Late-stage Functionalization for Improving Drug-like Molecular Properties.

The development of late-stage functionalization (LSF) methodologies, particularly C-H functionalization, has revolutionized the field of organic synthesis. Over the past decade, medicinal chemists have begun to implement LSF strategies into their drug discovery programs, allowing for the drug discovery process to become more efficient. Most reported applications of late-stage C-H functionalization of drugs and drug-like molecules have been to rapidly diversify screening libraries to explore structure-activity relationships. However, there has been a growing trend toward the use of LSF methodologies as an efficient tool for improving drug-like molecular properties of promising drug candidates. In this review, we have comprehensively reviewed recent progress in this emerging area. Particular emphasis is placed on case studies where multiple LSF techniques were implemented to generate a library of novel analogues with improved drug-like properties. We have critically analyzed the current scope of LSF strategies to improve drug-like properties and commented on how we believe LSF can transform drug discovery in the future. Overall, we aim to provide a comprehensive survey of LSF techniques as tools for efficiently improving drug-like molecular properties, anticipating its continued uptake in drug discovery programs.

Strategies for targeting the P2Y12 receptor in the central nervous system.

The purinergic 2Y type 12 receptor (P2Y12R) is a well-known biological target for anti-thrombotic drugs due to its role in platelet aggregation and blood clotting. While the importance of the P2Y12R in the periphery has been known for decades, much less is known about its expression and roles in the central nervous system (CNS), where it is expressed exclusively on microglia - the first responders to brain insults and neurodegeneration. Several seminal studies have shown that P2Y12 is a robust, translatable biomarker for anti-inflammatory and neuroprotective microglial phenotypes in models of degenerative diseases such as multiple sclerosis and Alzheimer's disease. An enduring problem for studying this receptor in vivo, however, is the lack of selective, high-affinity small molecule ligands that can bypass the blood-brain barrier and accumulate in the CNS. In this Digest, we discuss previous attempts by researchers to target the P2Y12R in the CNS and opine on strategies that may be employed to design and assess the suitability of novel P2Y12 ligands for this purpose going forward.

Understanding In Vitro Pathways to Drug Discovery for TDP-43 Proteinopathies.

The use of cellular models is a common means to investigate the potency of therapeutics in pre-clinical drug discovery. However, there is currently no consensus on which model most accurately replicates key aspects of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) pathology, such as accumulation of insoluble, cytoplasmic transactive response DNA-binding protein (TDP-43) and the formation of insoluble stress granules. Given this, we characterised two TDP-43 proteinopathy cellular models that were based on different aetiologies of disease. The first was a sodium arsenite-induced chronic oxidative stress model and the second expressed a disease-relevant TDP-43 mutation (TDP-43 M337V). The sodium arsenite model displayed most aspects of TDP-43, stress granule and ubiquitin pathology seen in human ALS/FTD donor tissue, whereas the mutant cell line only modelled some aspects. When these two cellular models were exposed to small molecule chemical probes, different effects were observed across the two models. For example, a previously disclosed sulfonamide compound decreased cytoplasmic TDP-43 and increased soluble levels of stress granule marker TIA-1 in the cellular stress model without impacting these levels in the mutant cell line. This study highlights the challenges of using cellular models in lead development during drug discovery for ALS and FTD and reinforces the need to perform assessments of novel therapeutics across a variety of cell lines and aetiological models.

Methyl-compounds driven benthic carbon cycling in the sulfate-reducing sediments of South China Sea.

Methane is a potent greenhouse gas; methane production and consumption within seafloor sediments has generated intense interest. Anaerobic oxidation of methane (AOM) and methanogenesis (MOG) primarily occur at the depth of the sulfate-methane transition zone or underlying sediment respectively. Methanogenesis can also occur in the sulfate-reducing sediments through the utilization of non-competitive methylated compounds; however, the occurrence and importance of this process are not fully understood. Here, we combined a variety of data, including geochemical measurements, rate measurements and molecular analyses to demonstrate the presence of a cryptic methane cycle in sulfate-reducing sediments from the continental shelf of the northern South China Sea. The abundance of methanogenic substrates as well as the high MOG rates from methylated compounds indicated that methylotrophic methanogenesis was the dominant methanogenic pathway; this conclusion was further supported by the presence of the methylotrophic genus Methanococcoides. High potential rates of AOM were observed in the sediments, indicating that methane produced in situ could be oxidized simultaneously by AOM, presumably by ANME-2a/b as indicated by 16S rRNA gene analysis. A significant correlation between the relative abundance of methanogens and methanotrophs was observed over sediment depth, indicating that methylotrophic methanogenesis could potentially fuel AOM in this environment. In addition, higher potential rates of AOM than sulfate reduction rates at in situ methane conditions were observed, making alternative electron acceptors important to support AOM in sulfate-reducing sediment. AOM rates were stimulated by the addition of Fe/Mn oxides, suggesting AOM could be partially coupled to metal oxide reduction. These results suggest that methyl-compounds driven methane production drives a cryptic methane cycling and fuels AOM coupled to the reduction of sulfate and other electron acceptors.

