Structural and functional analysis of engineered antibodies for cancer immunotherapy: insights into protein compactness and solvent accessibility.

Antibodies are crucial tools in various biomedical applications, including immunotherapy. In this study, we focused on designing and engineering antibodies to enhance their structural dynamics and functional properties. By employing advanced computational techniques and experimental validation, we gained crucial insights into the impact of specific mutations on the engineered antibodies. This study investigates the design and engineering of antibodies to improve their structural dynamics and functional properties. Structural attributes, such as protein compactness and solvent accessibility, were assessed, revealing interesting trends in anti-CD3 and anti-HER2 antibodies. Mutations in CD3 antibodies resulted in a more stable conformation, while mutant HER2 antibodies exhibited altered interaction with the target. Analysis of secondary structure assignments demonstrated significant changes in the folding and stability of the mutant antibodies compared to the wild-type counterparts. The conformational landscape of the engineered antibodies was explored, providing insights into folding pathways and binding mechanisms. Overall, the current study highlights the significance of antibody design and engineering in modulating structural dynamics and functional properties. The findings contribute to developing improved immunotherapeutic strategies by optimising antibody-based therapeutics for targeted diseases with enhanced efficacy and precision.Communicated by Ramaswamy H. Sarma.

Therapeutic Potential of HMF and Its Derivatives: a Computational Study.

Over the past century, chemicals and energy have increasingly been derived from non-renewable resources. The growing demand for essential chemicals and shrinking inventory make reliable, sustainable sources essential. Carbohydrates offer by far the greatest carbon supply. Furan compounds, a particular family of dehydration products, are believed to offer high chemical potential. Here, we analyze 5-HMF (5, hydroxymethylfurfural) and some of its derivatives in particular, a furan-type platform chemical. To analyze the therapeutic potential of HMF and its derivatives, this study utilized cutting-edge technologies such as computer-aided drug design, virtual screening, molecular docking, and molecular dynamic simulation. We conducted 189 docking simulations and examined some of the most promising dock poses using the molecular dynamic simulator. As for the receptors for our compounds, the leading candidates are human acetylcholinesterase, beta-lactamases, P. aeruginosa LasR, and S. aureus tyrosyl-tRNA synthetases. Out of all derivatives considered in this study, 2,5-furandicarboxylic acid (FCA) performed best.

Investigating the structural impact of Omicron RBD mutation on antibody escape and receptor management.

The SARS-CoV-2 Variant B.1.1.5291 evolved rapidly in late November 2021 from the existing mutants sparking fear worldwide owing to its infamous immune escape from a varied class of neutralising antibodies. To assess the structural behaviour of Omicron-Receptor Binding Domain (RBD) upon interacting with cross-reactive CR3022 antibody, we investigated the computational approach of structural engagement in B.1.1529 RBD and wild-type RBD with CR3022 antibody. The current study investigates the interacting interface between the RBDs and CR3022 to decipher the key residues accompanying the potential mutational landscape of SARS-CoV-2 variants. We conducted in-silico docking followed by molecular dynamics simulation analysis to examine the dynamic behaviour of protein-protein interactions. Furthermore, the study unleashed possible interactions post energy decomposition analysis via MM-GBSA. Conclusively, the mutational landscape of RBD eases in designing and discovering the effective neutralisation accompanied by development of a universal vaccine.Communicated by Ramaswamy H. Sarma.

Structure-activity relationships of dihydropyrimidone inhibitors against native and auto-processed human neutrophil elastase.

BACKGROUND: Human neutrophil elastase (HNE) is a key driver of systemic and cardiopulmonary inflammation. Recent studies have established the existence of a pathologically active auto-processed form of HNE with reduced binding affinity against small molecule inhibitors. METHOD: AutoDock Vina v1.2.0 and Cresset Forge v10 software were used to develop a 3D-QSAR model for a series of 47 DHPI inhibitors. Molecular Dynamics (MD) simulations were carried out using AMBER v18 to study the structure and dynamics of sc (single-chain HNE) and tcHNE (two-chain HNE). MMPBSA binding free energies of the previously reported clinical candidate BAY 85-8501 and the highly active BAY-8040 were calculated with sc and tcHNE. RESULTS: The DHPI inhibitors occupy the S1 and S2 subsites of scHNE. The robust 3D-QSAR model showed acceptable predictive and descriptive capability with regression coefficient of r2 = 0.995 and cross-validation regression coefficient q2 = 0.579 for the training set. The key descriptors of shape, hydrophobics and electrostatics were mapped to the inhibitory activity. In auto-processed tcHNE, the S1 subsite undergoes widening and disruption. All the DHPI inhibitors docked with the broadened S1'-S2' subsites of tcHNE with lower AutoDock binding affinities. The MMPBSA binding free energy of BAY-8040 with tcHNE reduced in comparison with scHNE while the clinical candidate BAY 85-8501 dissociated during MD. Thus, BAY-8040 may have lower inhibitory activity against tcHNE whereas the clinical candidate BAY 85-8501 is likely to be inactive. CONCLUSION: SAR insights gained from this study will aid the future development of inhibitors active against both forms of HNE.

