Off-target Binding Research Articles

Brainstem and cerebellar radiological findings in progressive supranuclear palsy

Abstract Progressive supranuclear palsy (PSP) is a sporadic neurodegenerative 4-repeat (4R) tauopathy associated with significant morbidity. Heterogeneity of symptom expression among this group is increasingly recognized, reflecting variable tau spread and neurodegeneration. Clinical manifestations consist of debilitating and rapidly progressive motor, oculomotor, speech, cognitive and affective impairments. Core pathological changes are noted with a predominance in the midbrain and basal ganglia, however spread to the more caudal brainstem and cerebellar regions is reported at various stages. Accordingly, whilst midbrain atrophy is the best recognized supportive imaging finding, quantitative neuroimaging studies using MRI and PET approaches have revealed a wider profile of brain abnormalities in cohorts of individuals with PSP. This expanded neurobiological scope of disease may account for individual heterogeneity and may highlight additional biological markers that are relevant to diagnosing and tracking the illness. Additionally, there is increasing understanding of the diverse cognitive, affective and speech functions of the cerebellum, which may be implicated in PSP beyond current recognition. In this review, we undertake a systematic literature search and summary of in vivo structural and functional neuroimaging findings in the brainstem and cerebellum in PSP to date. Novel and multimodal imaging techniques have emerged over recent years, which reveal several infratentorial alterations beyond midbrain atrophy in PSP. Most saliently, there is evidence for volume loss and microstructural damage in the pons, middle cerebellar peduncles and cerebellar cortex and deep nuclei, reported alongside recognised midbrain and superior cerebellar peduncle changes. Whilst the literature supporting the presence of these features is not unanimous, the evidence base is compelling, including correlations with disease progression, severity, or variant differences. A smaller number of studies report on abnormalities in MRI measures of iron deposition, neuromelanin, viscoelasticity, and the glymphatic system involving the infratentorial regions. Molecular imaging studies have also shown increased uptake of tau tracer in the midbrain and cerebellar dentate nucleus, although concern remains regarding possible off-target binding. Imaging of other molecular targets has been sparse, but reports of neurotransmitter, inflammatory and synaptic density alterations in cerebellar and brainstem regions are available. Taken together, there is an established evidence base of in vivo imaging alterations in the brainstem and cerebellum which highlight that midbrain atrophy is often accompanied by other infratentorial alterations in people with PSP. Further research examining the contribution of these features to clinical morbidity and inter-individual variability in symptom expression is warranted.

Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production

The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9. Cas9-BD and similarly modified dCas9-BD have been successfully employed for simultaneous biosynthetic gene cluster (BGC) refactoring, multiple BGC deletions, or multiplexed gene expression modulations in Streptomyces. We also demonstrate improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD is also used to capture large BGCs using a developed in vivo cloning method. The modified CRISPR-Cas9 system is successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome.

A Strategy for Simultaneous Engineering of Interspecies Cross-Reactivity, Thermostability, and Expression of a Bispecific 5T4 x CD3 DART® Molecule for Treatment of Solid Tumors.

Background: Bispecific antibodies represent a promising class of biologics for cancer treatment. However, their dual specificity and complex structure pose challenges in the engineering process, often resulting in molecules with good functional but poor physicochemical properties. Method: To overcome limitations in the properties of an anti-5T4 x anti-CD3 (α5T4 x αCD3) DART molecule, a phage-display method was developed, which succeeded in simultaneously engineering cross-reactivity to the cynomolgus 5T4 ortholog, improving thermostability and the elevating expression level. Results: This approach generated multiple DART molecules that exhibited significant improvements in all three properties. The lead DART molecule demonstrated potent in vitro and in vivo anti-tumor activity. Although its clearance in human FcRn-transgenic mice was comparable to that of the parental molecule, faster clearance was observed in cynomolgus monkeys. The lead α5T4 x αCD3 DART molecule displayed no evidence of off-target binding or polyspecificity, suggesting that the increased affinity for the target may account for its accelerated clearance in cynomolgus monkeys. Conclusions: This may reflect target-mediated drug disposition (TMDD), a potential limitation of targeting 5T4, despite its limited expression in healthy tissues.