DYRK1A Negatively Regulates CDK5-SOX2 Pathway and Self-Renewal of Glioblastoma Stem Cells.

Glioblastoma display vast cellular heterogeneity, with glioblastoma stem cells (GSCs) at the apex. The critical role of GSCs in tumour growth and resistance to therapy highlights the need to delineate mechanisms that control stemness and differentiation potential of GSC. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) regulates neural progenitor cell differentiation, but its role in cancer stem cell differentiation is largely unknown. Herein, we demonstrate that DYRK1A kinase is crucial for the differentiation commitment of glioblastoma stem cells. DYRK1A inhibition insulates the self-renewing population of GSCs from potent differentiation-inducing signals. Mechanistically, we show that DYRK1A promotes differentiation and limits stemness acquisition via deactivation of CDK5, an unconventional kinase recently described as an oncogene. DYRK1A-dependent inactivation of CDK5 results in decreased expression of the stemness gene SOX2 and promotes the commitment of GSC to differentiate. Our investigations of the novel DYRK1A-CDK5-SOX2 pathway provide further insights into the mechanisms underlying glioblastoma stem cell maintenance.

Design, synthesis and evaluation of carbamate-linked uridyl-based inhibitors of human ST6Gal I.

Sialic acid at the terminus of cell surface glycoconjugates is a critical element in cell-cell recognition, receptor binding and immune responses. Sialyltransferases (ST), the enzymes responsible for the biosynthesis of sialylated glycans are highly upregulated in cancer and the resulting hypersialylation of the tumour cell surface correlates strongly with tumour growth, metastasis and drug resistance. Inhibitors of human STs, in particular human ST6Gal I, are thus expected to be valuable chemical tools for the discovery of novel anticancer drugs. Herein, we report on the computationally-guided design and development of uridine-based inhibitors that replace the charged phosphodiester linker of known ST inhibitors with a neutral carbamate to improve pharmacokinetic properties and synthetic accessibility. A series of 24 carbamate-linked uridyl-based compounds were synthesised by coupling aryl and hetaryl α-hydroxyphosphonates with a 5'-amino-5'-deoxyuridine fragment. The inhibitory activities of the newly synthesised compounds against recombinant human ST6Gal I were determined using a luminescent microplate assay, and five promising inhibitors with Ki's ranging from 1 to 20 µM were identified. These results show that carbamate-linked uridyl-based compounds are a potential new class of readily accessible, non-cytotoxic ST inhibitors to be further explored.

Microbial metabolism of methanol and methylamine in the Gulf of Mexico: insight into marine carbon and nitrogen cycling.

One carbon (C1) metabolism plays an important role in marine carbon cycling but the dynamics and modes of C1 transformations are not fully understood. We made contemporaneous measurements of methylamine and methanol metabolism to elucidate the role of C1 compounds as sources of carbon, energy and nitrogen. Methanol and methylamine were predominantly used as an energy source in offshore waters (oxidation rate constant: kmethanol : 0.02-0.10 day-1 ; kmethylamine : 0.01-0.18 day-1 ), but were also important sources of biomass carbon in coastal waters (assimilation rate constant: kmethanol : 0.04-0.10 day-1 ; kmethylamine : 0.01-0.05 day-1 ). The relative extent of assimilation versus oxidation for these substrates correlated positively with chlorophyll, nutrients and heterotrophic bacterial production. Methanol oxidation and assimilation were stimulated significantly by nutrient addition. In contrast, methylamine metabolism was inhibited by ammonium or nitrate, suggesting that methylamine served as a nitrogen source. A preliminary metagenomic survey revealed a diverse population of putative C1-utilizing microorganisms. These results show that the remineralization of methylamine could provide both C and N sources for microbes. Both methanol and methylamine contribute to microbial energetic and carbon substrate demands with a distinctly different signature in nearshore versus offshore environments.

Computer-aided design of human sialyltransferase inhibitors of hST8Sia III.

