European Journal of Medicinal Chemistry

Ligand optimization is central to drug discovery as hundreds of analogs might be designed and synthesized between an initial hit and a therapeutic candidate. The efficiency of this process is unclear, at least partly because there is no random background for optimization against which to compare. Such a random background might emerge from synthetically accessible but otherwise systematic random small substitutions across starting ligands, measuring likelihood of achieving a substantial improvement in affinity/potency or other property by any single perturbation. Recent literature and ligand-affinity/potency databases suggest that perhaps 10% of analogs with minor modifications improve upon a parents potency substantially (by [≥]10-fold), but this number is clouded by reporting bias, intentional improvement, and inter-group reproducibility. To begin to establish a background expectation for ligand optimization, we comprehensively and systematically modified 18 lead molecules across six targets with single atom changes; 257 compounds were synthesized. Unexpectedly, 11.2% of these random small perturbation analogs improved potency by [≥]10-fold over their parents. Conversely, these more potent analogs typically had worse in vitro pharmacokinetics (e.g. reduced metabolic stability, lower plasma free fraction). While it was possible to find analogs where the potency increase compensated for inferior exposure and half-life, resulting in more potent compounds in vivo, overall a frustrated landscape for ligand optimization is revealed. This study begins to establish a background expectation for ligand potency optimization and offers a simple strategy to do so. It also begins to quantify the challenges confronting the field in moving beyond in vitro potency.

The use of covalent warheads targeting the catalytic cysteine has been a cornerstone in coronavirus main protease (Mpro) inhibitor development, where various electrophilic motifs have been used including aldehydes, nitriles, ketoamides, and hydroxymethyl ketones (HMKs). Recent efforts have been mostly centered around nitrile warheads, given the success of compounds like Nirmatrelvir and Ensitrelvir in the clinic. However, finding and advancing alternative chemotypes with differentiating chemical and pharmacological profiles is essential for future pandemic preparedness. Among such alternatives, HMKs hold special interest because they balance reduced intrinsic electrophilicity with an excellent selectivity profile. Nevertheless, early HMK-based compounds, such as the clinical-stage Mpro inhibitor PF-00835231, suffered from poor oral bioavailability and therefore required intravenous administration, with or without prodrug derivatization of the hydroxyl group. Here, we describe our efforts in advancing the HMK field via the discovery of mCMX110, a lead that has superior potency, increased unbound exposure in vivo, and favorable oral bioavailability in preclinical studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/725542v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@abe1c9org.highwire.dtl.DTLVardef@746a08org.highwire.dtl.DTLVardef@dd5861org.highwire.dtl.DTLVardef@1d572c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Alzheimers disease (AD) is a multifactorial disease with mixed pathologies. Consequentially, drugs targeting multiple pathological processes may offer synergistic benefits. While histone deacetylase (HDAC) inhibitors have demonstrated efficacy in alleviating AD-related pathologies in animal models, the neuroprotective Wnt/{beta}-catenin signaling pathway remains compromised in AD brain. CI-994 is a class I HDAC inhibitor containing N-(2-aminophenyl)-benzamide. Our recent studies indicate that CI-994 is also an activator of Wnt/{beta}-catenin signaling by stabilizing Wnt co-receptor LRP6. We herein use CI-994 as a scaffold to develop novel potent dual modulators of class I HDACs and Wnt/{beta}-catenin signaling for AD therapy. Our lead compound, W2A-28, selectively inhibits class I HDAC1, 2 and 3 with IC50 values of 0.51 M, 0.68 M, and 0.22 M, respectively, and shows no inhibitory activities on other HDACs. Furthermore, W2A-28 potently activates Wnt reporter activity with an EC50 value of 1.61 M in Wnt-3A-expressing HEK293 cells. As expected, activation of Wnt/{beta}-catenin signaling by W2A-28 is associated with elevated LRP6 protein level. Importantly, W2A-28 displays excellent microsomal stability in both mouse and human liver microsomal stability assays, alongside high permeability and a lack of active efflux in MDR1-MDCKII models. Critically, W2A-28 treatment significantly enhances histone acetylation, activates Wnt/{beta}-catenin signaling, and suppresses tau phosphorylation in AD patient-specific cerebral organoids carrying APOE {varepsilon}4/{varepsilon}4 or APOE {varepsilon}3/{varepsilon}4 with PSEN1 M146V mutation. Our findings position W2A-28 as a promising multi-target drug candidate for AD therapy.