Defining proteoform-specific interactions for drug targeting in a native cell signalling environment

Understanding the dynamics of membrane protein–ligand interactions within a native lipid bilayer is a major goal for drug discovery. Typically, cell-based assays are used, however, they are often blind to the effects of protein modifications. In this study, using the archetypal G protein-coupled receptor rhodopsin, we found that the receptor and its effectors can be released directly from retina rod disc membranes using infrared irradiation in a mass spectrometer. Subsequent isolation and dissociation by infrared multiphoton dissociation enabled the sequencing of individual retina proteoforms. Specifically, we categorized distinct proteoforms of rhodopsin, localized labile palmitoylations, discovered a Gβγ proteoform that abolishes membrane association and defined lipid modifications on G proteins that influence their assembly. Given reports of undesirable side-effects involving vision, we characterized the off-target drug binding of two phosphodiesterase 5 inhibitors, vardenafil and sildenafil, to the retina rod phosphodiesterase 6 (PDE6). The results demonstrate differential off-target reactivity with PDE6 and an interaction preference for lipidated proteoforms of G proteins. In summary, this study highlights the opportunities for probing proteoform–ligand interactions within natural membrane environments.

In Vivo Head-to-Head Comparison of [18F]GTP1 with [18F]MK-6240 and [18F]PI-2620 in Alzheimer Disease.

Alzheimer disease (AD) is characterized by the accumulation of tau neurofibrillary tangles that can be labeled with PET tracers. Multiple tau PET tracers have been used in clinical studies, including [18F]GTP1, [18F]PI-2620, and [18F]MK-6240. Standardized harmonization scales for comparing tau PET signals across tracers are currently under development and can be informed by comparisons of signals between tracers in both target and off-target regions of the brain. Methods: We conducted a head-to-head study comparing [18F]GTP1 with [18F]PI-2620 and [18F]MK-6240 in terms of dynamic range, magnitude of uptake, and correlation between tracers in participants with normal cognition and prodromal to mild AD. Results: [18F]GTP1 exhibited retention patterns that correlated with [18F]PI-2620 and [18F]MK-6240 for all Braak regions (except Braak II). Differences in tracer binding in AD target regions were relatively small, and off-target binding profiles were unique to each tracer. Conclusion: Our findings indicate that [18F]GTP1, [18F]PI-2620, and [18F]MK-6240 display similar uptake patterns in AD patients, suggesting that they detect the same tau pathology. However, the tracer-specific off-target signal distribution may impact their direct comparability, and for some use cases, tracer-specific considerations should be taken into account in the development of a standardized harmonization scale for tau PET.

Harnessing the potential of 2-methyl imidazolium dihydrogen phosphate as a protein stabilizer: Assessing its toxicity and impact on aggregation, structural integrity, and functional efficacy of monoclonal antibodies

The development of safe and effective biotherapeutics faces challenges due to protein aggregation. These aggregates, formed due to various stress factors, can lead to irreversible changes and increase the risk of immunogenicity and off-target binding. Ionic liquids (ILs) have recently gained significant attention due to their potential in stabilizing proteins and enzymes. In this investigation, we have assessed the thermal stability of bevacizumab (BmAb), in presence of 2-methyl imidazolium dihydrogen phosphate (2- MIDHP), both alone and in combination with other formulation excipients. SEC, DLS, FFF-MALS and AUC were used to study the protective effect of 2-MIDHP on BmAb aggregation. CD and fluorescence spectroscopy were used to evaluate the impact of 2-MIDHP on structural changes in BmAb, while SPR was used to assess the binding activity in the presence and absence of 2-MIDHP. Subsequently, a cell viability assay (MTT) was conducted to evaluate potential toxic effects of 2-MIDHP with BmAb. Overall, our findings suggested the potential of 2-MIDHP in enhancing the stability of BmAb without any negative impact on its structural and binding activity, and its compatibility with other formulation components, thus indicating its promising prospects for the development of improved biotherapeutics.

In silico identification of Nipah virus protein inhibitors from secondary metabolites of medicinal plants using a high-throughput virtual screening approach.

