further demonstrate the potential of this class of CDC42/RHOJ inhibitors for cancer treatment, with lead candidates ready for advanced preclinical studies.

Novel Insights into RAD52's Structure, Function, and Druggability for Synthetic Lethality and Innovative Anticancer Therapies.

In recent years, the RAD52 protein has been highlighted as a mediator of many DNA repair mechanisms. While RAD52 was initially considered to be a non-essential auxiliary factor, its inhibition has more recently been demonstrated to be synthetically lethal in cancer cells bearing mutations and inactivation of specific intracellular pathways, such as homologous recombination. RAD52 is now recognized as a novel and critical pharmacological target. In this review, we comprehensively describe the available structural and functional information on RAD52. The review highlights the pathways in which RAD52 is involved and the approaches to RAD52 inhibition. We discuss the multifaceted role of this protein, which has a complex, dynamic, and functional 3D superstructural arrangement. This complexity reinforces the need to further investigate and characterize RAD52 to solve a challenging mechanistic puzzle and pave the way for a robust drug discovery campaign.

The Mechanistic Understanding of RAD51 Defibrillation: A Critical Step in BRCA2-Mediated DNA Repair by Homologous Recombination.

The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a "domino" mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.

Identification of RAD51-BRCA2 Inhibitors Using N-Acylhydrazone-Based Dynamic Combinatorial Chemistry.

RAD51 is an ATP-dependent recombinase, recruited by BRCA2 to mediate DNA double-strand breaks repair through homologous recombination and represents an attractive cancer drug target. Herein, we applied for the first-time protein-templated dynamic combinatorial chemistry on RAD51 as a hit identification strategy. Upon design of N-acylhydrazone-based dynamic combinatorial libraries, RAD51 showed a clear templating effect, amplifying 19 N-acylhydrazones. Screening against the RAD51-BRCA2 protein-protein interaction via ELISA assay afforded 10 inhibitors in the micromolar range. Further 19F NMR experiments revealed that 7 could bind RAD51 and be displaced by BRC4, suggesting an interaction in the same binding pocket of BRCA2. These results proved not only that ptDCC could be successfully applied on full-length oligomeric RAD51, but also that it could address the need of alternative strategies toward the identification of small-molecule PPI inhibitors.

Identification of Novel GSK-3ß Hits Using Competitive Biophysical Assays.

Glycogen synthase kinase 3 beta (GSK-3ß) is an evolutionarily conserved serine-threonine kinase dysregulated in numerous pathologies, such as Alzheimer's disease and cancer. Even though GSK-3ß is a validated pharmacological target most of its inhibitors have two main limitations: the lack of selectivity due to the high homology that characterizes the ATP binding site of most kinases, and the toxicity that emerges from GSK-3ß complete inhibition which translates into the impairment of the plethora of pathways GSK-3ß is involved in. Starting from a 1D 19F NMR fragment screening, we set up several biophysical assays for the identification of GSK-3ß inhibitors capable of binding protein hotspots other than the ATP binding pocket or to the ATP binding pocket, but with an affinity able of competing with a reference binder. A phosphorylation activity assay on a panel of several kinases provided selectivity data that were further rationalized and corroborated by structural information on GSK-3ß in complex with the hit compounds. In this study, we identified promising fragments, inhibitors of GSK-3ß, while proposing an alternative screening workflow that allows facing the flaws that characterize the most common GSK-3ß inhibitors through the identification of selective inhibitors and/or inhibitors able to modulate GSK-3ß activity without leading to its complete inhibition.

Chloride intracellular channel 1 activity is not required for glioblastoma development but its inhibition dictates glioma stem cell responsivity to novel biguanide derivatives.

