Profiling cells with DELs: Small molecule fingerprinting of cell surfaces.

DNA-encoded small molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, it has been used to identify ligands against targets that are soluble or overexpressed on cell surfaces. Here, we report applying cell-based selection methods to profile surfaces of mouse C2C12 myoblasts and myotube cells in an unbiased, target agnostic manner. A panel of on-DNA compounds were identified and confirmed for cell binding selectivity. We optimized the cell selection protocol and employed a novel data analysis method to identify cell selective ligands against a panel of human B and T lymphocytes. We discuss the generality of using this workflow for DNA encoded small molecule library selection and data analysis against different cell types, and the feasibility of applying this method to profile cell surfaces for biomarker and target identification.

High-Throughput Binder Confirmation (HTBC).

In this chapter, we describe a DEL "recipe" approach to hit confirmation using on-DNA cleavable linkers which can be followed by direct AS-MS evaluation and identification of binder(s) from the released small-molecule mixture. When coupled with automation, this platform creates opportunity for enabling an even higher-throughput binder confirmation (HTBC) platform to allow full interrogation of selections results. Two on-DNA cleavable strategies of releasing small molecules from DNA are reported herein.

DNA-Encoded Library Hit Confirmation: Bridging the Gap Between On-DNA and Off-DNA Chemistry.

DNA-encoded library (DEL) technology is a powerful platform for hit identification in academia and the pharmaceutical industry. When conducting off-DNA resynthesis hit confirmation after affinity selection, PCR/sequencing, and data analysis, one typically assumes a "one-to-one" relationship between the DNA tag and the chemical structure of the attached small-molecule it encodes. Because library synthesis often yields a mixture, this approximation increases the risk of overlooking positive discoveries and valuable information. To address this issue, we apply a library synthesis "recipe" strategy for on-DNA resynthesis using a cleavable linker, followed by direct affinity selection mass spectrometry (AS-MS) evaluation and identification of binder(s) from the released small-molecule mixture. We validate and showcase this approach employing the receptor-interacting-protein kinase 2 (RIP2) DEL campaign. We also designed and developed two cleavable linkers to enable this method, a photocleavable linker (nitrophenyl-based) and acid-labile linker (tetrahydropyranyl ether). The strategy provides an effective means of hit identification and rapid determination of key active component(s) of the mixture.

Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase.

Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.

DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4-ones as Highly Potent and Monoselective Receptor Interacting Protein 1 Kinase Inhibitors.

The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

Discovery, SAR, and X-ray Binding Mode Study of BCATm Inhibitors from a Novel DNA-Encoded Library.

As a potential target for obesity, human BCATm was screened against more than 14 billion DNA encoded compounds of distinct scaffolds followed by off-DNA synthesis and activity confirmation. As a consequence, several series of BCATm inhibitors were discovered. One representative compound (R)-3-((1-(5-bromothiophene-2-carbonyl)pyrrolidin-3-yl)oxy)-N-methyl-2'-(methylsulfonamido)-[1,1'-biphenyl]-4-carboxamide (15e) from a novel compound library synthesized via on-DNA Suzuki-Miyaura cross-coupling showed BCATm inhibitory activity with IC50 = 2.0 µM. A protein crystal structure of 15e revealed that it binds to BCATm within the catalytic site adjacent to the PLP cofactor. The identification of this novel inhibitor series plus the establishment of a BCATm protein structure provided a good starting point for future structure-based discovery of BCATm inhibitors.

Orienting attention to locations in mental representations.

Many cognitive processes depend on our ability to hold information in mind, often well beyond the offset of the original sensory input. The capacity of this visual short-term memory (VSTM) is limited to around three to four items. Recent research has demonstrated that the content of VSTM can be modulated by top-down attentional biases. This has been demonstrated using retrodictive spatial cues, termed "retro-cues," which orient subjects' attention to spatial locations within VSTM. In the present article, we tested whether the use of these cues is modulated by memory load and cue delay. There are a number of important conclusions: (1) Top-down biases can operate on very brief iconic traces as well as on older VSTM representations (Exp. 1). (2) When operating within capacity, subjects use the cue to prioritise where they initiate their memory search, rather than to discard uncued items (Exps. 2 and 3). (3) When capacity is exceeded, there is little benefit to top-down biasing relative to a neutral condition; however, unattended items are lost, with there being a substantial cost of invalid spatial cueing (Exp. 3). (4) These costs and benefits of orienting spatial attention differ across iconic memory and VSTM representations when VSTM capacity is exceeded (Exp. 4).

Biasing perception by spatial long-term memory.