The Nipah virus (NiV), a highly pathogenic zoonotic virus of the Paramyxoviridae family, poses significant threats with its alarming mortality rates and pandemic potential. Despite historical cases, effective therapeutics remain elusive, prompting urgent exploration of potential antivirals. In this study, a structure-based virtual screening approach was employed to evaluate 690 metabolites sourced from ten medicinal plants (Allium sativum, Andrographis paniculata, Cocos nucifera, Euphorbia hirta, Euphorbia neriifolia, Moringa oreifera, Ocimum basilicum, Piper nigrum, Vitex negundo, and Zingiber officinale) for their antiviral activity against Nipah virus proteins. Through targeted and blind docking experiments, forty-three (43) compounds were found to exhibit high binding affinities (≤ -8 Kcal mol-1) and validated site-specificity. Subsequent analysis of the ADMET properties of these compounds, along with off-target docking to swine receptors, six (6) compounds with profiles akin to approved drugs and minimal off-target binding were identified. Stability screening via 100 ns and 300 ns molecular dynamics simulations identified two (2) of the six compounds that demonstrated sustained dynamic stability over an extended duration, coupled with favorable binding energies from MM-(GB/PB)SA calculations and biologically significant binding modes and residue interactions. Betulinic acid and CID 118716357 exhibited significant potential as inhibitors of Nipah virus fusion (F) glycoprotein trimer by targeting the oligomerization sites used to form the functional hexamer-of-trimer assembly. Coupled with their dynamic stability and favorable ADMET profiles in both human and swine conditions, these findings make them good candidates for subsequent invitro testing and further biological screening in the quest for potent antiviral drugs targeting Nipah virus proteins.

Parallel Proteomic and Transcriptomic Microenvironment Mapping (μMap) of Nuclear Condensates in Living Cells.

Cellular activity is spatially organized across different organelles. While several structures are well-characterized, many organelles have unknown roles. Profiling biomolecular composition is key to understanding function but is difficult to achieve in the context of small, dynamic structures. Photoproximity labeling has emerged as a powerful tool for mapping these interaction networks, yet maximizing catalyst localization and reducing toxicity remains challenging in live cell applications. Here, we disclose a new intracellular photocatalyst with minimal cytotoxicity and off-target binding, and we utilize this catalyst for HaloTag-based microenvironment-mapping (μMap) to spatially catalog subnuclear condensates in living cells. We also specifically develop a novel RNA-focused workflow (μMap-seq) to enable parallel transcriptomic and proteomic profiling of these structures. After validating the accuracy of our approach, we generate a spatial map across the nucleolus, nuclear lamina, Cajal bodies, paraspeckles, and PML bodies. These results provide potential new insights into RNA metabolism and gene regulation while significantly expanding the μMap platform for improved live-cell proximity labeling in biological systems.

Transmembrane protein 106B amyloid is a potential off-target molecule of tau PET tracers in the choroid plexus

PurposeTau positron emission tomography (PET) has become an essential tool for the clinical diagnosis of neurodegenerative diseases and the study of tau pathology in the brain. However, some tau tracers exhibit off-target binding in the basal ganglia, choroid plexus, and meninges. Recently, transmembrane protein 106B (TMEM106B) was identified to form novel amyloid filaments in the brain during aging. In this study, we explored the possibility that TMEM106B aggregates might be responsible for off-target binding of tau PET tracers in the choroid plexus. MethodsThe binding properties of 18F-labeled tau and amyloid tracers against choroid plexus tissues from postmortem human brains were evaluated through in vitro autoradiography and in vitro binding assays and compared with histochemical staining. ResultsAutoradiography showed strong binding of [18F]PM-PBB3 followed by [18F]flortaucipir in the choroid plexus. Immunostaining of the same sections revealed a high level of transmembrane protein 106B aggregates, which are thioflavin-S-labeled Biondi ring structures, in the choroid plexus epithelium and co-localization with PM-PBB3-stained structures. In contrast, co-localization of flortaucipir with TMEM106B immunoreactivity was not confirmed because flortaucipir had a low fluorescence intensity. In vitro binding assays for [18F]PM-PBB3 and [18F]flortaucipir demonstrated high affinities for collagenase A-treated choroid plexus homogenate containing transmembrane protein 106B aggregates. ConclusionThis study demonstrated high affinity of [18F]PM-PBB3 for TMEM106B aggregates in the choroid plexus. In vivo off-target binding of [18F]PM-PBB3 to the choroid plexus might result from binding to TMEM106B aggregates.

Classifying covalent protein binders by their targeted binding site.

