Within the fragment-based drug discovery (FBDD) community, there is growing interest in developing non-planar, 3D screening fragments to complement the predominantly 2D structures that currently populate fragment libraries. Molecular three-dimensionality is typically evaluated using metrics such as the fraction of sp3-hybridized carbon atoms (FCsp3), plane of best fit (PBF) and/or principal moment of inertia (PMI). In this study, we review these 3D metrics and perform systematic cheminformatic analyses. Furthermore, we explore the use of alternative metrics that consider atom hybridization for the assessment of molecular shape. Although our analysis is grounded in a fragment-based context, the insights gained are broadly applicable across the drug discovery phases.

The purinergic receptor P2Y12 (P2Y12R) is a well-recognized target for anti-thrombotic agents. This receptor is also expressed in microglia, the main immune cells of the brain, where it modulates microglial activation states and inflammatory responses. To investigate P2Y12R-mediated actions in the central nervous system (CNS), developing novel brain-penetrant ligands and further in vitro studies on brain tissues are essential. A radiolabeled, easily accessible tool compound would significantly advance such drug discovery efforts. Herein, we describe the 3H-labeling of a non-nucleotide P2Y12R antagonist AZ12464237, and its in vitro binding properties to the receptor in membrane preparations from transfected cells, as well as on mouse brain tissues. The radioligand shows high affinity toward both the human and rat P2Y12R, with Kd values of 3.12 ± 0.70 nM (human) and 16.6 ± 3.40 nM (rat), as determined by saturation binding studies. The binding kinetics of [3H]AZ12464237 are rapid with a short target residence time (∼1 min). We further confirmed the selectivity of the radioligand by performing competitive displacement studies, in which reported P2Y12R ligands and other P2Y receptors ligands were tested for binding against [3H]AZ12464237. Additionally, the radioligand proved valuable for in vitro autoradiography studies on mouse brain tissues, although limited off-target binding was observed in P2Y12R knock-out mouse brain. This could be traced to glycogen synthase kinase 3 α. Considering the growing interest in P2Y12R as a biomarker for anti-inflammatory microglia, [3H]AZ12464237 represents a promising tool for in vitro studies, including screening assays aimed at identifying novel P2Y12R ligands for CNS applications.

The purinergic receptor P2Y12 (P2Y12R) has emerged as a promising biomarker for selectively imaging the anti-inflammatory phenotype of microglia. Developing PET tracers for this target is an active area of research, as imaging of specific microglial phenotypes can provide valuable insights into their dynamics in neuroinflammation. A key challenge is identifying high affinity P2Y12R ligands with optimal properties for targeting this receptor in the central nervous system (CNS). In this study, we report the synthesis and evaluation of a series of fluorinated pyrazolidine-3,5-dione derivatives as potential P2Y12R PET tracers, designed based on a lead structure with favorable physicochemical properties for brain permeability. All synthesized derivatives exhibited strong affinity for P2Y12R in vitro, with Ki values ranging from 1.21 to 5.66 nM. One candidate was selected for radiolabeling with fluorine-18 ([18F]6d) and evaluated in healthy rats using dynamic PET imaging under baseline conditions and efflux transporter-blocking, in addition to ex vivo biodistribution and metabolism studies. Unfortunately, [18F]6d showed low brain uptake, potentially due to ionization of the pyrazolidine-3,5-dione core in vivo or poor tracer stability. These findings highlight the need for novel chemical entities as starting points for targeting P2Y12R in the CNS.

Clenbuterol is a potent partial agonist on the human β2-adrenergic receptor (β2-AR) and available for veterinary use to treat respiratory diseases. We executed an "azoextension" strategy to generate a small library of photoresponsive azobenzene derivatives of clenbuterol. Illumination with two complementary wavelengths allowed interconversion between isomeric trans and cis forms, as proven by UV/vis, NMR and LC-MS studies. The photoswitchable clenbuterol analogs were pharmacologically characterized using β2-AR radioligand binding and cAMP assays. Key compound 12b (VUF26034) has suitable photochemical properties and good thermal stability of the cis isomer (t1/2 ∼ 4 months), switching from a partial agonist to a >25-fold higher affinity antagonist upon illumination with 360 nm.