The rise of new, rapidly mutating viruses presents increasing challenges for drug developers. Traditional methods, such as high-throughput screening and drug repurposing against mutagenic viral targets, have recently shown their limitations. Our current rational molecular engineering approach offers a sustainable solution by targeting viral ion channels, which generally have low mutation rates. First, extending the amantadine molecule led to the development of new compounds that better match the alternating hydrophobic and hydrophilic patterns of the inner walls of ion channels--a common feature across many viruses. Then, simplifying the structure yielded a cyclohexylamine-based minimalist scaffold that effectively blocks the ion channel and demonstrates improved antiviral activity compared to well-known agents such as amantadine and arterolane. SARS-CoV-2 variants served as test systems in laboratory experiments. The new molecular scaffolds presented here provide a strong foundation for designing potent, broad-spectrum viral ion channel blockers.

Protein tyrosine kinases are important regulators of cell signaling, and aberrant kinase activity contributes to many human diseases, including cancers. All protein tyrosine kinases share a highly-conserved ATP binding pocket but diverge in their substrate binding sites in order to mediate distinct signaling events. Many potent and efficacious ATP-competitive tyrosine kinase inhibitors have been developed, however it remains challenging to achieve on-target selectivity across different kinases and target specific disease mutants, given the high degree of conservation in the ATP-binding pocket. By contrast, the variable substrate-binding site offers an opportunity for selective inhibition, provided molecules can be targeted to this site. Here, we present a modular strategy to design selective, peptide-based covalent inhibitors of tyrosine kinases with a distinct binding mode from existing ATP-competitive inhibitors. Using Src kinase as a model system, we demonstrate that Src-selective reactivity can be achieved by first designing an optimized substrate peptide and then strategically positioning an electrophile on the peptide to target a non-conserved cysteine on the kinase. We show that substrate-derived covalent peptides can inhibit kinase activity, bind simultaneously with an ATP-competitive inhibitor, and even inhibit the activity of kinases bearing a common drug resistance mutation. We further explore the application of this approach to develop an inhibitor of the cancer-relevant fibroblast growth factor receptor 1 kinase that shows selectivity for an oncogenic mutant over the wild-type enzyme. Our modular strategy to generate selective covalent peptides targeting protein tyrosine kinases provides a promising framework for future chemical probe and drug development efforts.

Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 {micro}M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 {micro}M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 M) for ACE2 were greater than that of Fvp (66.8 M), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 g/mL) compared to Fvp (IC50 > 239 g/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.

Antibiotic resistance in Mycobacterium tuberculosis is a pressing global health challenge demanding new therapeutic strategies. The bacterial respiratory chain comprises promising antibacterial targets, with dual inhibition of the terminal oxidases cytochrome bcc:aa3 and cytochrome bd (cyt bd) showing bactericidal activity. While bcc:aa3 inhibitors such as Q203 have advanced clinically, cyt bd remains underexplored due to difficulties in assigning activity of the purified enzyme and structurally resolving the quinol substrate binding site. Here, we report a rapid in vitro screening platform for cyt bd inhibitors by engineering a minimal respiratory system that couples the activity of cyt bd to that of a type 2 NADH dehydrogenase. This coupled assay enables spectroscopic monitoring of NADH oxidation as a proxy for cyt bd activity, allowing rapid screening of over 10,000 compounds. Screening identified WSL017, a fragment with low micromolar potency against both M. tuberculosis and E. coli cyt bd. Kinetic and structural analyses revealed competitive inhibition at the quinol-binding site, providing the first structural insights into cyt bd inhibition by a non-quinone scaffold. WSL017 displayed growth inhibition of M. tuberculosis H37ra, corroborating oxidase inhibition as a promising therapeutic strategy. This work establishes a pipeline for cyt bd inhibitor discovery and highlights new opportunities for structure-guided drug development against cytochrome bd oxidases.

