Exploring the Phenomena of Compound Aggregation and Nano-entities for Drug Discovery

This thesis explores the world of nano-entities. Nano-entities is a name given by our laboratory to describe the natural phenomenon that drugs can self-assemble in a multitude of particle sizes when placed in an aqueous environment. Surprisingly, this phenomenon remains relatively unexplored despite the widespread impact it has throughout the drug discovery and development processes. Given this, I became very interested in nano-entities and launched detailed investigations as described herein. These investigations indeed found that drugs can adopt some amazing free-state behaviors –which have been historically overlooked by the pharmaceutical industry. Perhaps one reason that nano-entities has been overlooked is the widely held assumption that compounds can only exist as lone molecules or as precipitates when placed into aqueous solution. However, recent studies are shedding light onto a non-negligible phenomenon where compounds can naturally adopt into a multi-state equilibrium. The principle states of this equilibrium can be classified as, (1) soluble-lone molecules, (2) soluble nano-entities, and (3) solid precipitates. Here, we focus on the nano-entity state. We and others noticed that many small-molecules can adopt a wide range of aggregate sizes and type, but to date, the size and types of these self-associated particles remain largely unexplored. This is due in part to the fact that each compound has its own fingerprints which are highly dependent on its environment such as, buffer, pH, etc. Also, our limited knowledge of these nano-entities is more complicated by the fact that there are only a few detection techniques to explore the existence of nano-entities and reveal their full range of sizes. As a result, the detection of nano-entities remains elusive. In my thesis, I wish to welcome you to this small world of drug self-assemblies. I prepared a detailed introduction along with the “hypothesis and objectives” for my investigations. I then provide three chapters which show how we observed these fascinating entities using specialized techniques that include Nuclear Magnetic Resonance (NMR), Transmission Electron Microscopy (TEM), Dynamic Light scattering (DLS) and Confocal Laser Scanning Microscopy (CLSM) assays. I also show that nano-entities can enter and accumulate within cells. The fourth chapter describes my discovery that nano-entities can be recognized by anti-bodies and induce an immune response. This is very interesting because it would be perhaps be first time that we correlate this drug property with drug side-effects. Given that “Knowledge is Power”, these findings may be used as tools to better observe and understand the phenomenon then perhaps identify potential solutions. La thèse explore le monde fascinant des nano-entités, nom donné par notre laboratoire décrivant le phénomène naturel selon lequel les médicaments peuvent réaliser en s’auto-assemblant en une multitude de particules de tailles différentes lorsqu’ils sont placés dans un environnement aqueux. Il est surprenant de constater que ce phénomène reste relativement peu exploré malgré l’impact grandissant qu’il a sur la découverte et le développement des médicaments. C’est pourquoi j’ai commencé à m’intéresser de près aux nanoparticules. Ces recherches ont révélé que les molécules peuvent adopter de surprenants comportements à l’état libre quand elles sont placées en solution. Cela peut être expliqué par cette hypothèse largement répandue, que les composés ne peuvent se solubiliser qu’en solution aqueuse, sous forme de molécules seules ou des précipités. Cependant, des études récentes mettent en lumière un phénomène non négligeable dans lesquels les composés peuvent adopter naturellement un équilibre entre plusieurs états. Ces principaux états sont classés comme suit : (1) des molécules solubles et uniques, (2) des nano-entités auto-assemblées solubles, et (3) des précipités. Ici, nous nous concentrons sur l’état des nano-entités. Nous avons remarqué, avec d’autres laboratoires, que plusieurs médicaments sous formes de petites molécules peuvent s’assembler pour former différents agrégats, dont les tailles et les types restent toujours peu caractérisés. Cela est dû en partie à la singularité de chaque composé dépendant fortement de leur environnement (tampon, pH, température, etc). De plus, les connaissances sur ces nano-entités sont limitées puisqu’il n’existe que quelques techniques de détection, nous permettant de déterminer leur existence, leur type et de caractériser leurs tailles, rendant le suivi des nano-entités insaisissable. Dans ma thèse, je souhaiterais donc vous inviter à découvrir ce petit monde des médicaments auto-assemblés. J’ai préparé une introduction détaillée suivie d’une partie « hypothèse et objectifs » décrivant mes investigations. Ces parties sont suivies de trois chapitres dans lesquels sont montrés comment mon laboratoire et moi avons observés ces fascinantes entités utilisant des techniques spécialisées incluant la dilution par RMN, la RMN-T2-CPMG, les essais de détergence par RMN, MET, DLS, CLSM. Je montre également que ces nano-entités peuvent pénétrer et s’accumuler dans les cellules. Le quatrième chapitre décrit ma découverte de nano-entités pouvant être reconnues par des anticorps, induisant une réponse immunitaire. Cette découverte nous permettrait alors d’établir une corrélation entre cette propriété des nano-entités et les effets secondaires des médicaments. Sachant que « la connaissance est le pouvoir », ces résultats pourront être utilisés comme outils afin d’observer et comprendre ces phénomènes et peut-être identifier des solutions potentielles.</br

