Theranostic Diagnostics.

Diagnosing and then treating disease defines theranostics. The approach holds promise by facilitating targeted disease outcomes. The simultaneous analysis of finding the presence of disease pathophysiology while providing a parallel in treatment is a novel and effective strategy for seeking improved medical care. We discuss how theranostics improves disease outcomes is discussed. The chapter reviews the delivery of targeted therapies. Bioimaging techniques are highlighted as early detection and tracking systems for microbial infections, degenerative diseases, and cancers.

Thiol-Mediated Enhancement of Nε-Acetyllysine Formation in Lens Proteins.

Lysine acetylation (AcK) is a prominent post-translational modification in eye lens crystallins. We have observed that AcK formation is preferred in some lysine residues over others in crystallins. In this study, we have investigated the role of thiols in such AcK formation. Upon incubation with acetyl-CoA (AcCoA), αA-Crystallin, which contains two cysteine residues, showed significantly higher levels of AcK than αB-Crystallin, which lacks cysteine residues. Incubation with thiol-rich γS-Crystallin resulted in higher AcK formation in αB-Crystallin from AcCoA. External free thiol (glutathione and N-acetyl cysteine) increased the AcK content in AcCoA-incubated αB-Crystallin. Reductive alkylation of cysteine residues significantly decreased (p < 0.001) the AcCoA-mediated AcK formation in αA-Crystallin. Introduction of cysteine residues within ∼5 Å of lysine residues (K92C, E99C, and V169C) in αB-Crystallin followed by incubation with AcCoA resulted in a 3.5-, 1.3- and 1.3-fold increase in the AcK levels when compared to wild-type αB-Crystallin, respectively. Together, these results suggested that AcK formation in α-Crystallin is promoted by the proximal cysteine residues and protein-free thiols through an S → N acetyl transfer mechanism.

Beyond Lipids: Exploring Advances in Polymeric Gene Delivery in the Lipid Nanoparticles Era.

The recent success of gene therapy during the COVID-19 pandemic has underscored the importance of effective and safe delivery systems. Complementing lipid-based delivery systems, polymers present a promising alternative for gene delivery. Significant advances have been made in the recent past, with multiple clinical trials progressing beyond phase I and several companies actively working on polymeric delivery systems which provides assurance that polymeric carriers can soon achieve clinical translation. The massive advantage of structural tunability and vast chemical space of polymers is being actively leveraged to mitigate shortcomings of traditional polycationic polymers and improve the translatability of delivery systems. Tailored polymeric approaches for diverse nucleic acids and for specific subcellular targets are now being designed to improve therapeutic efficacy. This review describes the recent advances in polymer design for improved gene delivery by polyplexes and covalent polymer-nucleic acid conjugates. The review also offers a brief note on novel computational techniques for improved polymer design. The review concludes with an overview of the current state of polymeric gene therapies in the clinic as well as future directions on their translation to the clinic.

CCR5 Decorated Rilpivirine Lipid Nanoparticles Build Myeloid Drug Depots Which Sustains Antiretroviral Activities.

Antiretroviral therapy (ART) improves the quality of life for those living with the human immunodeficiency virus type one (HIV-1). However, poor compliance reduces ART effectiveness and leads to immune compromise, viral mutations, and disease co-morbidities. A novel drug formulation is made whereby a lipid nanoparticle (LNP) carrying rilpivirine (RPV) is decorated with the C-C chemokine receptor type 5 (CCR5). This facilitates myeloid drug depot deposition. Particle delivery to viral reservoirs is tracked by positron emission tomography. The CCR5-mediated RPV LNP cell uptake and retention reduce HIV-1 replication in human monocyte-derived macrophages and infected humanized mice. Focused ultrasound allows the decorated LNP to penetrate the blood-brain barrier and reach brain myeloid cells. These findings offer a role for CCR5-targeted therapeutics in antiretroviral delivery to optimize HIV suppression.