Homeodomain-interacting protein kinase 4 (HIPK4) is a dual-specificity kinase that is predominantly expressed in differentiating spermatids, required for sperm development, and a promising target for nonhormonal male contraception. Genetic and functional studies have established an essential role for HIPK4 in spermiogenesis, where it acts at least in part through regulation of the F-actin-scaffolded acroplaxome during spermatid head shaping. The direct molecular targets of HIPK4 and their downstream effectors remain poorly defined, and small-molecule probes would be versatile tools for further investigating HIPK4 functions. Synthetic HIPK4 ligands could also be valuable leads for the development of nonhormonal male contraceptives. Here, we report the discovery of a cyanoquinoline-based series of HIPK4 inhibitors with nanomolar potency. Our lead compounds are selective for HIPK4, both within the HIPK family and across the broader kinome, establishing this scaffold as a useful starting point for probe and lead development. Unexpectedly, we found that a subset of these cyanoquinolines also perturbs HIPK4 proteostasis in a cell type-specific manner. In spermatids, these compounds induce the formation of detergent-insoluble HIPK4 aggregates and promote interactions between this kinase and the autophagy receptor Tax1-binding protein 1 (TAX1BP1). Together, our findings establish cyanoquinoline ligands as a new chemotype for probing HIPK4 biology and advancing male contraceptive discovery.

Temporal lobe epilepsy (TLE) has an unmet need for precision treatments targeting the seizure focus while avoiding effects on other body parts to minimise side effects. Photopharmacology could enable precision treatment by combining systemic administration of a photoswitchable drug with implantation of an optic fibre in the epileptic focus to induce light-dependent drug conversion from an inactive to an active configuration that interacts with its target receptor to suppress seizures. The photoswitchable {Delta}9-tetrahydrocannabinol ({Delta}9-THC) derivative, azo-THC-3, transitions from an inactive trans to an active cis configuration upon UV irradiation. We demonstrate that local or systemic administration of azo-THC-3 and local UV irradiation in the hippocampus supresses difficult-to-treat seizures in the intrahippocampal kainic acid mouse model of TLE. Furthermore, our findings illustrate that the photoswitch strategy avoids hypolocomotion, a common side effect of systemic {Delta}9-THC administration. As such, we provide the first demonstration of seizure suppression with the systemic administration of a photoswitchable compound and its local photoactivation in the seizure focus. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/720358v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1e42794org.highwire.dtl.DTLVardef@1e26891org.highwire.dtl.DTLVardef@13f2b6forg.highwire.dtl.DTLVardef@3c8e48_HPS_FORMAT_FIGEXP M_FIG C_FIG

c-Myc is a transcription factor that drives tumorigenesis in many cancers. It is notoriously difficult to directly target c-Myc, mainly due to its lack of well-defined druggable pockets. O-linked {beta}-N-acetylglucosamine modification (O-GlcNAcylation) is a post-translational modification (PTM) playing an important role in regulating c-Myc functions in cancer. However, previous studies have primarily relied on global perturbations to investigate c-Myc O-GlcNAcylation, making it difficult to determine its direct functional consequences due to concurrent cellular effects. Here, we report a bifunctional O-GlcNAcylation TArgeting Chimera (OGTAC) molecule, which can induce the proximity of c-Myc and O-GlcNAc transferase (OGT) in living cells, thereby enhancing the O-GlcNAcylation of c-Myc. The c-Myc-targeting OGTAC exhibits anti-proliferation effect against cancer cells. Mapping of c-Myc occupancy on genome indicates that OGTAC rewires c-Myc transcriptional activity and reprograms expression of the downstream oncogene MALAT1, in an O-GlcNAcylation-dependent manner. Overall, OGTAC presents a novel chemically induced proximity (CIP)-based tool to target and rewire c-Myc activity in cancer. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/722559v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1c640org.highwire.dtl.DTLVardef@2eb70corg.highwire.dtl.DTLVardef@f38970org.highwire.dtl.DTLVardef@c421c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Selective inhibition of phosphodiesterase 4B (PDE4B) remains a promising strategy for preserving the anti-inflammatory benefit of PDE4 inhibition in chronic obstructive pulmonary disease while reducing PDE4D-associated tolerability liabilities. This study integrated SHAP-interpretable machine learning, natural product virtual screening, hierarchical docking, post-docking MM-GBSA, isoform cross-docking, binding-pocket comparison, ADMET prediction, and 100 ns molecular dynamics simulations to identify PDE4B-selective inhibitors from the LOTUS natural product database. A Random Forest classifier trained on curated ChEMBL PDE4B bioactivity data achieved an external performance with AUC-ROC = 0.955, accuracy = 0.893, F1-score = 0.896, MCC = 0.785, and prioritized 119,698 predicted actives from 276,518 LOTUS compounds. SHAP analysis identified BertzCT and TPSA as major contributors to predicted activity. Sequential Lipinski, PAINS, and QED filtering retained 14,210 candidates for structure-based evaluation. Extra precision docking identified four leads with PDE4B docking scores of -9.123 to -12.080 kcal/mol, all outperforming roflumilast (-7.658 kcal/mol). Cross-docking and post-docking MM-GBSA supported preferential PDE4B binding for three candidates. The top lead, LTS0048837, maintained a stable PDE4B-bound pose during simulation, with comparatively stronger interaction persistence than its PDE4D complex and the roflumilast reference. These findings nominate LTS0048837 as a computationally prioritized PDE4B-selective natural product lead requiring experimental enzyme, cellular, and pharmacokinetic validation.