Exploring the role of framework mutations in enabling breadth of a cross-reactive antibody (CR3022) against the SARS-CoV-2 RBD and its variants of concern.

Cross-reactive and broadly neutralizing antibodies against surface proteins of diverse strains of rapidly evolving viral pathogens like SARS-CoV-2 can prevent infection and therefore are crucial for the development of effective universal vaccines. While antibodies typically incorporate mutations in their complementarity determining regions during affinity maturation, mutations in the framework regions have been reported as players in determining properties of broadly neutralizing antibodies against HIV and the Influenza virus. We propose an increase in the cross-reactive potential of CR3022 against the emerging SARS- CoV-2 variants of concern through enhanced conformational flexibility. In this study, we use molecular dynamics simulations, in silico mutagenesis, structural modeling, and docking to explore the role of light chain FWR mutations in CR3022, a SARS-CoV anti-spike (S)-protein antibody cross-reactive to the S-protein receptor binding domain of SARS-CoV-2. Our study shows that single substitutions in the light chain framework region of CR3022 with conserved epitopes across SARS-CoV strains allow targeting of diverse antibody epitope footprints that align with the epitopes of recently-categorized neutralizing antibody classes while enabling binding to more than one strain of SARS-CoV-2. Our study has implications for rapid and evolution-based engineering of broadly neutralizing antibodies and reaffirms the role of framework mutations in effective change of antibody orientation and conformation via improved flexibility.Communicated by Ramaswamy H. Sarma.

Enhanced Algal Biomass Production in a Novel Electromagnetic Photobioreactor (E-PBR).

Enhancement in algal biomass production is a crucial parameter for generating value-added chemicals from renewable carbon sources. This study aimed to fabricate a novel photobioreactor with integrated and innovative electromagnets to assess microalgae's growth under the magnetic field's effect. The present study observed the effect of magnetic field exposure on the microalgae Chlorella sorokiniana cultivation and estimated the change in pigments with growth. In addition, the carbohydrate content of Chlorella sorokiniana was also evaluated. Chlorella sorokiniana culture, when exposed to a magnetic field strength of 10 mT d-1 resulted in a greater biomass concentration and productivity of about 12% and 8%, respectively, higher than the control culture. A positive effect of magnetic field exposure was also observed in the microalga's pigments concentration and carbohydrate content. The chlorophyll-a content increased twofold exponentially with the growth and the carbohydrate content of the microalgae cells also increased by 15% compared to the control culture. Therefore, the novel Electromagnetic Photobioreactor (E-PBR) can be used to produce enhanced biomass and other value-added products.

Immunotherapeutics for AD: A Work in Progress.

Alzheimer's Disease (AD), often called the 'Plague of the 21st Century,' is a progressive, irreversible neurodegenerative disorder that leads to the degeneration and death of neurons. Multiple factors, such as genetic defects, epigenetic regulations, environmental factors, or cerebrovascular damage, are a manifestation of the neurodegenerative process that begins to occur decades before the onset of disease. To date, no treatment or therapeutic strategy has proven to be potent in inhibiting its progress or reversing the effects of the disease. The ever-increasing numbers and lack of sufficient therapies that can control or reverse the effects of the disease have propelled research in the direction of devising efficient therapeutic strategies for AD. This review comprehensively discusses the active and passive immunotherapies against Amyloid-ß and Tau protein, which remain the popular choice of targets for AD therapeutics. Some of the prospective immunotherapies against Aß plaques have failed due to various reasons. Much of the research is focused on targeting Tau, specifically, targeting the mid-region of extracellular Tau due to their potential to prevent seeding and hence the spread of neurofibrillary tangles (NFTs). Thus, there is a need to thoroughly understand the disease onset mechanisms and discover effective therapeutic strategies.

TLR-Mediated Signal Transduction and Neurodegenerative Disorders.

