Small Molecule Drug Development Research Articles

Aryloxypropanolamine targets amyloid aggregates and reverses Alzheimer-like phenotypes in Alzheimer mouse models

BackgroundAggregated amyloid-β (Aβ) is considered a pathogenic initiator of Alzheimer’s disease (AD), in strong association with tau hyperphosphorylation, neuroinflammation, synaptic dysfunction, and cognitive decline. As the removal of amyloid burden from AD patient brains by antibodies has shown therapeutic potential, the development of small molecule drugs inducing chemical dissociation and clearance of Aβ is compelling as a therapeutic strategy. In this study, we synthesized and screened aryloxypropanolamine derivatives and identified 1-(3-(2,4-di-tert-pentylphenoxy)-2-hydroxypropyl)pyrrolidin-1-ium chloride, YIAD002, as a strong dissociator of Aβ aggregates.MethodsThe dissociative activity of aryloxypropanolamine derivatives against Aβ aggregates were evaluated through in vitro assays. Immunohistochemical staining, immunoblot assays, and the Morris water maze were used to assess the anti-Alzheimer potential in YIAD002-treated 5XFAD and transgenic APP/PS1 mice. Target-ligand interaction mechanism was characterized via a combination of peptide mapping, fluorescence dissociation assays, and constrained docking simulations.ResultsAmong 11 aryloxypropanolamine derivatives, YIAD002 exerted strongest dissociative activity against β-sheet-rich Aβ aggregates. Upon oral administration, YIAD002 substantially reduced amyloid burden and accordingly, improved cognitive performance in the Morris water maze and attenuated major pathological hallmarks of AD including tauopathy, neuroinflammation, and synaptic protein loss. Mechanism studies suggest that YIAD002 interferes with intermolecular β-sheet fibrillation by directly interacting with KLVFFA and IGLMVG domains of Aβ. In addition, YIAD002 was found to possess dissociative activity against aggregates of pyroglutamate-modified Aβ and tau.ConclusionsCollectively, our results evince the potential of chemical-driven dissociation of Aβ aggregates by aryloxypropanolamines as a therapeutic modality of the amyloid clearance approach.

Cell and Tissue Specific Metabolism of Nucleoside and Nucleotide Drugs: Case Studies and Implications for Precision Medicine.

Many clinically used antiviral drugs are nucleoside or nucleotide analog drugs, which have a unique mechanism of action that requires intracellular phosphorylation. This dependence on intracellular activation presents novel challenges for the discovery and development of nucleoside/nucleotide analog drugs. Contrary to many small molecule drug development programs that rely on plasma pharmacokinetics and systemic exposures, the precise mechanisms that result in efficacious intracellular nucleoside triphosphate concentrations must be understood in the process of nucleoside/nucleotide drug development. The importance is highlighted here, using the following as case studies: the herpes treatment acyclovir, the cytomegalovirus therapy ganciclovir, and human immunodeficiency virus (HIV) treatments based on tenofovir, which are also in use for HIV prophylaxis. For each drug, the specificity of metabolism that results in its activation in different cells or tissues is discussed, and the implications explored. Acyclovir's dependence on a viral enzyme for activation provides selective pressure for resistance mutations. Ganciclovir is also dependent on a viral enzyme for activation, and suicide gene therapy capitalizes on that for a novel oncology treatment. The tissue of most relevance for tenofovir activation depends on its use as treatment or as prophylaxis, and the pharmacogenomics and drug-drug interactions in those tissues must be considered. Finally, differential metabolism of different tenofovir prodrugs and its effects on toxicity risk are explored. Taken together, these examples highlight the importance of understanding tissue specific metabolism for optimal use of nucleoside/nucleotide drugs in the clinic. SIGNIFICANCE STATEMENT: Nucleoside and nucleotide analogue drugs are cornerstones in current antiviral therapy and prevention efforts that require intracellular phosphorylation for activity. Understanding their cell and tissue specific metabolism enables their rational, precision use for maximum efficacy.