Glycyrrhiza uralensis is a widely used medicinal plant present in more than 70% of Kampo formulations in Japan owing to its diverse pharmacological activities, including immunomodulatory, antitumor, and antioxidant effects. Isoliquiritigenin (ILG), a major chalcone constituent of G. uralensis, exhibits anti-inflammatory activity; however, its molecular mechanism remains unclear. Here, we employed an activity-based protein profiling approach to identify the molecular targets of ILG. Given that the ,{beta}-unsaturated carbonyl moiety of ILG can covalently react with reactive cysteine residues via nucleophilic addition, we used an iodoacetamide-based probe to globally profile cysteine-reactive proteomes. The comparative analysis between ILG- and vehicle-treated RAW 264.7 macrophages identified cysteine 65 (Cys65) of lipocalin-type prostaglandin D2 synthase (L-PGDS) as a potential covalent target. ILG treatment did not alter L-PGDS expression levels, indicating that reduced probe labeling reflects direct covalent competition rather than changes in expression. Consistently, ILG significantly suppressed prostaglandin D2 (PGD2) production, comparable to the selective L-PGDS inhibitor AT-56. Although both ILG and AT-56 reduced interleukin-6 expression, ILG exerted a stronger inhibitory effect. Our results demonstrate that covalent inhibition of L-PGDS and subsequent suppression of PGD2 production represent a key mechanism underlying the anti-inflammatory activity of ILG.

Arylmalonate decarboxylase (AMDase) stereoselectively converts disubstituted malonates to chiral carboxylic acids, but its substrate spectrum is very limited regarding the size of the smaller substituent. Inspired by the observation that (S)-selective AMDase variants also convert larger substrates, we unlocked the synthesis of the (R)-enantiomers of -aryl and -alkenyl n-butanoic and n-pentanoic acids, respectively, in exquisite enantiopurity.

Catechinopyranocyanidins (Cpcs) which consist of diastereomers A and B are pigments derived from adzuki beans and are compounds in which the catechin and cyanidin skeletons are condensed to a pyrano ring. While catechins and anthocyanidins possess high antioxidant capacity, the physiological functions of Cpcs remains unclear. In this study, the antioxidant capacity of Cpcs was evaluated by in vitro antioxidant assays and by assessing their cytoprotective activity against oxidative stress in normal human dermal fibroblasts (NHDFs). Antioxidant capacity based on the hydrogen atom transfer (HAT) mechanism, as assessed by the ORAC assay revealed that Cpcs exhibit 14.1 mol TE/mol (Trolox equivalent antioxidant capacity: TEAC). Meanwhile, capacity based on the single electron transfer (SET) mechanism, as assessed by the DPPH, ABTS and CUPRAC assays revealed, they exhibit 2.1-3.6 mol TE/mol. Since TEAC value of Cpcs demonstrated by the HAT based mechanism higher than its SET based oxidative capacity suggesting that the antioxidant capacity of Cpcs is driven by the HAT mechanism. In cell culture experiments, Cpcs ameliorate cell toxicity in rotenone-induced injury model, suggesting to cytoprotective activity against mitochondrial dysfunction-dependent apoptosis. These results reveal novel physiological functions of Cpcs which may serve as a design guideline for elucidating in vivo dynamics based on antioxidant mechanisms.