A special class of proteins called Toll-like receptors (TLRs) are an essential part of the innate immune system, connecting it to the adaptive immune system. There are 10 different Toll-Like Receptors that have been identified in human beings. TLRs are part of the central nervous system (CNS), showing that the CNS is capable of the immune response, breaking the long-held belief of the brain's "immune privilege" owing to the blood-brain barrier (BBB). These Toll-Like Receptors are present not just on the resident macrophages of the central nervous system but are also expressed by the neurons to allow them for the production of proinflammatory agents such as interferons, cytokines, and chemokines; the activation and recruitment of glial cells; and their participation in neuronal cell death by apoptosis. This study is focused on the potential roles of various TLRs in various neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD), namely TLR2, TLR3, TLR4, TLR7, and TLR9 in AD and PD in human beings and a mouse model.

Cytolethal distending toxin: from genotoxin to a potential biomarker and anti-tumor target.

Cytolethal Distending Toxin (CDT) belongs to the AB toxin family and is produced by a plethora of Gram-negative bacteria. Eight human-affecting enteropathogens harbor CDT that causes irritable bowel syndrome (IBS), dysentery, chancroid, and periodontitis worldwide. They have a novel molecular mode of action as they interfere in the eukaryotic cell-cycle progression leading to G2/M arrest and apoptosis. CDT, the first bacterial genotoxin described, is encoded in a single operon possessing three proteins, CdtA, CdtB, and CdtC. CdtA and CdtC are needed for the binding of the CDT toxin complex to the cholesterol-rich lipid domains of the host cell while the CdtB is the active moiety. Sequence and 3D structural-based analysis of CdtB showed similarities with nucleases and phosphatases, it was hypothesized that CdtB exercises a biochemical function identical to both these enzymes. CDT is secreted through the outer membrane vesicles from the producing bacteria. It is internalized in the target cells via clathrin-dependent endocytosis and translocated to the host cell nucleus through the Golgi complex and ER. This study discusses the virulence role of CDT, causing pathogenicity by acting as a tri-perditious complex in the CDT-producing species with an emphasis on its potential role as a biomarker and an anti-tumor agent.

Bispecific antibodies: a novel approach for targeting prominent biomarkers.

Many types of cancers are prevalent in India and worldwide. Monoclonal antibodies (MAbs) are one of the major types of cancer therapeutics, which have included MAbs of hybridoma, chimeric, humanized, or human origin. MAbs are mostly generated currently by direct cloning from B cells. Bispecific antibodies (BAbs), as the name suggests, have two different antigen-binding domains in a single molecule and thus have dual functionality/specificity combined in a single antibody. In addition to the detection of two different antigenic molecules, the dual functionality of BAbs can be utilized to mount T-cell-mediated killing of tumor cells wherein one Fv binds to the tumor-specific antigen and the another recruits T cells to the site of action. Breast cancer and prostate cancer are among the most prevalent cancers in women and men, respectively. Biomarkers such as HER2 and ER/PR are expressed in breast cancer, while overexpression of hepsin and prostate-specific membrane antigen is observed in prostate cancer. Developing BAbs against these biomarkers may be a potent therapeutic option to target breast and prostate cancer, respectively. Therefore, an efficient method using recombinant DNA technology and mammalian cell culture platform is required to generate BAbs against specific diseases as biomarkers as well as for the generation of antibody-based therapeutics.

Nature's Derivative(s) as Alternative Anti-Alzheimer's Disease Treatments.

Alzheimer's disease (AD), the 'Plague of Twenty-First Century,' is a crippling neurodegenerative disease that affects a majority of the older population globally. By 2050, the incidence of AD is expected to rise to 135 million, while no treatment(s) that can reverse or control the progression of AD are currently available. The treatment(s) in use are limited in their ability to manage the symptoms or slow the progression of the disease and can lead to some severe side effects. The overall care is economically burdensome for the affected individuals as well as the caretakers or family members. Thus, there is a pressing need to identify and develop much safer alternative therapies that can better manage AD. This review discusses a multitude of such treatments borrowed from Ayurveda, traditional Chinese practices, meditation, and exercising for AD treatment. These therapies are in practice since ancient times and reported to be beneficial as anti-AD therapies. Ayurvedic drugs like turmeric, Brahmi, Ashwagandha, etc., management of stress by meditation, regular exercising, and acupuncture have been reported to be efficient in their anti-AD usage. Besides, a combination of vitamins and natural dietary intakes is likely to play a significant role in combating AD. We conclude that the use of such alternative strategies will be a stepping-stone in preventing, treating, curing, or managing the disease.