Sialyltransferase (ST) upregulation and the resultant hypersialylation of tumour cell surfaces is an established hallmark of many cancers including lung, breast, ovarian, pancreatic and prostate cancer. The role of ST enzymes in tumour cell growth and metastasis, as well as links to multi-drug resistance, has seen ST inhibition emerge as a target for potential antimetastatic cancer treatments. The most potent of these reported inhibitors are transition-state analogues. Although there are several examples of these in the literature, many have suspected poor pharmacokinetic properties and are not readily synthetically accessible. A proposed solution to these problems is the use of a neutral carbamate or 1,2,3-triazole linker instead of the more commonly used phosphodiester linker, and replacing the traditionally utilised cytidine nucleotide with uridine. Another issue in this area is the paucity of structural information of human ST enzymes. However, in late 2015 the structure of human ST8Sia III was reported (only the second human ST described so far), creating the opportunity for structure-based design of selective ST8 inhibitors for the first time. Herein, molecular docking and molecular dynamics simulations with the newly published crystal structure of hST8Sia III were performed for the first time with selected ST transition state analogues. Simulations showed that these compounds could participate in many of the key interactions common with the natural donor and acceptor substrates, and reveals some key insights into the synthesis of potentially selective ST inhibitors.

Computational characterisation of the interactions between human ST6Gal I and transition-state analogue inhibitors: insights for inhibitor design.

Human ß-galactoside α-2,6-sialyltransferase I (hST6Gal I) catalyses the synthesis of sialylated glycoconjugates involved in cell-cell interactions. Overexpression of hST6Gal I is observed in many different types of cancers, where it promotes metastasis through altered cell surface sialylation. A wide range of sialyltransferase (ST) inhibitors have been developed based on the natural donor, cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). Of these, analogues that are structurally similar to the transition state exhibit the highest inhibitory activity. In order to design inhibitors that are readily accessible synthetically and with favourable pharmacokinetic properties, an investigation of the replacement of the charged phosphodiester-linker, present in many ST inhibitors, with a potential neutral isostere such as a carbamate or a 1,2,3-triazole has been undertaken. To investigate this, molecular docking and molecular dynamics simulations were performed. These simulations provided an insight into the binding mode of previously reported phosphodiester-linked ST inhibitors and demonstrated that targeting the proposed sialyl acceptor site is a viable option for producing selective inhibitors. The potential for a carbamate- or triazole-linker as an isosteric replacement for the phosphodiester in transition-state analogue ST inhibitors was established using molecular docking. Molecular dynamics simulations of carbamate- and phosphodiester-linked compounds revealed that both classes exhibit consistent interactions with hST6Gal I. Overall, the results obtained from this study provide a rationale for synthetic and biological evaluation of triazole- and carbamate-linked transition-state analogue ST inhibitors as potential new antimetastatic agents.

Structure-based drug design for TSPO: Challenges and opportunities.

The translocator protein 18 kDa (TSPO) is an evolutionarily conserved mitochondrial transmembrane protein implicated in various neuropathologies and inflammatory conditions, making it a longstanding diagnostic and therapeutic target of interest. Despite the development of various classes of TSPO ligand chemotypes, and the elucidation of bacterial and non-human mammalian experimental structures, many unknowns exist surrounding its differential structural and functional features in health and disease. There are several limitations associated with currently used computational methodologies for modelling the native structure and ligand-binding behaviour of this enigmatic protein. In this perspective, we provide a critical analysis of the developments in the uses of these methods, outlining their uses, inherent limitations, and continuing challenges. We offer suggestions of unexplored opportunities that exist in the use of computational methodologies which offer promise for enhancing our understanding of the TSPO.

Synthesis of α-Hydroxy-1,2,3-Triazole-linked Sialyltransferase Inhibitors and Evaluation of Selectivity Towards ST3GAL1, ST6GAL1 and ST8SIA2.

Tumour-derived sialoglycans, bearing the charged nonulosonic sugar sialic acid at their termini, play a critical role in tumour cell adhesion and invasion, as well as evading cell death and immune surveillance. Sialyltransferases (ST), the enzymes responsible for the biosynthesis of sialylated glycans, are highly upregulated in cancer, with tumour hypersialylation strongly correlated with tumour growth, metastasis and drug resistance. As a result, desialylation of the tumour cell surface using either targeted delivery of a pan-ST inhibitor (or sialidase) or systemic delivery of a non-toxic selective ST inhibitors are being pursued as potential new anti-metastatic strategies against multiple cancers including pancreatic, ovarian, breast, melanoma and lung cancer. Herein, we have employed molecular modelling to give insights into the selectivity observed in a series of selective ST inhibitors that incorporate a uridyl ring in place of the cytidine of the natural donor (CMP-Neu5Ac) and replace the charged phosphodiester linker of classical ST inhibitors with a neutral α-hydroxy-1,2,3-triazole linker. The inhibitory activities of the nascent compounds were determined against recombinant human ST enzymes (ST3GAL1, ST6GAL1, ST8SIA2) showing promising activity and selectivity towards specific ST sub-types. Our ST inhibitors are non-toxic and show improved synthetic accessibility and drug-likeness compared to earlier nucleoside-based ST inhibitors.