Polymer Delivery Systems for Long-Acting Antiretroviral Drugs.

The success of long-acting (LA) drug delivery systems (DDSs) is linked to their biocompatible polymers. These are used for extended therapeutic release. For treatment or prevention of human immune deficiency virus type one (HIV-1) infection, LA DDSs hold promise for improved regimen adherence and reduced toxicities. Current examples include Cabenuva, Apretude, and Sunlenca. Each is safe and effective. Alternative promising DDSs include implants, prodrugs, vaginal rings, and microarray patches. Each can further meet patients' needs. We posit that the physicochemical properties of the formulation chemical design can optimize drug release profiles. We posit that the strategic design of LA DDS polymers will further improve controlled drug release to simplify dosing schedules and improve regimen adherence.

Biophysical evolution of the receptor-binding domains of SARS-CoVs.

With hundreds of coronaviruses (CoVs) identified in bats that can infect humans, it is essential to understand how CoVs that affected the human population have evolved. Seven known CoVs have infected humans, of which three CoVs caused severe disease with high mortalities: severe acute respiratory syndrome (SARS)-CoV emerged in 2002, Middle East respiratory syndrome-CoV in 2012, and SARS-CoV-2 in 2019. SARS-CoV and SARS-CoV-2 belong to the same family, follow the same receptor pathway, and use their receptor-binding domain (RBD) of spike protein to bind to the angiotensin-converting enzyme 2 (ACE2) receptor on the human epithelial cell surface. The sequence of the two RBDs is divergent, especially in the receptor-binding motif that directly interacts with ACE2. We probed the biophysical differences between the two RBDs in terms of their structure, stability, aggregation, and function. Since RBD is being explored as an antigen in protein subunit vaccines against CoVs, determining these biophysical properties will also aid in developing stable protein subunit vaccines. Our results show that, despite RBDs having a similar three-dimensional structure, they differ in their thermodynamic stability. RBD of SARS-CoV-2 is significantly less stable than that of SARS-CoV. Correspondingly, SARS-CoV-2 RBD shows a higher aggregation propensity. Regarding binding to ACE2, less stable SARS-CoV-2 RBD binds with a higher affinity than more stable SARS-CoV RBD. In addition, SARS-CoV-2 RBD is more homogenous in terms of its binding stoichiometry toward ACE2 compared to SARS-CoV RBD. These results indicate that SARS-CoV-2 RBD differs from SARS-CoV RBD in terms of its stability, aggregation, and function, possibly originating from the diverse receptor-binding motifs. Higher aggregation propensity and decreased stability of SARS-CoV-2 RBD warrant further optimization of protein subunit vaccines that use RBD as an antigen by inserting stabilizing mutations or formulation screening.

Promotion of Protein Solubility and Reduction in Stiffness in Human Lenses by Aggrelyte-1: Implications for Reversing Presbyopia.

With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. An increase in stiffness or decrease in lens elasticity due to protein aggregation and insolubilization are the primary reasons for presbyopia. In this study, we tested aggrelyte-1 (S,N-diacetyl glutathione diethyl ester) for its ability to promote protein solubility and decrease the stiffness of lenses through its dual property of lysine acetylation and disulfide reduction. Treatment of water-insoluble proteins from aged human lenses (58-75 years) with aggrelyte-1 significantly increased the solubility of those proteins. A control compound that did not contain the S-acetyl group (aggrelyte-1C) was substantially less efficient in solubilizing water-insoluble proteins. Aggrelyte-1-treated solubilized protein had significant amounts of acetyllysine, as measured by Western blotting and LC-MS/MS. Aggrelytes increased the protein-free thiol content in the solubilized protein. Aged mouse (7 months) and human (44-66 years) lenses treated with aggrelyte-1 showed reduced stiffness accompanied by higher free thiol and acetyllysine levels compared with those treated with aggrelyte-1C or untreated controls. Our results suggested that aggrelyte-1 reduced lens stiffness through acetylation followed by disulfide reduction. This proof-of-concept study paves the way for developing aggrelyte-1 and related compounds to reverse presbyopia.