Axodendritic sorting and pathological missorting of Tau are isoform-specific and determined by axon initial segment architecture.

Subcellular mislocalization of the microtubule-associated protein Tau is a hallmark of Alzheimer disease (AD) and other tauopathies. Six Tau isoforms, differentiated by the presence or absence of a second repeat or of N-terminal inserts, exist in the human CNS, but their physiological and pathological differences have long remained elusive. Here, we investigated the properties and distributions of human and rodent Tau isoforms in primary forebrain rodent neurons. We found that the Tau diffusion barrier (TDB), located within the axon initial segment (AIS), controls retrograde (axon-to-soma) and anterograde (soma-to-axon) traffic of Tau. Tau isoforms without the N-terminal inserts were sorted efficiently into the axon. However, the longest isoform (2N4R-Tau) was partially retained in cell bodies and dendrites, where it accelerated spine and dendrite growth. The TDB (located within the AIS) was impaired when AIS components (ankyrin G, EB1) were knocked down or when glycogen synthase kinase-3ß (GSK3ß; an AD-associated kinase tethered to the AIS) was overexpressed. Using superresolution nanoscopy and live-cell imaging, we observed that microtubules within the AIS appeared highly dynamic, a feature essential for the TDB. Pathomechanistically, amyloid-ß insult caused cofilin activation and F-actin remodeling and decreased microtubule dynamics in the AIS. Concomitantly with these amyloid-ß-induced disruptions, the AIS/TDB sorting function failed, causing AD-like Tau missorting. In summary, we provide evidence that the human and rodent Tau isoforms differ in axodendritic sorting and amyloid-ß-induced missorting and that the axodendritic distribution of Tau depends on AIS integrity.

A Disease-associated Mutant of NLRC4 Shows Enhanced Interaction with SUG1 Leading to Constitutive FADD-dependent Caspase-8 Activation and Cell Death.

Nod-like receptor family card containing 4 (NLRC4)/Ipaf is involved in recognition of pathogen-associated molecular patterns leading to caspase-1 activation and cytokine release, which mediate protective innate immune response. Point mutations in NLRC4 cause autoinflammatory syndromes. Although all the mutations result in constitutive caspase-1 activation, their phenotypic presentations are different, implying that these mutations cause different alterations in properties of NLRC4. NLRC4 interacts with SUG1 and induces caspase-8-mediated cell death. Here, we show that one of the autoinflammatory syndrome-causing mutants of NLRC4, H443P, but not T337A and V341A, constitutively activates caspase-8 and induces apoptotic cell death in human lung epithelial cells. Compared with wild type NLRC4, the H443P mutant shows stronger interaction with SUG1 and with ubiquitinated cellular proteins. Phosphorylation of NLRC4 at Ser533 plays a crucial role in caspase-8 activation and cell death. However, H443P mutant does not require Ser533 phosphorylation for caspase-8 activation and cell death. Caspase-8 activation by NLRC4 and its H443P mutant are dependent on the adaptor protein FADD. A phosphomimicking mutant of NLRC4, S533D does not require SUG1 activity for inducing cell death. Ubiquitin-tagged NLRC4 could induce cell death and activate caspase-8 independent of Ser533 phosphorylation. Our work suggests that SUG1-mediated signaling results in enhanced ubiquitination and regulates FADD-dependent caspase-8 activation by NLRC4. We show that the autoinflammation-associated H443P mutant is altered in interaction with SUG1 and ubiquitinated proteins, triggering constitutive caspase-8-mediated cell death dependent on FADD but independent of Ser533 phosphorylation.

Amyloid-ß oligomers induce synaptic damage via Tau-dependent microtubule severing by TTLL6 and spastin.

Mislocalization and aggregation of Aß and Tau combined with loss of synapses and microtubules (MTs) are hallmarks of Alzheimer disease. We exposed mature primary neurons to Aß oligomers and analysed changes in the Tau/MT system. MT breakdown occurs in dendrites invaded by Tau (Tau missorting) and is mediated by spastin, an MT-severing enzyme. Spastin is recruited by MT polyglutamylation, induced by Tau missorting triggered translocalization of TTLL6 (Tubulin-Tyrosine-Ligase-Like-6) into dendrites. Consequences are spine loss and mitochondria and neurofilament mislocalization. Missorted Tau is not axonally derived, as shown by axonal retention of photoconvertible Dendra2-Tau, but newly synthesized. Recovery from Aß insult occurs after Aß oligomers lose their toxicity and requires the kinase MARK (Microtubule-Affinity-Regulating-Kinase). In neurons derived from Tau-knockout mice, MTs and synapses are resistant to Aß toxicity because TTLL6 mislocalization and MT polyglutamylation are prevented; hence no spastin recruitment and no MT breakdown occur, enabling faster recovery. Reintroduction of Tau re-establishes Aß-induced toxicity in TauKO neurons, which requires phosphorylation of Tau's KXGS motifs. Transgenic mice overexpressing Tau show TTLL6 translocalization into dendrites and decreased MT stability. The results provide a rationale for MT stabilization as a therapeutic approach.