Synergistic effect of conformational changes in phosphoglycerate kinase 1 product release

In the glycolysis pathway, phosphoglycerate kinase 1 (PGK1) transfers one phosphoryl-group from 1,3-diphosphoglycerate (1,3BPG) to ADP to product 3-phosphoglycerate (3PG) and ATP. The catalytic process is accompanied with the conversion between the open conformation and the closed conformation of PGK1. However, the dynamic collaboration mechanism between the PGK1 conformation transition and the products releasing process remains poorly understood. Here using molecular dynamics simulations combined with molecular mechanics generalized born surface area (MM/GBSA) analysis, we demonstrated that PGK1 in the closed conformation first releases the product ATP to reach a semi-open conformation, and releases the product 3PG to achieve the full open conformation, which could accept new substrates ADP and 1,3BPG for the next cycle. It is noteworthy that the phosphorylation of PGK1 at T243 causes the loop region (residues L248-E260) flip outside the protein, and the phosphorylation of Y324 leads PGK1 become looser. Both modifications cause the exposure of the ADP/ATP binding site, which was beneficial for the substrates/products binding/releasing of PGK1. In addition, the other post translational modifications (PTMs) were also able to regulate the ligands binding/releasing with different effects. Our results revealed the dynamic cooperative molecular mechanism of PGK1 conformational transition with products releasing, as well as the influence of PTMs, which would contribute to the understanding of PGK1 substrates/products conversion process and the development of small molecule drugs targeting PGK1. Communicated by Ramaswamy H. Sarma.

Two-in-one combination therapy of fluoronucleoside analogues and triplex forming oligonucleotides

Antigene technology based on triplex-forming oligonucleotide (TFO) has been widely used in gene-specific regulation, serving as a potential gene therapy method. The optimization of TFO backbone and base can significantly improve the formation and stability of the triplex DNA. Fluorination is widely used in small molecule drug development, but its application in TFO optimization has not been well characterized. Herein, we developed a ‘in silico-in vitro-in vivo’ screening strategy to optimize fluorine atom (F) substitution of TFO. Molecular dynamics simulations are applied to screen single-site F substitutions in the TFO bases that retained stable triplex formation ability with the Ets2-targeted duplex DNA. Combining existing nucleotide analogue floxuridine, the synergistic effect is realized via a two-in-one chemo-TFO including gene regulation and chemotherapeutic functions, which is verified through in vitro and in vivo anti-tumor experiments.

Intranasal Peptide Therapeutics: A Promising Avenue for Overcoming the Challenges of Traditional CNS Drug Development.

The central nervous system (CNS) has, among all organ systems in the human body, the highest failure rate of traditional small-molecule drug development, ranging from 80-100% depending on the area of disease research. This has led to widespread abandonment by the pharmaceutical industry of research and development for CNS disorders, despite increased diagnoses of neurodegenerative disorders and the continued lack of adequate treatment options for brain injuries, stroke, neurodevelopmental disorders, and neuropsychiatric illness. However, new approaches, concurrent with the development of sophisticated bioinformatic and genomic tools, are being used to explore peptide-based therapeutics to manipulate endogenous pathways and targets, including "undruggable" intracellular protein-protein interactions (PPIs). The development of peptide-based therapeutics was previously rejected due to systemic off-target effects and poor bioavailability arising from traditional oral and systemic delivery methods. However, targeted nose-to-brain, or intranasal (IN), approaches have begun to emerge that allow CNS-specific delivery of therapeutics via the trigeminal and olfactory nerve pathways, laying the foundation for improved alternatives to systemic drug delivery. Here we review a dozen promising IN peptide therapeutics in preclinical and clinical development for neurodegenerative (Alzheimer's, Parkinson's), neuropsychiatric (depression, PTSD, schizophrenia), and neurodevelopmental disorders (autism), with insulin, NAP (davunetide), IGF-1, PACAP, NPY, oxytocin, and GLP-1 agonists prominent among them.