BACKGROUND: Chloride intracellular channel-1 (CLIC1) activity controls glioblastoma proliferation. Metformin exerts antitumor effects in glioblastoma stem cells (GSCs) inhibiting CLIC1 activity, but its low potency hampers its translation in clinical settings. METHODS: We synthesized a small library of novel biguanide-based compounds that were tested as antiproliferative agents for GSCs derived from human glioblastomas, in vitro using 2D and 3D cultures and in vivo in the zebrafish model. Compounds were compared to metformin for both potency and efficacy in the inhibition of GSC proliferation in vitro (MTT, Trypan blue exclusion assays, and EdU labeling) and in vivo (zebrafish model), migration (Boyden chamber assay), invasiveness (Matrigel invasion assay), self-renewal (spherogenesis assay), and CLIC1 activity (electrophysiology recordings), as well as for the absence of off-target toxicity (effects on normal stem cells and toxicity for zebrafish and chick embryos). RESULTS: We identified Q48 and Q54 as two novel CLIC1 blockers, characterized by higher antiproliferative potency than metformin in vitro, in both GSC 2D cultures and 3D spheroids. Q48 and Q54 also impaired GSC self-renewal, migration and invasion, and displayed low systemic in vivo toxicity. Q54 reduced in vivo proliferation of GSCs xenotransplanted in zebrafish hindbrain. Target specificity was confirmed by recombinant CLIC1 binding experiments using microscale thermophoresis approach. Finally, we characterized GSCs from GBMs spontaneously expressing low CLIC1 protein, demonstrating their ability to grow in vivo and to retain stem-like phenotype and functional features in vitro. In these GSCs, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC1-expressing tumors. However, in 3D cultures, metformin and Q48 (but not Q54) inhibited proliferation, which was dependent on the inhibition dihydrofolate reductase activity. CONCLUSIONS: These data highlight that, while CLIC1 is dispensable for the development of a subset of glioblastomas, it acts as a booster of proliferation in the majority of these tumors and its functional expression is required for biguanide antitumor class-effects. In particular, the biguanide-based derivatives Q48 and Q54, represent the leads to develop novel compounds endowed with better pharmacological profiles than metformin, to act as CLIC1-blockers for the treatment of CLIC1-expressing glioblastomas, in a precision medicine approach.

ARN25068, a versatile starting point towards triple GSK-3ß/FYN/DYRK1A inhibitors to tackle tau-related neurological disorders.

The human kinome plays a crucial role in several pathways. Its dysregulation has been linked to diverse central nervous system (CNS)-related disorders with a drastic impact on the aging population. Among them, tauopathies, such as Alzheimer's Disease (AD) and Frontotemporal Lobar degeneration (FTLD-tau), are neurodegenerative disorders pathologically defined by the presence of hyperphosphorylated tau-positive intracellular inclusions known as neurofibrillary tangles (NFTs). Compelling evidence has reported the great potential of the simultaneous modulation of multiple protein kinases (PKs) involved in abnormal tau phosphorylation through a concerted pharmacological approach to achieve a superior therapeutic effect relative to classic "one target, one drug" approaches. Here, we report on the identification and characterization of ARN25068 (4), a low nanomolar and well-balanced dual GSK-3ß and FYN inhibitor, which also shows inhibitory activity against DYRK1A, an emerging target in AD and tauopathies. Computational and X-Ray studies highlight compound 4's typical H-bonding pattern of ATP-competitive inhibitors at the binding sites of all three PKs. In a tau phosphorylation assay on Tau0N4R-TM-tGFP U2OS cell line, 4 reduces the extent of tau phosphorylation, promoting tau-stabilized microtubule bundles. In conclusion, this compound emerges as a promising prototype for further SAR explorations to develop potent and well-balanced triple GSK-3ß/FYN/DYRK1A inhibitors to tackle tau hyperphosphorylation.

Design, Synthesis, Dynamic Docking, Biochemical Characterization, and in Vivo Pharmacokinetics Studies of Novel Topoisomerase II Poisons with Promising Antiproliferative Activity.