Kinesin Regulation in the Proximal Axon is Essential for Dendrite-selective Transport.

Neurons are polarized and typically extend multiple dendrites and one axon. To maintain polarity, vesicles carrying dendritic proteins are arrested upon entering the axon. To determine whether kinesin regulation is required for terminating anterograde axonal transport, we overexpressed the dendrite-selective kinesin KIF13A. This caused mistargeting of dendrite-selective vesicles to the axon and a loss of dendritic polarity. Polarity was not disrupted if the kinase MARK2/Par1b was coexpressed. MARK2/Par1b is concentrated in the proximal axon, where it maintains dendritic polarity-likely by phosphorylating S1371 of KIF13A, which lies in a canonical 14-3-3 binding motif. We probed for interactions of KIF13A with 14-3-3 isoforms and found that 14-3-3ß and 14-3-3ζ bound KIF13A. Disruption of MARK2 or 14-3-3 activity by small molecule inhibitors caused a loss of dendritic polarity. These data show that kinesin regulation is integral for dendrite-selective transport. We propose a new model in which KIF13A that moves dendrite-selective vesicles in the proximal axon is phosphorylated by MARK2. Phosphorylated KIF13A is then recognized by 14-3-3, which causes dissociation of KIF13A from the vesicle and termination of transport. These findings define a new paradigm for the regulation of vesicle transport by localized kinesin tail phosphorylation, to restrict dendrite-selective vesicles from entering the axon.

Polarized transport requires AP-1-mediated recruitment of KIF13A and KIF13B at the trans-Golgi.

Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family members KIF13A and KIF13B are thought to mediate dendrite-selective transport, but the mechanism by which they are recruited to polarized vesicles and the differences in the specific trafficking role of each KIF13 have not been defined. We performed live-cell imaging in cultured hippocampal neurons and found that KIF13A is a dedicated dendrite-selective kinesin. KIF13B confers two different transport modes, dendrite- and axon-selective transport. Both KIF13s are maintained at the trans-Golgi network by interactions with the heterotetrameric adaptor protein complex AP-1. Interference with KIF13 binding to AP-1 resulted in disruptions to both dendrite- and axon-selective trafficking. We propose that AP-1 is the molecular link between the sorting of polarized cargoes into vesicles and the recruitment of kinesins that confer polarized transport.

Fragment-based drug discovery for disorders of the central nervous system: designing better drugs piece by piece.

Fragment-based drug discovery (FBDD) has emerged as a powerful strategy to confront the challenges faced by conventional drug development approaches, particularly in the context of central nervous system (CNS) disorders. FBDD involves the screening of libraries that comprise thousands of small molecular fragments, each no greater than 300 Da in size. Unlike the generally larger molecules from high-throughput screening that limit customisation, fragments offer a more strategic starting point. These fragments are inherently compact, providing a strong foundation with good binding affinity for the development of drug candidates. The minimal elaboration required to transition the hit into a drug-like molecule is not only accelerated, but also it allows for precise modifications to enhance both their activity and pharmacokinetic properties. This shift towards a fragment-centric approach has seen commercial success and holds considerable promise in the continued streamlining of the drug discovery and development process. In this review, we highlight how FBDD can be integrated into the CNS drug discovery process to enhance the exploration of a target. Furthermore, we provide recent examples where FBDD has been an integral component in CNS drug discovery programs, enabling the improvement of pharmacokinetic properties that have previously proven challenging. The FBDD optimisation process provides a systematic approach to explore this vast chemical space, facilitating the discovery and design of compounds piece by piece that are capable of modulating crucial CNS targets.

An update on late-stage functionalization in today's drug discovery.

INTRODUCTION: Late-stage functionalization (LSF) allows for the introduction of new chemical groups toward the end of a synthetic sequence, which means new molecules can be rapidly accessed without laborious de novo chemical synthesis. Over the last decade, medicinal chemists have begun to implement LSF strategies into their drug discovery programs, affording benefits such as efficient access to diverse libraries to explore structure-activity relationships and the improvement of physicochemical and pharmacokinetic properties. AREAS COVERED: An overview of the key advancements in LSF methodology development from 2019 to 2022 and their applicability to drug discovery is provided. In addition, several examples from both academia and industry where LSF methodologies have been applied by medicinal chemists to their drug discovery programs are presented. EXPERT OPINION: Utilization of LSF by medicinal chemists is on the rise, both in academia and in industry. The maturation of the LSF field to produce methodologies bearing increased regioselectivity, scope, and functional group tolerance is envisaged to narrow the gap between methodology development and medicinal chemistry research. The authors predict that the sheer versatility of these techniques in facilitating challenging chemical transformations of bioactive molecules will continue to increase the efficiency of the drug discovery process.