The chemical constituents and cytoprotective potential of Cyathea podophylla, a Vietnamese fern, remain poorly investigated. This study aimed to isolate its compounds and evaluate their in vitro cytoprotective activity against 6-hydroxydopamine (6-OHDA)-induced toxicity in F11 cells. Compounds were chromatographically isolated and structurally characterized using NMR and HR-ESI-MS. Seven compounds were identified: five phenolics (trans-cinnamic acid, (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one, p-coumaric acid, 3,4-dihydroxybenzoic acid, 4-O-acetyl-caffeic acid), 5-hydroxymethylfurfural, and butyl-{beta}-D-fructofuranoside. Six of these are newly reported for the Cyathea genus. In MTT assays, butyl-{beta}-D-fructofuranoside exhibited the strongest cytoprotective effect (69.6% cell protection at 10 {micro}M, p < 0.001), followed by (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one (39.2% at 10 {micro}M). The remaining compounds lacked significant activity. These findings expand the phytochemical profile of Cyathea podophylla and provide preliminary evidence of its cytoprotective properties against 6-OHDA-induced injury, warranting further mechanistic and in vivo validation.

The influenza virus poses a significant global health threat due to its continuous evolution, immune evasion, and zoonotic spillover. The rise of drug resistance, reduced susceptibility to existing antiviral medications, and the limited effectiveness of annual vaccines underscore the need for new antiviral strategies. To infect, the influenza virus binds to sialic acid (SA)-containing molecules on host cell membranes through hemagglutinin (HA). Blocking this interaction represents a promising antiviral approach. Herein, we report that SA containing plasma membrane-derived vesicles (PMV) efficiently inhibits in vitro Influenza A virus (IAV) infection. Using orthogonal methods, we demonstrate that PMV derived from A549, MDCK, and HEK cells competitively bind to H1N1 (WSN) and H3N2 (X-31) IAV strains, block entry and infection in human respiratory epithelial cells in a dose-dependent manner, without causing significant toxicity. When the size of the vesicles was reduced through extrusion, the antiviral activity was enhanced, and this was found to be correlated with a size-dependent increase in hemagglutination inhibition and reduced IAV internalisation. Plasma membrane-derived vesicles may serve as a novel antiviral strategy against influenza virus infections due to their simple production method and conserved SA binding site on HA.

In this work we present antibody-metal conjugate as a new subclass of antibody-drug conjugates (ADC) for the chemodynamic therapy of cancer based on the rapid generation of reactive oxygen species (ROS) upon copper reduction. We used conventional therapeutic antibody trastuzumab and DOTA-NHS ester for the design and initial proof-of-concept. Thus, trastuzumab-DOTA-copper conjugate (TDCC) was synthesized. We demonstrate that TDCC retains specific binding to HER2-positive cancer cells with approximately native immunoreactivity and achieves stable copper incorporation with an average drug-to-antibody ratio of up to [~]8. In the presence of physiological reducing agents such as N-acetylcysteine or cysteine, TDCC generates substantial reactive oxygen species (ROS), leading to pronounced cytotoxicity and long-term suppression of clonogenic survival in HER2-positive SK-BR-3 and BT-474 cells. Notably, HER2-negative MDA-MB-231 cells and non-malignant HS5 fibroblasts remain largely unaffected, confirming target-dependent activity. The conjugate remains stable under storage conditions for up to 30 days, and the DOTA linker itself does not interfere with copper-mediated redox chemistry. Our findings identify TDCC as a novel class of targeted oxidative stress inducers that exploit the vulnerability of HER2-positive tumors to copper-mediated cytotoxicity. This strategy not only preserves the specificity of antibody-based delivery but also introduces a distinct mechanism of action capable of bypassing conventional resistance pathways, warranting further preclinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/721915v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@7ed6bdorg.highwire.dtl.DTLVardef@1442b2aorg.highwire.dtl.DTLVardef@6dff28org.highwire.dtl.DTLVardef@18aba16_HPS_FORMAT_FIGEXP M_FIG C_FIG