We previously reported a first set of hybrid topoisomerase II (topoII) poisons whose chemical core merges key pharmacophoric elements of etoposide and merbarone, which are two well-known topoII blockers. Here, we report on the expansion of this hybrid molecular scaffold and present 16 more hybrid derivatives that have been designed, synthesized, and characterized for their ability to block topoII and for their overall drug-like profile. Some of these compounds act as topoII poison and exhibit good solubility, metabolic (microsomal) stability, and promising cytotoxicity in three cancer cell lines (DU145, HeLa, A549). Compound 3f (ARN24139) is the most promising drug-like candidate, with a good pharmacokinetics profile in vivo. Our results indicate that this hybrid new chemical class of topoII poisons deserves further exploration and that 3f is a favorable lead candidate as a topoII poison, meriting future studies to test its efficacy in in vivo tumor models.

Synthetic Lethality in Pancreatic Cancer: Discovery of a New RAD51-BRCA2 Small Molecule Disruptor That Inhibits Homologous Recombination and Synergizes with Olaparib.

Synthetic lethality is an innovative framework for discovering novel anticancer drug candidates. One example is the use of PARP inhibitors (PARPi) in oncology patients with BRCA mutations. Here, we exploit a new paradigm based on the possibility of triggering synthetic lethality using only small organic molecules (dubbed "fully small-molecule-induced synthetic lethality"). We exploited this paradigm to target pancreatic cancer, one of the major unmet needs in oncology. We discovered a dihydroquinolone pyrazoline-based molecule (35d) that disrupts the RAD51-BRCA2 protein-protein interaction, thus mimicking the effect of BRCA2 mutation. 35d inhibits the homologous recombination in a human pancreatic adenocarcinoma cell line. In addition, it synergizes with olaparib (a PARPi) to trigger synthetic lethality. This strategy aims to widen the use of PARPi in BRCA-competent and olaparib-resistant cancers, making fully small-molecule-induced synthetic lethality an innovative approach toward unmet oncological needs.

Investigating Drug-Target Residence Time in Kinases through Enhanced Sampling Simulations.

It is widely accepted that drug-target association and dissociation rates directly affect drug efficacy and safety. To rationally optimize drug binding kinetics, one must know the atomic arrangement of the protein-ligand complex during the binding/unbinding process in order to detect stable and metastable states. Whereas experimental approaches can determine kinetic constants with fairly good accuracy, computational approaches based on molecular dynamics (MD) simulations can deliver the atomistic details of the unbinding process. Furthermore, they can also be utilized prospectively to predict residence time (i.e., the inverse of unbinding kinetics constant, koff) with an acceptable level of accuracy. Here, we report a novel method based on adiabatic bias MD with an electrostatics-like collective variable (dubbed elABMD) for sampling protein-ligand dissociation events in two kinases. elABMD correctly ranked a ligand series on glucokinase, in agreement with experimental data and previous calculations. Subsequently, we applied the new method prospectively to a congeneric series of GSK-3ß inhibitors. For this series, new crystal structures were generated and the residence time was experimentally measured with surface plasmon resonance (SPR). There was good agreement between computational predictions and experimental measures, suggesting that elABMD is an innovative and efficient tool for calculating residence times.

Rad51/BRCA2 disruptors inhibit homologous recombination and synergize with olaparib in pancreatic cancer cells.

Olaparib is a PARP inhibitor (PARPi). For patients bearing BRCA1 or BRCA2 mutations, olaparib is approved to treat ovarian cancer and in clinical trials to treat breast and pancreatic cancers. In BRCA2-defective patients, PARPi inhibits DNA single-strand break repair, while BRCA2 mutations hamper double-strand break repair. Recently, we identified a series of triazole derivatives that mimic BRCA2 mutations by disrupting the Rad51-BRCA2 interaction and thus double-strand break repair. Here, we have computationally designed, synthesized, and tested over 40 novel derivatives. Additionally, we designed and conducted novel biological assays to characterize how they disrupt the Rad51-BRCA2 interaction and inhibit double-strand break repair. These compounds synergized with olaparib to target pancreatic cancer cells with functional BRCA2. This supports the idea that small organic molecules can mimic genetic mutations to improve the profile of anticancer drugs for precision medicine. Moreover, this paradigm could be exploited in other genetic pathways to discover innovative anticancer targets and drug candidates.