Analysis of ligand-induced structural changes in proteins is challenging due to the lack of experimental methods suited for detection and characterisation of both ligand binding and induced structural changes. We have explored biosensors with different detection principles to study interactions between ligands and acetylcholine binding proteins (AChBPs), soluble homologues of Cys-loop ligand gated ion channels (LGICs) that undergo similar structural changes as LGICs upon ligand binding. X-ray crystallography was used to identify binding sites and establish if the detected conformational changes involved small changes in loop C or major structural changes in the pentamer associated with ion channel opening. Experiments were initially focused on ligands exhibiting complex surface plasmon resonance (SPR) biosensor sensorgrams or detected by second harmonic generation (SHG) biosensor analysis. Surface acoustic wave (SAW) and SHG biosensors confirmed that complexities in SPR data were indeed due to ligand-induced conformational changes. Grating coupled interferometry (GCI) biosensor sensorgrams were less complex, despite similar detection principles. switchSENSE biosensor analysis revealed that ligands resulted in either a compaction or expansion of the protein structure. X-ray crystallography of the protein-ligand complexes was only successful for 7 out of 12 ligands, despite nM-μM affinities. Crystals were not obtained for the two compounds shown by SHG analysis to induce large structural changes, while electron densities were not seen in the structures for some ligands. The work presented herein shows that several biosensor technologies have a unique capability to detect and discriminate binding and ligand induced conformational changes in proteins, also when interactions are rapid, weak and structural changes are small. However, they are complementary and provide different information.

Photopharmacology offers the promise of optical modulation of cellular signaling in a spatially and temporally controlled fashion with light-sensitive molecules. This study presents the first small-molecule photoswitchable agonist for an atypical G protein-coupled receptor (GPCR), the atypical chemokine receptor 3 (ACKR3). Inspired by a known benzylpiperidine-based ACKR3 agonist scaffold, 12 photoswitchable azobenzene-containing analogs were synthesized and characterized for their interaction with ACKR3. After analysis of concise Structure-Photochemistry and Structure-Affinity Relationships (SAR), compound 3e was selected as the best photoswitchable ACKR3 agonist in the series. Compound 3e can be effectively switched from its thermodynamically stable trans state to the less active cis-isomer with a photostationary state of 96%. The thermodynamically less stable cis- 3e only slowly switches back to the trans state (t 1/2,37°C ∼ 15 days), and trans-3e binds and activates ACKR3 at 10-fold lower concentrations compared to its cis-isomer. Compound 3e demonstrates selectivity for ACKR3 within in a wide panel of chemokine receptors. Using the recently published ACKR3 cryo-EM structures in computational studies, a binding mode for trans-3e is proposed that is perfectly in line with the observed SAR and the reduction in interaction with ACKR3 upon photoswitching. ACKR3 agonist 3e (VUF25471) is the first photoswitchable ligand for an atypical GPCR and will be a useful tool to investigate the role of ACKR3 in biological settings.

This Perspective summarizes successful fragment-to-lead (F2L) studies that were published in 2023 and is the ninth installment in an annual series. A tabulated summary of the relevant articles published in 2023 is provided (17 entries from 16 articles), and a comparison of the target classes, screening methods, and overall fragment or lead property trends for 2023 examples and for the combined entries over the years 2015–2023 is discussed. In addition, we identify several trends and innovations in the 2023 literature that promise to further increase the success of fragment-based drug discovery (FBDD), particularly in the areas of NMR and virtual screening, fragment library design, and fragment linking.

Photopharmacology offers powerful opportunities to control protein signaling using photoresponsive ligands. Despite the vast potential of photoswitchable ligands for spatiotemporal target protein control, research on ligand-protein binding kinetics of these ligands remains limited. Herein, we describe the discovery of the first radiolabeled photoswitchable ligand, [3H]VUF26063 ([3H]3f), to assess light-dependent ligand-protein binding kinetics in real time. The key compound (3f) is an arylazopyrazole-based antagonist targeting a prototypic family A G protein-coupled receptor (GPCR), the histamine H3 receptor (H3R), and enabled convenient radiolabeling via a growth vector on the pyrazole. Its photochemical properties, subnanomolar affinity of the trans isomer and a 50-fold decrease in affinity upon switching, allowed for reversible photochemical control of H3R binding kinetics in real time. The kinetic binding data obtained with this radiolabeled ligand indicate that 3f isomerizes in the H3R extended binding pocket upon illumination. Our results shed light on the binding kinetics of photoswitchable ligands and will have relevance beyond GPCRs as targets.