Synthesis and Preclinical Evaluation of Fluorinated 5-Azaindoles as CB2 PET Radioligands.

Several classes of cannabinoid receptor type 2 radioligands have been evaluated for imaging of neuroinflammation, with successful clinical translation yet to take place. Here we describe the synthesis of fluorinated 5-azaindoles and pharmacological characterization and in vivo evaluation of 18F-radiolabeled analogues. [18F]2 (hCB2 Ki = 96.5 nM) and [18F]9 (hCB2 Ki = 7.7 nM) were prepared using Cu-mediated 18F-fluorination with non-decay-corrected radiochemical yields of 15 ± 6% and 18 ± 2% over 85 and 80 min, respectively, with high radiochemical purities (>97%) and molar activities (140-416 GBq/µmol). In PET imaging studies in rats, both [18F]2 and [18F]9 demonstrated specific binding in CB2-rich spleen after pretreatment with CB2-specific GW405833. Moreover, [18F]9 exhibited higher brain uptake at later time points in a murine model of neuroinflammation compared with a healthy control group. The results suggest further evaluation of azaindole based CB2 radioligands is warranted in other neuroinflammation models.

Visualizing Vesicle-Bound Kinesins in Cultured Hippocampal Neurons.

Eukaryotic cells use microtubule-based vesicle transport to exchange molecules between compartments. Kinesin family members mediate all microtubule plus end-directed vesicle transport. Of the 45 kinesins expressed in humans, some 20 mediate microtubule plus-end directed vesicle transport. Here we describe a technique to visualize vesicle-bound kinesins in cultured hippocampal neurons. The method involves the expression of the vesicle-binding tail domain while minimizing the cytoplasmic pool. Using this approach drastically improves vesicle labeling compared to full-length kinesins. This tool is useful for systematically comparing the localization of different kinesins in the same cell type and for identifying cargo proteins that reside in vesicles moved by a specific kinesin family member. While we describe the assay in cultured hippocampal neurons, we expect it to be easily transferable to other eukaryotic cell types.

KAP is the neuronal organelle adaptor for Kinesin-2 KIF3AB and KIF3AC.

Kinesin-driven organelle transport is crucial for neuron development and maintenance, yet the mechanisms by which kinesins specifically bind their organelle cargoes remain undefined. In contrast to other transport kinesins, the neuronal function and specific organelle adaptors of heterodimeric Kinesin-2 family members KIF3AB and KIF3AC remain unknown. We developed a novel microscopy-based assay to define protein-protein interactions in intact neurons. The experiments found that both KIF3AB and KIF3AC bind kinesin-associated protein (KAP). These interactions are mediated by the distal C-terminal tail regions and not the coiled-coil domain. We used live-cell imaging in cultured hippocampal neurons to define the localization and trafficking parameters of KIF3AB and KIF3AC organelle populations. We discovered that KIF3AB/KAP and KIF3AC/KAP bind the same organelle populations and defined their transport parameters in axons and dendrites. The results also show that ∼12% of KIF3 organelles contain the RNA-binding protein adenomatous polyposis coli. These data point toward a model in which KIF3AB and KIF3AC use KAP as their neuronal organelle adaptor and that these kinesins mediate transport of a range of organelles.

Recent Scaffold Hopping Applications in Central Nervous System Drug Discovery.

The concept of bioisosterism and the implementation of bioisosteric replacement is fundamental to medicinal chemistry. The exploration of bioisosteres is often used to probe key structural features of candidate pharmacophores and enhance pharmacokinetic properties. As the understanding of bioisosterism has evolved, capabilities to undertake more ambitious bioisosteric replacements have emerged. Scaffold hopping is a broadly used term in the literature referring to a variety of different bioisosteric replacement strategies, ranging from simple heterocyclic replacements to topological structural overhauls. In this work, we have highlighted recent applications of scaffold hopping in the central nervous system drug discovery space. While we have highlighted the benefits of using scaffold hopping approaches in central nervous system drug discovery, these are also widely applicable to other medicinal chemistry fields. We also recommend a shift toward the use of more refined and meaningful terminology within the realm of scaffold hopping.