Kinases have proven to be one of the most fertile target classes for new drug approvals. However, classical reversible inhibitors may not be capable of the levels of specificity or target modulation required across a broad spectrum of disease areas. Approaches that chemically modify kinase inhibitors in solvent exposed regions are unveiling a swathe of mechanisms to address kinase function in new ways. For example, by either covalently recruiting nucleophilic residues outside of the ATP-binding pocket to inhibit, or by recruiting secondary effector proteins to degrade. Here, we systematically assessed the impact of minimal electrophilic modifications to ATP-site binding scaffolds, leading us to identify molecules that can control the activity and abundance of the master autophagy regulator, Unc-51-like autophagy activating kinase 1 (ULK1).

Fv-HSP72 is a rapid cell-penetrating human heat shock protein for the treatment of traumatic organ injuries. We have shown this re-engineered protein (HSP72) is capable of crossing the blood brain barrier (BBB) of rats suffering a controlled cortical impact (CCI) and remains in brain tissue for up to 12 hours; long after clearance from the cortex of uninjured rats. Peptide sequences unique to Fv-HSP72 allow for its differential detection from endogenous HSP72. Male Sprague-Dawley rats were divided into 10 groups of n=10 with those animals receiving a CCI subjected to a unilateral cortical contusion simulating a moderate to severe brain injury using an electronically controlled pneumatic impact device. Control groups were either uninjured (Sham), injured (TBI Only), or injured and given buffer (TBI+Vehicle). Rats treated with one of three Fv-HSP72 variants were dosed at 10 or 30mg/kg 15m post-impact, then sacrificed 48 hours later. Cortical tissues were extracted from the ipsilateral and contralateral hemispheres for biomarker analysis. Here we report results of our drug inhibiting neurodegeneration based on five biomarkers (NF-L, pNF-H, pTau [T181, T231, S396]). These results were statistically significant, especially for one of the Fv-HSP72 variants, when comparing differences both between treatment groups and within groups (i.e. when comparing ipsi-vs. contralateral hemispheres). Significant inhibition of oxidative stress (3-NT) and inflammatory (IL-6) biomarkers were also observed (both p<0.0001). With similar results obtained for a blast injury model being published elsewhere, the analyses suggest Fv-HSP72 is neuroprotective following a direct impact brain injury. One sentence summaryThis study describes the effectiveness of a biologic agent, Fv-HSP72, in significantly inhibiting neuronal tissue damage in the brain when administered after a direct cortical impact.

Colony stimulating factor 1 receptor (CSF1R) is a tyrosine kinase receptor that is expressed exclusively in microglia within the CNS. Its endogenous ligands, colony stimulating factor-1 (CSF1) and interleukin-34 (IL-34), are released from neurons, positioning CSF1R as a key mediator receptor of neuron-glia communication. CSF1R is considered not only a potential drug target, but also a biomarker of neuroinflammation. From that perspective, selective radioligands for neuroimaging are of great interest for imaging neuroinflammation and determining drug occupancy. In this study, we have validated the binding characteristics of a CSF1R inhibitor, 4-((5-MethOxy-6-((5-methoxypyridin-2-yl)methoxy)pyridin-3-yl)methyl)-2-(1-methyl-1H-pyrazol-4-yl)pyrimidine (5-MOP) as a novel CSF1R radioligand, by performing in vitro saturation binding experiments in human and murine tissues. 5-MOP was found to be selective for CSF1R among a broad range of kinases. Autoradiography revealed that [3H]5-MOP binds with high affinity (KD = 9.8 nM) to a single saturable binding site in human meningioma tissues, and this binding was displaced with known CSF1R inhibitors, including CPPC, sCSF1inh and GW-2580. In contrast, CPPC, which has been extensively used as a CSF1R radioligand showed substantial cross-reactivity to other brain kinases, including Trk A/B/C, and [3H]CPPC could only be displaced with CPPC itself, not by other ligands, including 5-MOP. These results identify [3H]5-MOP as the most selective radioligand currently available, enabling accurate detection of drug occupancy and activated microglia. Significance of the studyThis study identifies and validates a novel selective radioligand that binds CSF1R with high selectivity and low nanomolar affinity. Because CSF1R is selectively expressed in activated microglia, this radioligand could be useful for detecting neuroinflammatory activity.