The atypical chemokine receptor 3 (ACKR3) has emerged as a promising drug target for the treatment of cancer, cardiovascular, and autoimmune diseases. In this study, we present the pharmacological characterization of VUF16840, the first small-molecule inverse agonist of ACKR3. VUF16840 effectively displaces CXC chemokine ligand 12 binding to ACKR3 and inhibits chemokine-induced β-arrestin2 recruitment in a concentration-dependent manner. Furthermore, VUF16840 stabilizes the inactive conformation of ACKR3, as demonstrated by its ability to suppress constitutive recruitment of downstream effector proteins. This inverse agonism alters ACKR3 constitutive trafficking, leading to receptor enrichment at the plasma membrane and inhibition of intracellular CXC chemokine ligand 12 uptake. Importantly, VUF16840 exhibits high selectivity for ACKR3 over a broad panel of human chemokine receptors. These findings establish VUF16840 as a potent and selective ACKR3 inverse agonist capable of modulating constitutive and chemokine-induced signaling and internalization events. As such, VUF16840 represents a valuable pharmacological tool for exploring the molecular and translational roles of ACKR3 in both physiologic and pathologic contexts. SIGNIFICANCE STATEMENT: A small molecule inverse agonist of the atypical chemokine receptor 3 (ACKR3), named VUF16840, is characterized in this work. It was shown that VUF16840 was able to inhibit basal as well as ligand-induced ACKR3 activation and, moreover, inhibits the scavenging function of ACKR3.

Despite the pharmacological relevance of the histamine H1 receptor (H1R), the second most therapeutically targeted G protein-coupled receptor (GPCR), an effective photoswitchable ligand to optically control this receptor remains elusive. In this work, we aimed to identify a suitable photoswitchable H1R ligand by performing an 'azoscan' on the H1R antagonist desloratadine. Taking advantage of the synthetic toolbox available for the desloratadine scaffold, aniline groups were regioselectively installed on the aromatic positions of this scaffold to enable the synthesis of azobenzene analogs targeting the orthosteric binding pocket of H1R. Additionally, we functionalized the piperidine ring of desloratadine with azobenzene moieties. These two strategies resulted in a total of nine photoswitchable compounds, displaying efficient trans to cis isomerization (PSS cis > 87%) and a broad range of thermal relaxation half-lives. Pharmacological evaluation revealed the 2-position (10a) to be most suitable for accommodation of a photoswitchable group, as it exhibits the most balanced profile in absolute affinity (K i trans = 2 nM) and a 3.2-fold light-induced affinity shift. Computational docking studies provide a rationale, with the binding pose of the trans and cis isomer in the H1R binding pocket potentially being inverted. While the development of effective photoswitchable ligands for H1R remains challenging, this study provides promising opportunities for future optimization to achieve optical control of this GPCR.

Surface plasmon resonance (SPR) biosensor methods are ideally suited for fragment -based lead discovery. However, generally applicable experimental procedures and detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are not available. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode -of -action, and availability of orthogonal methods capable of confirming fragment hits. The range of targets and libraries amenable to an SPR biosensor -based approach for identifying hits is considerably expanded by adopting multiplexed strategies, using multiple complementary surfaces or experimental conditions. Here we illustrate principles and multiplexed approaches for using flow-based SPR biosensor systems for screening fragment libraries of different sizes (90 and 1056 compounds) against a selection of challenging targets. It shows strategies for the identification of fragments interacting with 1) large and structurally dynamic targets, represented by acetyl choline binding protein (AChBP), a Cys-loop receptor ligand gated ion channel homologue, 2) targets in multi protein complexes, represented by lysine demethylase 1 and a corepressor (LSD1/CoREST), 3) structurally variable or unstable targets, represented by farnesyl pyrophosphate synthase (FPPS), 4) targets containing intrinsically disordered regions, represented by protein tyrosine phosphatase 1B (PTP1B), and 5) aggregation-prone proteins, represented by an engineered form of human tau (tau K18M). Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted. The study shows that the challenges for addressing these types of targets is not identification of potentially useful fragments per se, but establishing methods for their validation and evolution into leads.

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this β-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based β-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.

In search of new opportunities to develop Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) inhibitors that have selectivity over the off-target human PDE4 (hPDE4), different stages of a fragment-growing campaign were studied using a variety of biochemical, structural, thermodynamic, and kinetic binding assays. Remarkable differences in binding kinetics were identified and this kinetic selectivity was explored with computational methods, including molecular dynamics and interaction fingerprint analyses. These studies indicate that a key hydrogen bond between GlnQ.50 and the inhibitors is exposed to a water channel in TbrPDEB1, leading to fast unbinding. This water channel is not present in hPDE4, leading to inhibitors with a longer residence time. The computer-aided drug design protocols were applied to a recently disclosed TbrPDEB1 inhibitor with a different scaffold and our results confirm that shielding this key hydrogen bond through disruption of the water channel represents a viable design strategy to develop more selective inhibitors of TbrPDEB1. Our work shows how computational protocols can be used to understand the contribution of solvent dynamics to inhibitor binding, and our results can be applied in the design of selective inhibitors for homologous PDEs found in related parasites.