Human perception is highly flexible and adaptive. Selective processing is tuned dynamically according to current task goals and expectations to optimize behavior. Arguably, the major source of our expectations about events yet to unfold is our past experience; however, the ability of long-term memories to bias early perceptual analysis has remained untested. We used a noninvasive method with high temporal resolution to record neural activity while human participants detected visual targets that appeared at remembered versus novel locations within naturalistic visual scenes. Upon viewing a familiar scene, spatial memories changed oscillatory brain activity in anticipation of the target location. Memory also enhanced neural activity during early stages of visual analysis of the target and improved behavioral performance. Both measures correlated with subsequent target-detection performance. We therefore demonstrated that memory can directly enhance perceptual functions in the human brain.

Differential engagement of brain regions within a 'core' network during scene construction.

Reliving past events and imagining potential future events engages a well-established "core" network of brain areas. How the brain constructs, or reconstructs, these experiences or scenes has been debated extensively in the literature, but remains poorly understood. Here we designed a novel task to investigate this (re)constructive process by directly exploring how naturalistic scenes are built up from their individual elements. We "slowed-down" the construction process through the use of auditorily presented phrases describing single scene elements in a serial manner. Participants were required to integrate these elements (ranging from three to six in number) together in their imagination to form a naturalistic scene. We identified three distinct sub-networks of brain areas, each with different fMRI BOLD response profiles, favouring specific points in the scene construction process. Areas including the hippocampus and retrosplenial cortex had a biphasic profile, activating when a single scene element was imagined and when 3 elements were combined together; regions including the intra-parietal sulcus and angular gyrus steadily increased activity from 1 to 3 elements; while activity in areas such as lateral prefrontal cortex was observed from the second element onwards. Activity in these sub-networks did not increase further when integrating more than three elements. Participants confirmed that three elements were sufficient to construct a coherent and vivid scene, and once this was achieved, the addition of further elements only involved maintenance or small changes to that established scene. This task offers a potentially useful tool for breaking down scene construction, a process that may be key to a range of cognitive functions such as episodic memory, future thinking and navigation.

Tracking the emergence of conceptual knowledge during human decision making.

Concepts lie at the very heart of intelligence, providing organizing principles with which to comprehend the world. Surprisingly little, however, is understood about how we acquire and deploy concepts. Here, we show that a functionally coupled circuit involving the hippocampus and ventromedial prefrontal cortex (vMPFC) underpins the emergence of conceptual knowledge and its effect on choice behavior. Critically, the hippocampus alone supported the efficient transfer of knowledge to a perceptually novel setting. These findings provide compelling evidence that the hippocampus supports conceptual learning through the networking of discrete memories and reveal the nature of its interaction with downstream valuation modules such as the vMPFC. Our study offers neurobiological insights into the remarkable capacity of humans to discover the conceptual structure of related experiences and use this knowledge to solve exacting decision problems.

Cortical midline involvement in autobiographical memory.

Recollecting autobiographical memories of personal past experiences is an integral part of our everyday lives and relies on a distributed set of brain regions. Their occurrence externally in the real world ('realness') and their self-relevance ('selfness') are two defining features of these autobiographical events. Distinguishing between personally experienced events and those that happened to other individuals, and between events that really occurred and those that were mere figments of the imagination, is clearly advantageous, yet the respective neural correlates remain unclear. Here we experimentally manipulated and dissociated realness and selfness during fMRI using a novel paradigm where participants recalled self (autobiographical) and non-self (from a movie or television news clips) events that were either real or previously imagined. Distinct sub-regions within dorsal and ventral medial prefrontal cortex, retrosplenial cortex and along the parieto-occipital sulcus preferentially coded for events (real or imagined) involving the self. By contrast, recollection of autobiographical events that really happened in the external world activated different areas within ventromedial prefrontal cortex and posterior cingulate cortex. In addition, recall of externally experienced real events (self or non-self) was associated with increased activity in areas of dorsomedial prefrontal cortex and posterior cingulate cortex. Taken together our results permitted a functional deconstruction of anterior (medial prefrontal) and posterior (retrosplenial cortex, posterior cingulate cortex, precuneus) cortical midline regions widely associated with autobiographical memory but whose roles have hitherto been poorly understood.

Orienting attention based on long-term memory experience.

Attentional orienting and memory are intrinsically bound, but their interaction has rarely been investigated. Here we introduce an experimental paradigm using naturalistic scenes to investigate how long-term memory can guide spatial attention and thereby enhance identification of events in the perceptual domain. In the task, stable memories of objects embedded within complex scenes guide spatial orienting. We compared the behavioral effects and neural systems of memory-guided orienting with those in a more traditional attention-orienting task in which transient spatial cues guide attention. Memory-guided attention operated within surprisingly short intervals and conferred reliable and sizeable advantages for detection of objects embedded in scenes. Event-related functional magnetic resonance imaging showed that memory-guided attention involves the interaction between brain areas participating in retrieval of memories for spatial context with the parietal-frontal network for visual spatial orienting. Activity in the hippocampus was specifically engaged in memory-guided spatial attention and correlated with the ensuing behavioral advantage.