Inhibition of RAS-driven signaling and tumorigenesis with a pan-RAS monobody targeting the Switch I/II pocket

RAS mutants are major therapeutic targets in oncology with few efficacious direct inhibitors available. The identification of a shallow pocket near the Switch II region on RAS has led to the development of small-molecule drugs that target this site and inhibit KRAS(G12C) and KRAS(G12D). To discover other regions on RAS that may be targeted for inhibition, we have employed small synthetic binding proteins termed monobodies that have a strong propensity to bind to functional sites on a target protein. Here, we report a pan-RAS monobody, termed JAM20, that bound to all RAS isoforms with nanomolar affinity and demonstrated limited nucleotide-state specificity. Upon intracellular expression, JAM20 potently inhibited signaling mediated by all RAS isoforms and reduced oncogenic RAS-mediated tumorigenesis invivo. NMR and mutation analysis determined that JAM20 bound to a pocket between Switch I and II, which is similarly targeted by low-affinity, small-molecule inhibitors, such as BI-2852, whose invivo efficacy has not been demonstrated. Furthermore, JAM20 directly competed with both the RAF(RBD) and BI-2852. These results provide direct validation of targeting the Switch I/II pocket for inhibiting RAS-driven tumorigenesis. More generally, these results demonstrate the utility of tool biologics as probes for discovering and validating druggable sites on challenging targets.

Small-Molecule PROTACs for Cancer Immunotherapy.

Unsatisfactory physicochemical properties of macromolecular drugs seriously hinder their application in tumor immunotherapy. However, these problems can be effectively solved by small-molecule compounds. In the promising field of small-molecule drug development, proteolysis targeting chimera (PROTAC) offers a novel mode of action in the interactions between small molecules and therapeutic targets (mainly proteins). This revolutionary technology has shown considerable impact on several proteins related to tumor survival but is rarely exploited in proteins associated with immuno-oncology up until now. This review attempts to comprehensively summarize the well-studied and less-developed immunological targets available for PROTAC technology, as well as some targets to be explored, aiming to provide more options and opportunities for the development of small-molecule-based tumor immunotherapy. In addition, some novel directions that can magnify and broaden the protein degradation efficiency are mentioned to improve PROTAC design in the future.

Rapid identification of short oligonucleotide impurities using lithium adduct consolidated MALDI-TOF mass spectrometry

Nucleic acids are an increasingly popular platform for the development of pharmaceuticals to treat and prevent multiple diseases including those where traditional small molecule and protein-based drug development efforts have failed. Short oligonucleotide therapeutics, which consist of antisense oligonucleotides (ASOs) and short interfering ribonucleic acids (siRNAs), are prepared by solid phase chemical synthesis, which can generate various impuriies that have the potential to lower drug safety and efficacy. Drug substance-related impurities can be especially difficult to identify and characterize. To address this short coming, here we apply lithium adduct consolidation with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to afford a simplified, quick, and facile method for identification of a known drug substance impurity, isobutyryl (iBu) groups arising from incomplete deprotection during solid phase synthesis. We further employ high-resolution nuclear magnetic resonance (NMR) spectroscopy to confirm assessment of the iBu impurity. This lithium adduction consolidation method should find general applicability for routine quality assessment of synthetic oligonucleotides in both academic and industrial laboratories.

Stem cells from human exfoliated deciduous teeth relieves Alzheimer's disease symptoms in SAMP8 mice by up-regulating the PPARγ pathway.

The pathology of Alzheimer's disease (AD) is complex and heterogeneous, and there are currently no drugs that can stop its progression. The failure of traditional chemical small-molecule drug development showed the weakness of single target and made researchers look to cell therapy with multiple regulatory effects. Stem cells from human exfoliated deciduous teeth (SHED) are a kind of neural crest-derived mesenchymal stem cells which have broad prospects in the treatment of neurodegenerative diseases. In this study, we demonstrated the therapeutic effects of SHED in AD mice, including behavioral improvement, neuronal protection, and alleviation of neuroinflammation. Tracking experiments on SHED showed that some of the transplanted cells could enter the brain. To elucidate the role played by the majority of cells transplanted into veins, blood proteomic assays were performed. Data are available via ProteomeXchange with identifier PXD030313. Among the altered proteins, the PPAR pathway related to energy metabolism was considered to be an important signaling pathway involved in regulation through gene ontology analysis and pathway analysis. Western blot showed that the transplantation of SHED improved the glucose metabolism in AD mice by increasing the PPARγ signaling pathway. These results suggested that SHED have a potential in relieving AD pathological symptoms and improving behavioral cognition. The therapeutic mechanism of SHED is related to up-regulating PPARγ signaling pathway and reducing neuronal damage.

Key Considerations in Targeted Protein Degradation Drug Discovery and Development.