SQUAD: Combining Sketching and Sampling Is Better than Either for Per-item Quantile Estimation

Latency quantiles measurements are essential as they often capture the user's utility. For example, if a video connection has high tail latency, the perceived quality will suffer, even if the average and median latencies are low. In this work, we consider the problem of approximating the per-item quantiles. Elements in our stream are (ID, latency) tuples, and we wish to track the latency quantiles for each ID. Existing quantile sketches are designed for a single number stream (e.g., containing just the latency). While one could allocate a separate sketch instance for each ID, this may require an infeasible amount of memory. Instead, we consider tracking the quantiles for the heavy hitters (most frequent items), which are often considered particularly important, without knowing them beforehand. We first present a simple sampling algorithm that serves as a benchmark. Then, we design an algorithm that augments a quantile sketch within each entry of a heavy hitter algorithm, resulting in similar space complexity but with a deterministic error guarantee. Finally, we present SQUAD, a method that combines sampling and sketching while improving the asymptotic space complexity. Intuitively, SQUAD uses a background sampling process to capture the behaviour of the latencies of an item before it is allocated with a sketch, thereby allowing us to use fewer samples and sketches. Our solutions are rigorously analyzed, and we demonstrate the superiority of our approach using extensive simulations

Revealing dye and dye-drug aggregation into nano-entities using NMR

It is becoming increasingly apparent that small molecules can self-assemble into a wide-range of nano-entities in solution that have intriguing properties. The recently introduced NMR aggregation assay is playing an important role in revealing these nano-entities. Here, we employ the NMR aggregation assay to expose the self-aggregation tendencies of dyes in solution. This dilution-based assay demonstrates that some dyes can exist as single-molecule entities whereas others can adopt aggregates of distinct sizes. Interestingly, dyes with highly related chemical structures can adopt largely different sized aggregates - demonstrating the existence of structure-nanoentity relationships – which suggests that they can assume and/or be designed to have distinct properties. One property was evaluated where the drug Quetiapine (Seroquel) was added to the dye Congo red which resulted in the absorption of the drug into the dye nano-entity. This showed a direct drug-dye interaction, and it demonstrated that dye aggregates can have influences on drug solution behaviors. The NMR method described in this study provides a practical and valuable tool to monitor dye aggregates and to better understand their associated properties (e.g. toxicity, off-target activity) and potential utility (e.g. drug encapsulation, drug delivery systems)

Revealing Drug Self-Associations into Nano-Entities

International audienceThe aqueous properties of the drugs Sorafenib, Lapatinib, Gefitinib, Fulvestrant, and Clofazimine were explored to monitor their tendency to self-associate. A combination of nuclear magnetic resonance, dynamics light scattering, and electron and confocal microscopies found that they tended to form large nano-entities having distinct types and sizes and were capable of entering cells. The combination of strategies employed serves to detect and reveal nano-entities along with their three-state equilibria and behaviors in buffers, media, and cells

Jumping from Fragment to Drug via Smart Scaffolds

A focused drug repurposing approach is described where an FDA-approved drug is rationally selected for biological testing based on structural similarities to a fragment compound found to bind a target protein by an NMR screen. The approach is demonstrated by first screening a curated fragment library using 19 F NMR to discover a quality binder to ACE2, the human receptor required for entry and infection by the SARS-CoV-2 virus. Based on this binder, a highly related scaffold was derived and used as a "smart scaffold" or template in a computer-aided finger-print search of a library of FDA-approved or marketed drugs. The most interesting structural match involved the drug vortioxetine which was then experimentally shown by NMR spectroscopy to bind directly to human ACE2. Also, an ELISA assay showed that the drug inhibits the interaction of human ACE2 to the SARS-CoV-2 receptor-binding-domain (RBD). Moreover, our cell-culture infectivity assay confirmed that vortioxetine is active against SARS-CoV-2 and inhibits viral replication. Thus, the use of "smart scaffolds" based on binders from fragment screens may have general utility for identifying candidates of FDA-approved or marketed drugs as a rapid repurposing strategy. Similar approaches can be envisioned for other fields involving small-molecule chemical applications

Robust Strategy for Hit-to-Lead Discovery: NMR for SAR

Establishing robust structure–activity relationships (SARs) is key to successful drug discovery campaigns, yet it often remains elusive due to screening and hit validation artifacts (false positives and false negatives), which frequently result in unproductive downstream expenditures of time and resources. To address this issue, we developed an integrative biophysics-driven strategy that expedites hit-to-lead discovery, mitigates false positives/negatives and common hit validation errors, and provides a robust approach to obtaining accurate binding and affinity measurements. The advantage of this method is that it vastly improves the clarity and reproducibility for affinity-driven SAR by monitoring and eliminating confounding factors. We demonstrate the ease at which high-quality micromolar binders can be generated from the initial millimolar fragment screening hits against an “undruggable” protein target, HRas