Covalent protein targeting represents a powerful tool for protein characterization, identification, and activity modulation. The safety of covalent therapeutics was questioned for many years due to the possibility of off-target binding and subsequent potential toxicity. Researchers have recently, however, demonstrated many covalent binders as safe, potent, and long-acting therapeutics. Moreover, they have achieved selective targeting among proteins with high structural similarities, overcome mutation-induced resistance, and obtained higher potency compared to non-covalent binders. In this review, we highlight the different classes of binding sites on a target protein that could be addressed by a covalent binder. Upon folding, proteins generate various concavities available for covalent modifications. Selective targeting to a specific site is driven by differences in the geometry and physicochemical properties of the binding pocket residues as well as the geometry and reactivity of the covalent modifier "warhead". According to the warhead reactivity and the nature of the binding region, covalent binders can alter or lock a targeted protein conformation and inhibit or enhance its activity. We survey these various modification sites using case studies of recently discovered covalent binders, bringing to the fore the versatile application of covalent protein binders with respect to drug discovery approaches.

Head-to-head comparison of tau PET tracers [18F]PI-2620 and [18F]RO948 in non-demented individuals with brain amyloid deposition: the TAU-PET FACEHBI cohort

BackgroundSecond-generation tau tracers for positron emission tomography (PET) show high affinity for paired helical filaments tau deposits characteristic of Alzheimer´s disease and low off-target binding. Differences in their chemical structure though may lead to variations in their regional tau uptake and off-target signal. In this work, we aimed to compare the in-vivo uptake of tau tracers [18F]PI-2620 and [18F]RO948 in the early stages of the AD continuum.MethodsData from the TAU-PET FACEHBI clinical trial (EUDRA-CT 2021–000473-83) were analyzed. All participants were non-demented and underwent tau imaging with [18F]PI-2620 and [18F]RO948 PET within 3 months, amyloid imaging with [18F]Florbetaben and brain magnetic resonance imaging. Tau PET standardized uptake values ratios (SUVR) were calculated in Braak and typical off-target regions using the inferior cerebellar cortex as a reference region.ResultsThe cohort consisted of 18 individuals with subjective cognitive decline (n = 13) and mild cognitive impairment (n = 5), with centiloid values ranging from 17 to 159. Both tau tracers showed similar tau pathology distribution but presented a distinct off-target signal pattern on visual read. SUVR measurements for [18F]PI-2620 and [18F]RO948 were highly correlated in all Braak regions (R2 range [0.65–0.80]). Regarding off-target signal, [18F]PI-2620 had higher SUVRs in vascular structures, and [18F]RO948 had higher SUVRs in the skull/meninges.ConclusionsIn a cohort of individuals at early stages of the AD continuum, tau PET tracers [18F]PI-2620 and [18F]RO948 showed similar in-vivo uptake in all Braak regions and distinct off-target signal. These preliminary results support the development of standardized quantification scales for tau deposition that are tracer-independent.Trial registrationAEMPS EudraCT 2021–000473-83. Registered 30 December 2021.

Abstract 4119327: Tyrosine Kinase Inhibitor and Their Cardiotoxic Profile in the African American Population at a Community Safety Net Hospital

Intro: Tyrosine Kinase inhibitors (TKI) have been an effective treatment option for multiple cancer types. Unfortunately, TKIs have shown to have off-target Kinase binding to cardiac tissue. The examination of these cardiotoxic side effects have not been sufficiently explored in minority populations and studies such as DASISION and ENESTnd with high NAA homogeneous demographics reflect this issue. AA tend to have higher rates of HTN and HF at baseline, so further evaluation of TKI cardiotoxicity in this population is warranted. Methods: A total of 181 patients (pts) charts were reviewed in this study, who were exposed to either Ibrutinib, Imatinib and Sorafenib. First, Patients’ demographic data, such as age, gender, race, malignancy type were obtained along with their corresponding exposure to TKIs discussed above. Next, each patient (pt) chart was examined for any cardiotoxic etiologies after starting a TKI, such as arrhythmias, new- onset heart failure (nOHF) , cardiac arrest (CA), and Myocardial infarction (MI). Echocardiography reports were examined for Ejection fraction ( EF). In summary, this is an observational study looking at Cardiotoxicity secondary to TKI use in the AA population. Results: This study's demographics consisted of 149 AA and 32 NAA (non- african american), with the average age of 64.2, and had 128 males and 53 females. A total of 89 pts were on imatinib, 57 on Ibrutinib, 35 on sorafenib. In our population, 64 pts received cardiac echography while on TKIs, with 9 of them demonstrating nOHF. In terms of cardiac arrhythmias, a total of 67 pts experienced this while on TKI, with about 34% (51/149) of AA compared to 50% (16/32) of NAA. A total of 18 pts in the study required hospital management as a sequelae of their cardiotoxicity. Conclusion: There were a few trends that diverged from most studies regarding cardiotoxicity associated with TKIs in our study. When looking at individual items, such as rate of Atrial Fibrillation (AF), seen commonly in patients on Ibrutinib, our rate seen is 4.6% which is lower compared to earlier pooled homogenous population studies (6-7%). Earlier clinical trials of Sorafenib had a 2 % rate of HF in mostly homogenous NAA populations, and our study had 0% rate in total. Overall, several cardiotoxicity trends seen with the TKIs in this observational study warrant need for future studies to enable improved cardiac surveillance strategies post treatment in heterogeneous populations.