Targeting proteins’ enzymatic functions with small molecule inhibitors, as well as functions of receptor proteins with small-molecule agonists and antagonists, were the major forms of small-molecule drug development. These small-molecule modulators are based on a conventional occupancy-driven pharmacological approach. For proteome space traditionally considered undruggable by small-molecule modulators, such as enzymes with scaffolding functions, transcription factors, and proteins that lack well-defined binding pockets for small molecules, targeted protein degraders offer the opportunity to drug the proteome with an event-driven pharmacological approach. A degrader molecule, either PROTAC or molecular glue, brings the protein of interest (POI) and E3 ubiquitin ligase in close proximity and engages the ubiquitin-proteasome system (UPS), the cellular waste disposal system for the degradation of the POI. For the development of targeted protein degraders to meet therapeutic needs, several aspects will be considered, namely, the selective degradation of disease-causing proteins, the oral bioavailability of degraders beyond Lipinski’s rule of five (bRo5) scope, demands of new E3 ubiquitin ligases and molecular glue degraders, and drug resistance of the new drug modality. This review will illustrate several under-discussed key considerations in targeted protein degradation drug discovery and development: 1) the contributing factors for the selectivity of PROTAC molecules and the design of PROTACs to selectively degrade synergistic pathological proteins; 2) assay development in combination with a multi-omics approach for the identification of new E3 ligases and their corresponding ligands, as well as molecular glue degraders; 3) a molecular design to improve the oral bioavailability of bRo5 PROTACs, and 4) drug resistance of degraders.

Metformin: A promising drug for human cancers.

Small-molecule chemical drugs are of great significance for tumor-targeted and individualized therapies. However, the development of new small-molecule drugs, from basic experimental research and clinical trials to final application in clinical practice, is a long process that has a high cost. It takes at least 5 years for most drugs to be developed in the laboratory to prove their effectiveness and safety. Compared with the development of new drugs, repurposing traditional non-tumor drugs can be a shortcut. Metformin is a good model for a new use of an old drug. In recent years, the antitumor efficacy of metformin has attracted much attention. Epidemiological data and in vivo, and in vitro experiments have shown that metformin can reduce the incidence of cancer in patients with diabetes and has a strong antagonistic effect on metabolism-related tumors. Recent studies have shown that metformin can induce autophagy in esophageal cancer cells, mainly by inhibiting inflammatory signaling pathways. In recent years, studies have shown that the antitumor functions and mechanisms of metformin are multifaceted. The present study aims to review the application of metformin in tumor prevention and treatment.

New Insights into the Structure‐Function Relationships of G Proteins Containing Disease‐Associated Mutations

G proteins regulate intracellular signaling processes through their interactions with guanine nucleotides, GDP and GTP, and initiate numerous physiological responses. In normal conditions, G protein functions are regulated in a tightly controlled signaling network through complex formation with partner proteins, especially G protein‐coupled receptors (GPCRs). However, loss‐of‐function or gain‐of‐function mutations in the Gα subunit of G proteins disrupt this process, circumventing the usual control mechanisms and causing changes in G protein activity that result in serious illnesses. Our studies focused on the stimulatory class of trimeric G proteins, Gs, and mutations in Gαs associated with multiple cancers1. While the structure of one Gαs variant has been determined and additional Gαs variants have been characterized biochemically, there remains a need for an overall mechanistic framework for relating mutations in Gαs to changes in G protein signaling.To fill in this gap in knowledge, we investigated the interactions of Gαs variants with nucleotides using saturation transfer difference nuclear magnetic resonance (STD‐NMR) spectroscopy in aqueous solutions. In STD‐NMR experiments, we observed interactions between nucleotides and the Gαs variant proteins at atomic resolution, which provided information on nucleotide binding modes. STD‐NMR data of one Gαs variant closely correlated with literature data of the structure and activity of the same variant protein2, establishing a foundation for extending the analysis of NMR data to seven additional disease‐associated Gαs variant proteins for which no structures are available. These NMR data were combined with correlative biophysical data that measured the overall fold of the Gαs‐nucleotide complexes and with functional data reporting on the relative affinities of the Gαs variants for GDP and GTP. Integrating these data together provided a conceptual framework for categorizing variations in Gαs amino acid sequences with changes in signaling activity. Surprisingly, differences in the binding modes of the Gαs variants did not always directly correlate with their different functions, suggesting that mutations in Gαs that impact nucleotide exchange are nuanced and complex. These results imply that strategies for the development of small molecule drugs may need to be specifically tailored for each disease associated Gαs variant.References O’Hayre, M. et. al. The emerging mutational landscape of G proteins and G protein‐coupled receptors in cancer. Nat. Rev. Cancer. 13, 412‐424 (2013). Hu, Q. & Shokat, K. M. Disease‐Causing Mutations in the G Protein Gαs Subvert the Roles of GDP and GTP. Cell. 173, 1254–1264 (2018).