Analysis of structure-kinetic relationships (SKR) can contribute to an improved understanding of receptor-ligand interactions. Here, fragment 1 (4-(2-benzylphenoxy)-1-methylpiperidine) was used in different fragment growing approaches to mimic the putative binding mode of the long residence time (RT) ligands olopatadine, acrivastine, and levocetirizine at the histamine H1 receptor (H1R). SKR analyses reveal that introduction of a carboxylic acid moiety can increase RT at H1R up to 11-fold. Ligand efficiency (LE) decreases upon the introduction of the negatively charged group, whereas kinetic efficiency (KE) increases up to 8.5-fold. The olopatadine/acrivastine mimics give up to 15-fold differences in the RT, while the levocetirizine mimics afford similar RTs with only a 3-fold difference. Therefore, the levocetirizine mimics are less sensitive to structural changes. This study illustrates that for H1R, there are several ways to increase RT but the different strategies differ significantly in SKR.

Human African trypanosomiasis (HAT) still faces few therapeutic options and emerging drug resistance, stressing an urgency for novel antitrypanosomal drug discovery. Here, we describe lead optimization efforts aiming at improving antitrypanosomal efficacy and better physicochemical properties based on our previously reported optimized hit NPD-2975 (pIC50 7.2). Systematic modification of the 5-phenylpyrazolopyrimidinone NPD-2975 led to the discovery of a R4-substituted analogue 31c (NPD-3519), showing higher in vitro potency (pIC50 7.8) against Trypanosoma brucei and significantly better metabolic stability. Further, in vivo pharmacokinetic evaluation of 31c and experiments in an acute T. brucei mouse model confirmed improved oral bioavailability and antitrypanosomal efficacy at 50 mg/kg with no apparent toxicity. With good physicochemical properties, low toxicity, improved pharmacokinetic features, and in vivo efficacy, 31c may serve as a promising candidate for future drug development for HAT.

This Perspective is the eighth in an annual series that summarizes successful fragment-to-lead (F2L) case studies published each year. A tabulated summary of relevant articles published in 2022 is provided, and features such as target class, screening methods, and ligand efficiency are discussed both for the 2022 examples and for the combined examples over the years 2015-2022. In addition, trends and new developments in the field are summarized. In 2022, 18 publications described successful fragment-to-lead studies, including the development of three clinical compounds (MTRX1719, MK-8189, and BI-823911).

Public-private partnerships (PPPs) for neglected tropical diseases (NTDs) are often studied as an organizational form that facilitates the management and control of the huge costs of drug research and development. Especially the later stages of drug development, including clinical trials, become very expensive. This present study investigates whether and how the type of PPPs influences the initiation and duration of NTD clinical trials. Using the ClinicalTrials.gov database, a dataset of 1175 NTD clinical studies that started between 2000 and 2021 is analyzed based on affiliation information and project duration. For the NTD clinical trials that resulted from PPPs, the collaborating types were determined and analyzed, including the public sector-, private sector-, governmental sector-, and nongovernmental organization-led collaborations. The determinants for the discontinuation of all stopped clinical trials were categorized into scientific-, funding-, political-, and logistic dimensions. The results reveal that public sector-led PPPs were the most common collaborative types, and logistic and scientific issues were the most frequent determinants of stopped clinical trials. Trial registration: ClinicalTrials.gov.

Schistosomiasis is a neglected tropical disease with high morbidity. Recently, the Schistosoma mansoni phosphodiesterase SmPDE4A was suggested as a putative new drug target. To support SmPDE4A targeted drug discovery, we cloned, isolated, and biochemically characterized the full-length and catalytic domains of SmPDE4A. The enzymatically active catalytic domain was crystallized in the apo-form (PDB code: 6FG5) and in the cAMP- and AMP-bound states (PDB code: 6EZU). The SmPDE4A catalytic domain resembles human PDE4 more than parasite PDEs because it lacks the parasite PDE-specific P-pocket. Purified SmPDE4A proteins (full-length and catalytic domain) were used to profile an in-house library of PDE inhibitors (PDE4NPD toolbox). This screening identified tetrahydrophthalazinones and benzamides as potential hits. The PDE inhibitor NPD-0001 was the most active tetrahydrophthalazinone, whereas the approved human PDE4 inhibitors roflumilast and piclamilast were the most potent benzamides. As a follow-up, 83 benzamide analogs were prepared, but the inhibitory potency of the initial hits was not improved. Finally, NPD-0001 and roflumilast were evaluated in an in vitro anti-S. mansoni assay. Unfortunately, both SmPDE4A inhibitors were not effective in worm killing and only weakly affected the egg-laying at high micromolar concentrations. Consequently, the results with these SmPDE4A inhibitors strongly suggest that SmPDE4A is not a suitable target for anti-schistosomiasis therapy.