An antibody against human interleukin-2 showing minimal binding to neonatal Fc receptor: potent anti-tumor activity and reduced systemic toxicity in mice

The anti-tumor efficacy of immune checkpoint inhibitors can be enhanced by combining them with interleukin-2 (IL-2), which promotes the clonal expansion of antigen-activated CD8+ T cells and natural killer cells. However, IL-2 can simultaneously bind to endothelial cells and regulatory T cells to induce adverse and immunosuppressive effects. Such off-target binding can be inhibited by co-administering IL-2 with anti-IL-2 antibodies, but these antibodies can interact with neonatal Fc receptor to protect the IL-2 from lysosomal degradation, leading to substantial toxicity. Here we developed and humanized a mouse monoclonal antibody against human IL-2 and introduced two mutations (H310A and H435Q) in the Fc segment in order to weaken binding to the neonatal Fc receptor. The humanized antibody bound tightly to IL-2 but minimally to neonatal Fc receptor. Hydrogen deuterium exchange mass spectrometry indicated that the antibody binds to IL-2 at the site where the cytokine binds to subunit α (CD25) of the trimeric IL-2 receptor. Co-administering the antibody with human IL-2 improved the cytokine's ability to slow growth of two types of colorectal tumor xenografts (CT26, MC38) in immunocompetent mice. Co-administration to mice with CT26 xenografts strongly expanded CD8+ T cells, without expanding regulatory T cells in spleen or blood. Co-administration to mice with MC38 xenografts downregulated PD-1 and CD44, while upregulating CD62L and CD69, on tumor-infiltrating CD8+ T cells. Notably, co-administration synergized with anti-PD-1 antibody to slow growth of MC38 xenografts. Our results suggest that the combination of human IL-2 and our novel antibody shows promise for providing more effective, less toxic cancer immunotherapy.

Seed sequences mediate off-target activity in the CRISPR-interference system

The CRISPR interference (CRISPRi) system is a powerful tool for selectively and efficiently silencing genes in functional genomics research applications. However, its off-target activity has not been systematically investigated. Here, we utilized a genome-wide CRISPRi-Cas9 single-guide RNA (sgRNA) library to investigate the presence of off-target activity and its effects on gene expression. Our findings suggest that off-target effects in CRISPRi are quite pervasive and have direct and indirect impacts on gene expression. Most of the identified off-targets can be accounted for by complementarity of the protospacer adjacent motif (PAM)-proximal genomic sequence with the 3' half of the sgRNA spacer sequence, the seed sequence. We also report that while the stability of off-target binding is primarily driven by the PAM-proximal seed sequences, variations in the length of these seed sequences and the degree of mismatch tolerance at various positions can differ across different sgRNAs.

Lysine deacetylase inhibitors have low selectivity in cells and exhibit predominantly off-target effects.