Novel diffusion barrier for axonal retention of Tau in neurons and its failure in neurodegeneration.

Missorting of Tau from axons to the somatodendritic compartment of neurons is a hallmark of Alzheimer's disease, but the mechanisms underlying normal sorting and pathological failure are poorly understood. Here, we used several Tau constructs labelled with photoconvertible Dendra2 to analyse its mobility in polarized neurons. This revealed a novel mechanism of sorting-a retrograde barrier in the axon initial segment (AIS) operating as cellular rectifier. It allows anterograde flow of axonal Tau but prevents retrograde flow back into soma and dendrites. The barrier requires binding of Tau to microtubules but does not require F-actin and thus is distinct from the sorting of membrane-associated proteins at the AIS. The barrier breaks down when Tau is phosphorylated in its repeat domain and detached from microtubules, for example, by the kinase MARK/Par1. These observations link the pathological hallmarks of Tau missorting and hyperphosphorylation in neurodegenerative diseases.

Influence of carvedilol on anticonvulsant effect of gabapentin.

The present study was performed to investigate whether or not carvedilol (a beta-adrenoreceptor antagonist) potentiates the anticonvulsive activity of gabapentin against ICES (Increasing current electroshock) and PTZ (Pentylenetetrazole) induced seizures in mice. Further the effect of combination of both the drugs on spatial working memory and locomotor activity on rotarod was also evaluated. The biochemical estimation was done by measuring the lipid peroxidation and reduced glutathione in brain tissue. The results indicate that carvedilol significantly potentiates the anticonvulsive activity of gabapentin in both the models of epilepsy. The combination of both the drugs has no effect on spatial working memory and locomotor activity. In addition carvedilol in combination with gabapentin significantly decreased the level of the lipid peroxidation and increased the level of reduced glutathione (GSH) in brain. So, the present study showed that carvedilol potentiates the anticonvulsive activity of gabapentin, which can be useful for the treatment of epilepsy in patients with hypertension.

Interaction with Sug1 enables Ipaf ubiquitination leading to caspase 8 activation and cell death.

Activation of initiator caspases is dependent on interacting proteins, and Ipaf [ICE (interleukin-1beta-converting enzyme)-protease activating factor] {NLRC4 [NLR (Nod-like receptor) family CARD (caspase activation and recruitment domain)-containing 4]} an inflammasome component, is involved in caspase 1 activation and apoptosis. Investigating the mechanisms of Ipaf activation, we found that the C-terminal LRR (leucine-rich repeat) domain of Ipaf, through intramolecular interaction, negatively regulates its apoptosis-inducing function. In A549 lung carcinoma cells, expression of Ac-Ipaf (LRR-domain-deleted Ipaf) induced cell death that was dependent on caspase 8, but not on caspase 1. A yeast two-hybrid screen using Ac-Ipaf as bait identified human Sug1 (suppressor of gal 1), a component of the 26S proteasome, as an interacting protein. In mammalian cells Sug1 interacts and co-localizes with Ipaf. Sug1 binds to amino acids 91-253 of Ipaf, which is also the region that the LRR domain binds to. It potentiates cell death induced by Ipaf and Ac-Ipaf, and co-expression of Sug1 and Ipaf induces caspase-8-dependent cell death. Cellular complexes formed by Ipaf and Sug1 contain caspase 8. Expression of Ac-Ipaf or co-expression of Sug1 with Ipaf results in the formation of cytoplasmic aggregates and caspase 8 activation. Sug1 co-expression enabled modification of Ipaf by ubiquitination. Tagging ubiquitin molecules to Ipaf led to aggregate formation, enhanced caspase 8 interaction and activation, resulting in induction of cell death. Using RNAi (RNA interference) and dominant-negative approaches, we have shown that cell death induced by Ac-Ipaf expression or by treatment with TNF-alpha (tumour necrosis factor alpha) or doxorubicin is dependent on Sug1. Our results suggest a role for ubiquitination of Ipaf that is enabled by its interaction with Sug1, leading to caspase 8 activation and cell death.