Resolving the deceptive isoform and complex selectivity of HDAC1/2 inhibitors

The histone deacetylase paralogs HDAC1/2/3 and their corepressor complexes serve as epigenetic master regulators of chromatin function. Over the past decades, HDACs have been widely pursued as pharmacological targets, and considerable efforts have been invested in the development of small molecule drugs. Specifically, ortho-aminoanilide-derived inhibitors, including CI-994 and Cpd-60, stand out with their attractive selectivity profiles and have been used extensively as tools to delineate the biological roles of specific HDAC isoforms and complexes. Here, we apply a suite of activity-independent strategies to investigate how dynamic processes that regulate HDAC complexes govern the isoform and complex selectivity of HDAC inhibitors. Importantly, we find that overreliance on static and simplified biochemical activity assays has confounded the determination of the biological selectivity of these ligands. Our data urge a comprehensive reinterpretation of numerous studies utilizing these tool compounds for the interrogation of epigenetic and other cellular processes.

Precision Medicine Based on CFTR Genotype for People with Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive genetic condition that is caused by variants in the cystic fibrosis transmembrane conductance regulator gene. This causes multisystem disease due to dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel at the apical surface of epithelia. Until recently, treatment was directed at managing the downstream effects in affected organs, principally improving airway clearance and treating infection in the lungs and improving malabsorption in the gastrointestinal tract. Care delivered by multidisciplinary teams has yielded incremental improvements in outcomes. However, the development of small-molecule CFTR modulator drugs over the last decade has heralded a new era of CF therapeutics. Modulators target the underlying defect and improve CFTR function. Either monotherapy or a combination of modulators is used depending on the specific genotype and class of CFTR disease-causing variants that an individual has. Both ivacaftor and the ivacaftor/tezacaftor/elexacaftor combination have been demonstrated to be associated with clinically very significant benefits in randomised trials and have rapidly been made available as part of standard care in many countries. CFTR modulators represent one of the best examples of precision medicine to date. They are expensive, however, and equity of access to them worldwide remains an issue. Studies and approvals are also ongoing for children under the age of 6 years for ivacaftor/tezacaftor/elexacaftor. Furthermore, no modulators are available for around 10% of the people with CF. In this review, we firstly summarise the genetics, pathophysiology and clinical problems associated with CF. We then discuss the development of CFTR modulators and key clinical trials to support their use along with other potential future therapeutic approaches.

A surface plasmon resonance database for better experimental planning and execution

Surface plasmon resonance (SPR) is a label-free biophysical technique that is widely used to investigate biomolecular interactions and a very powerful tool useful in different stages of small molecule drug development and antibody characterization. Direct molecular binding on the SPR chip surface is recorded as a response in real time. Several experimental variables, including buffer composition, type of sensor chip, coupling chemistry of molecules on the sensor surface, and surface regeneration conditions, contribute to quality and accuracy of the SPR data.

Combating Viral Diseases in the Era of Systems Medicine.

Viruses can cause many diseases resulting in disabilities and death. Fortunately, advances in systems medicine enable thedevelopment of effective therapies for treating viral diseases, of vaccines to prevent viral infections, as well as of diagnostic tools to mitigate the risk and reduce the death toll. Characterizing the SARS-CoV-2 gene sequence and the role of its spike protein in infection informs development of small molecule drugs, antibodies, and vaccines to combat infection and complication, as well as to end the pandemic. Drug repurposing of small molecule drugs is a viable strategy to combat viral diseases; the key concepts include (1) linking a drug candidate's pharmacological network to its pharmacodynamic response in patients; (2) linking a drug candidate's physicochemical properties to its pharmacokinetic characteristics; and (3) optimizing the safe and effective dosing regimen within its therapeutic window. Computational integration of drug-induced signaling pathways with clinical outcomes is useful to inform selection of potential drug candidates with respect to safety and effectiveness. Key pharmacokinetic and pharmacodynamic principles for computational optimization of drug development include a drug candidate's Cminss/IC95 ratio, pharmacokinetic characteristics, and systemic exposure-response relationship, where Cminss is the trough concentration following multiple dosing. In summary, systems medicine approaches play a vital role in global success in combating viral diseases, including global real-time information sharing, development of test kits, drug repurposing, discovery and development of safe, effective therapies, detection of highly transmissible and deadly variants, and development of vaccines.