Pharmacophore Identification and Scaffold Exploration to Discover Novel, Potent, and Chemically Stable Inhibitors of Acid Ceramidase in Melanoma Cells.

Acid ceramidase (AC) hydrolyzes ceramides, which are central lipid messengers for metabolism and signaling of sphingolipids. A growing body of evidence links deregulation of sphingolipids to several diseases, including cancer. Indeed, AC expression is abnormally high in melanoma cells. AC inhibition may thus be key to treating malignant melanoma. Here, we have used a systematic scaffold exploration to design a general pharmacophore for AC inhibition. This pharmacophore comprises a 6 + 5 fused ring heterocycle linked to an aliphatic substituent via a urea moiety. We have thus identified the novel benzimidazole derivatives 10, 21, 27, and 30, which are highly potent AC inhibitors. Their chemical and metabolic stabilities are comparable or superior to those of previously reported AC inhibitors. Moreover, they are potent against endogenous AC in intact melanoma cells. These novel inhibitors merit further characterization and can serve as a promising starting point for the discovery of new antimelanoma therapeutics.

Lid domain plasticity and lipid flexibility modulate enzyme specificity in human monoacylglycerol lipase.

Human monoacylglycerol lipase (MAGL) is a membrane-interacting enzyme that generates pro-inflammatory signaling molecules. For this reason, MAGL inhibition is a promising strategy to treat pain, cancer, and neuroinflammatory diseases. MAGL can hydrolyze monoacylglycerols bearing an acyl chain of different lengths and degrees of unsaturation, cleaving primarily the endocannabinoid 2-arachidonoylglycerol. Importantly, the enzymatic binding site of MAGL is confined by a 75-amino-acid-long, flexible cap domain, named 'lid domain', which is structurally similar to that found in several other lipases. However, it is unclear how lid domain plasticity affects catalysis in MAGL. By integrating extensive molecular dynamics simulations and free-energy calculations with mutagenesis and kinetic experiments, we here define a lid-domain-mediated mechanism for substrate selection and binding in MAGL catalysis. In particular, we clarify the key role of Phe159 and Ile179, two conserved residues within the lid domain, in regulating substrate specificity in MAGL. We conclude by proposing that other structurally related lipases may share this lid-domain-mediated mechanism for substrate specificity.

Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets.

The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the "membrane-access" and the "acyl chain-binding" pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH's mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.

NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells.

α-Synuclein (aS) aggregation has been amply investigated for its involvement in Parkinson's disease because its amyloid fibrils are the main constituent of Lewy bodies, one of the hallmarks of the disease. aS aggregation was studied here in vitro and in cellular models to correlate aggregation products with toxicity mechanisms. Independent results published elsewhere suggested that aS overexpression and/or aggregation may impair cellular metabolism and cause mitochondrial damage. In this context, we report the characterization of changes in NADH fluorescence properties in vitro and in human embryonic kidney 293 cells upon aS aggregation. The application of the phasor approach to study NADH fluorescence lifetime and emission allowed us to identify changes that correlate with aS aggregation. In particular, the fraction of bound NADH, characterized by longer lifetimes in comparison to free NADH, is increased, and the maximum of the NADH emission is shifted toward shorter wavelengths in the presence of aggregating aS both in vitro and in cells. These data suggest that NADH binds to aggregated aS. NMR experiments in vitro substantiate such binding, which occurs during aggregation. NADH fluorescence is thus useful to detect aS aggregation and by extension the associated oxidative stress.