Lysine deacetylases (KDACs or HDACs) are metal-dependent enzymes that regulate lysine acetylation, a post-translational modification that is present on thousands of human proteins, essential for many cellular processes, and often misregulated in diseases. The selective inhibition of KDACs would allow for understanding of the biological roles of individual KDACs and therapeutic targeting of individual enzymes. Recent studies have suggested that purportedly specific KDAC inhibitors have significant off-target binding, but the biological consequences of off-target binding were not evaluated. We compared the effects of treatment with two of the reportedly most KDAC-selective inhibitors, Tubastatin A and PCI-34051, in HT1080 cells in which the endogenous KDAC6 or KDAC8 gene has been mutated to inactivate enzyme catalysis while retaining enzyme expression. Genetic inactivation results in much stronger deacetylation defects on known targets compared to inhibitor treatment. Gene expression analysis revealed that both inhibitors have extensive and extensively overlapping off-target effects in cells, even at low inhibitor doses. Furthermore, Tubastatin A treatment led to increased histone acetylation, while inactivation of KDAC6 or KDAC8 did not. Genetic inactivation of KDAC6, but not KDAC8, impaired tumor formation in a xenograft model system, in contrast to previous reports with KDAC inhibitors suggesting the reverse. We conclude that the majority of observed biological effects of treatment with KDAC inhibitors are due to off-target effects rather than the intended KDAC inhibition. Developing a truly specific KDAC6 inhibitor could be a promising therapeutic avenue, but it is imperative to develop new inhibitors that selectively mimic genetic inactivation of individual KDACs.

Zibotentan in microvascular angina: a randomized, placebo-controlled, crossover trial

Abstract Background/Introduction Microvascular angina is a chronic condition characterized by abnormal myocardial blood flow leading to ischemic symptoms. Endothelin-1, a peptide secreted by endothelial cells, is a highly potent constrictor of the human coronary arterioles. Microvascular angina is associated with elevated circulating concentrations of endothelin-1, and prolonged exposure to ‘excess’ endothelin causes vasoconstriction and vascular remodelling. The chronic elevation of circulating endothelin-1 in microvascular angina may be influenced by genetic factors. Purpose Zibotentan, the most selective antagonist of the endothelin A receptor with no off-target binding to the endothelin B receptor, was evaluated in oncology trials and did not improve survival. We identified zibotentan as a potential disease-modifying therapy for patients suffering from microvascular angina. We hypothesized that zibotentan 10 mg daily for 12 weeks in addition to background medical therapy could be an efficacious and safe treatment for patients with microvascular angina. We further hypothesized that the SNP regulator of EDN1 gene expression, rs9349379 (G allele), could be a novel theragnostic biomarker. Methods The trial involved a prospective, multicentre, randomized, double-blind, placebo-controlled, sequential crossover design to assess the effects of zibotentan 10 mg or matched placebo, once daily for 12 weeks. The primary outcome was treadmill exercise duration (seconds) using the Bruce protocol. The secondary outcomes included exercise test parameters, health status questionnaires, safety (frequency and severity of severe adverse events (SAEs) and adverse events), feasibility (withdrawal rate), and biomarkers of efficacy (pharmacokinetics, pharmacodynamics). Results The study found no significant difference in exercise duration with zibotentan (-4.26 seconds; 95% CI: -19.60 to 11.06; P=0.5871). There was no effect on the secondary outcomes. However, for exploratory (mechanistic) outcomes, zibotentan increased plasma big endothelin-1, endothelin-1, and global myocardial blood flow, while reducing hemoglobin, diastolic, and systolic blood pressure (all p<0.001). Adverse events were more common during the zibotentan period (60.2%) compared to placebo (14.4%, p<0.001). Conclusion(s) In conclusion, daily administration of 10 mg zibotentan for 12 weeks did not enhance exercise duration and was commonly associated with adverse effects related to fluid retention. Further trials exploring lower zibotentan doses in combination with agents to mitigate fluid retention, and longer treatment durations, are warranted.

18F]PI-2620 Tau PET signal across the aging and Alzheimer’s disease clinical spectrum