A longitudinal and cross-sectional study of plasma neurofilament light chain concentration in Charcot-Marie-Tooth disease.

Advances in genetic technology and small molecule drug development have paved the way for clinical trials in Charcot-Marie-Tooth disease (CMT); however, the current FDA-approved clinical trial outcome measures are insensitive to detect a meaningful clinical response. There is, therefore, a need to identify sensitive outcome measures or clinically relevant biomarkers. The aim of this study was to further evaluate plasma neurofilament light chain (NFL) as a disease biomarker in CMT. Plasma NFL was measured using SIMOA technology in both a cross-sectional study of a US cohort of CMT patients and longitudinally over 6 years in a UK CMT cohort. In addition, plasma NFL was measured longitudinally in two mouse models of CMT2D. Plasma concentrations of NFL were increased in a US cohort of patients with CMT1B, CMT1X and CMT2A but not CMT2E compared with controls. In a separate UK cohort, over a 6-year interval, there was no significant change in plasma NFL concentration in CMT1A or HSN1, but a small but significant reduction in patients with CMT1X. Plasma NFL was increased in wild type compared to GARSC201R mice. There was no significant difference in plasma NFL in GARSP278KY compared to wild type mice. In patients with CMT1A, the small difference in cross-sectional NFL concentration vs healthy controls and the lack of change over time suggests that plasma NFL may lack sufficient sensitivity to detect a clinically meaningful treatment response in adulthood.

Glossary and tutorial of xenobiotic metabolism terms used during small molecule drug discovery and development (IUPAC Technical Report)

Article Glossary and tutorial of xenobiotic metabolism terms used during small molecule drug discovery and development (IUPAC Technical Report) was published on October 1, 2021 in the journal Chemistry International (volume 43, issue 4).

Flow radiocytometry using droplet optofluidics

Flow-based cytometry methods are widely used to analyze heterogeneous cell populations. However, their use for small molecule studies remains limited due to bulky fluorescent labels that often interfere with biochemical activity in cells. In contrast, radiotracers require minimal modification of their target molecules and can track biochemical processes with negligible interference and high specificity. Here, we introduce flow radiocytometry (FRCM) that broadens the scope of current cytometry methods to include beta-emitting radiotracers as probes for single cell studies. FRCM uses droplet microfluidics and radiofluorogenesis to translate the radioactivity of single cells into a fluorescent signal that is then read out using a high-throughput optofluidic device. As a proof of concept, we quantitated [18F]fluorodeoxyglucose radiotracer uptake in single human breast cancer cells and successfully assessed the metabolic flux of glucose and its heterogeneity at the cellular level. We believe FRCM has potential applications ranging from analytical assays for cancer and other diseases to development of small-molecule drugs.

TNF-α: The shape of small molecules to come?

In 2020, the anti-tumor necrosis factor (TNF) monoclonal antibody Humira® generated US$165.8 billion in cumulative sales and snatched the crown for the industry’s most successful drug from Lipitor (atorvastatin). TNF-α is a major component in beneficial and disease-related inflammation and TNF-α-inhibitor biologics have gained widespread use in autoimmune diseases, such as rheumatoid arthritis (RA). Many more diseases could benefit from TNF-α inhibitors, such as Alzheimer’s disease (AD) or major depression. However, the nature of TNF-α-inhibitor biologics prohibits central nervous system (CNS) applications. Moreover, high drug production costs and pricing, together with antidrug immune reactions and insufficient patient coverage, argue for the development of small-molecule drugs. Recently, drug-like orally available small molecules were described with high activity in animal disease models with activities comparable to those of antibodies.