Abstract [18F]PI-2620 is a second generation tracer that has shown high binding affinity for tau aggregation in Alzheimer’s disease (AD). However, [18F]PI-2620 signal in a large sample spanning the healthy aging and AD clinical spectrum as well as the stability of signal across different acquisition time windows has not yet been examined. Here, amyloid negative (Aβ-) cognitively unimpaired (CU; n = 49), amyloid positive (Aβ+) CU (n = 37), CU individuals with unknown amyloid status (n = 5), mild cognitive impairment (MCI; n = 14), dementia due to AD (n = 19), and non-AD neurodegenerative disorder (n = 54) participants were scanned with [18F]PI-2620 using a 45–75 min and/or 60–90 min acquisition time window. The impact of acquisition time on standardized uptake value ratio (SUVR) magnitude was first quantified with linear mixed models, and in participants and regions with high [18F]PI-2620 signal, SUVRs increased linearly up to 0.04 SUVR with each additional 5 min past injection time. We then accounted for differences in acquisition time using a voxel-wise correction approach and showed high correlations (all r’s ≥ 0.986) between SUVRs calculated from 45–75 min data and SUVRs from 60–90 min data that were interpolated to the 45–75 min scale in 15 participants who were scanned across both time windows. Using real and interpolated 45–75 min data, we next examined [18F]PI-2620 signal in Braak regions of interest and an off-target binding region (putamen) in Aβ- and Aβ+ CU, Aβ+ MCI, and Aβ+ AD dementia (n = 115) and showed that SUVRs in all Braak regions increased with greater disease severity. Within CU, higher Braak I SUVR was significantly associated with greater CSF pTau-181 (n = 35), and higher SUVRs were significantly associated with worse memory and language (n = 57). Thus, voxel-wise acquisition time corrections can be applied to combine [18F]PI-2620 datasets collected at different post-injection times, and once acquisition time is accounted for, [18F]PI-2620 signal shows the expected increases across the AD spectrum and can be used for detection of early tau elevations.

Precious Cargo: The Role of Polymeric Nanoparticles in the Delivery of Covalent Drugs

Covalent drugs can offer significant advantages over non-covalent drugs in terms of pharmacodynamics (i.e., target-binding properties). However, the development of covalent drugs is sometimes hampered by pharmacokinetic limitations (e.g., low bioavailability, rapid metabolism and toxicity due to off-target binding). Polymeric nanoparticles offer a potential solution to these limitations. Delivering covalent drugs via polymeric nanoparticles provides myriad benefits in terms of drug solubility, permeability, lifetime, selectivity, controlled release and the opportunity for synergistic administration alongside other drugs. In this short review, we examine each of these benefits in turn, illustrated through multiple case studies.

The genetic architecture of protein interaction affinity and specificity

The encoding and evolution of specificity and affinity in protein-protein interactions is poorly understood. Here, we address this question by quantifying how all mutations in one protein, JUN, alter binding to all other members of a protein family, the 54 human basic leucine zipper transcription factors. We fit a global thermodynamic model to the data to reveal that most affinity changing mutations equally affect JUN’s affinity to all its interaction partners. Mutations that alter binding specificity are relatively rare but distributed throughout the interaction interface. Specificity is determined both by features that promote on-target interactions and by those that prevent off-target interactions. Approximately half of the specificity-defining residues in JUN contribute both to promoting on-target binding and preventing off-target binding. Nearly all specificity-altering mutations in the interaction interface are pleiotropic, also altering affinity to all partners. In contrast, mutations outside the interface can tune global affinity without affecting specificity. Our results reveal the distributed encoding of specificity and affinity in an interaction interface and how coiled-coils provide an elegant solution to the challenge of optimizing both specificity and affinity in a large protein family.

A Small-molecule Antagonist Radiotracer for Positron Emission Tomography Imaging of the Mu Opioid Receptor.

The opioid crisis is a catastrophic health emergency catalyzed by the misuse of opioids that target and activate the mu opioid receptor. Traditional radioligands used to study the mu opioid receptor are often tightly regulated owing to their abuse and respiratory depression potential. In the present study, we sought to design and characterize a library of 24 non-agonist ligands for the mu opioid receptor. Ligands were evaluated for the binding affinity, intrinsic activity, and predicted blood-brain barrier permeability. Several ligands demonstrated single-digit nM binding affinity for the mu opioid receptor while also demonstrating selectivity over the delta and kappa opioid receptors. The antagonist behavior of 1A and 3A at the mu opioid receptor indicate that these ligands would likely not induce opioid-dependent respiratory depression. Therefore, these ligands can enable a safer means to interrogate the endogenous opioid system. Based on binding affinity, selectivity, and potential off-target binding, [ 11 C] 1A was prepared via metallophotoredox of the aryl-bromide functional group to [ 11 C]methyl iodide. The nascent radiotracer demonstrated brain uptake in a rhesus macaque model and accumulation in the caudate and putamen. Naloxone was able to reduce [ 11 C] 1A binding, though the interactions were not as pronounced as naloxone's ability to displace [ 11 C]carfentanil. These results suggest that GSK1521498 and related congeners are amenable to radioligand design and can offer a safer way to query opioid neurobiology.