Aggrelyte-2 promotes protein solubility and decreases lens stiffness through lysine acetylation and disulfide reduction: Implications for treating presbyopia.

Aging proteins in the lens become increasingly aggregated and insoluble, contributing to presbyopia. In this study, we investigated the ability of aggrelyte-2 (N,S-diacetyl-L-cysteine methyl ester) to reverse the water insolubility of aged human lens proteins and to decrease stiffness in cultured human and mouse lenses. Water-insoluble proteins (WI) of aged human lenses (65-75 years) were incubated with aggrelyte-2 (500 µM) for 24 or 48 h. A control compound that lacked the S-acetyl group (aggrelyte-2C) was also tested. We observed 19%-30% solubility of WI upon treatment with aggrelyte-2. Aggrelyte-2C also increased protein solubility, but its effect was approximately 1.4-fold lower than that of aggrelyte-2. The protein thiol contents were 1.9- to 4.9-fold higher in the aggrelyte-2- and aggrelyte-2C-treated samples than in the untreated samples. The LC-MS/MS results showed Nε -acetyllysine (AcK) levels of 1.5 to 2.1 nmol/mg protein and 0.6 to 0.9 nmol/mg protein in the aggrelyte-2- and aggrelyte-2C-treated samples. Mouse (C57BL/6J) lenses (incubated for 24 h) and human lenses (incubated for 72 h) with 1.0 mM aggrelyte-2 showed significant decreases in stiffness with simultaneous increases in soluble proteins (human lenses) and protein-AcK levels, and such changes were not observed in aggrelyte-2C-treated lenses. Mass spectrometry of the solubilized protein revealed AcK in all crystallins, but more was observed in α-crystallins. These results suggest that aggrelyte-2 increases protein solubility and decreases lens stiffness through acetylation and disulfide reduction. Aggrelyte-2 might be useful in treating presbyopia in humans.

Oral Delivery of Nucleic Acid Therapies for Local and Systemic Action.

Nucleic acid (NA) therapy has gained importance over the past decade due to its high degree of selectivity and minimal toxic effects over conventional drugs. Currently, intravenous (IV) or intramuscular (IM) formulations constitute majority of the marketed formulations containing nucleic acids. However, oral administration is traditionally preferred due to ease of administration as well as higher patient compliance. To leverage the benefits of oral delivery for NA therapy, the NA of interest must be delivered to the target site avoiding all degrading and inhibiting factors during its transition through the gastrointestinal tract. The oral route presents myriad of challenges to NA delivery, making formulation development challenging. Researchers in the last few decades have formulated various delivery systems to overcome such challenges and several reviews summarize and discuss these strategies in detail. However, there is a need to differentiate between the approaches based on target so that in future, delivery strategies can be developed according to the goal of the study and for efficient delivery to the desired site. The goal of this review is to summarize the mechanisms for target specific delivery, list and discuss the formulation strategies used for oral delivery of NA therapies and delineate the similarities and differences between local and systemic targeting oral delivery systems and current challenges.

Advances in Lipid-Based Codelivery Systems for Cancer and Inflammatory Diseases.

Combination therapy targeting multiple therapeutic targets is a favorable strategy to achieve better therapeutic outcomes in cancer and inflammatory diseases. Codelivery is a subfield of drug delivery that aims to achieve combined delivery of diverse therapeutic cargoes within the same delivery system, thereby ensuring delivery to the same site and providing an opportunity to tailor the release kinetics as desired. Among the wide range of materials being investigated in the design of codelivery systems, lipids have stood out on account of their low toxicity, biocompatibility, and ease of formulation scale-up. This review highlights the advances of the last decade in lipid-based codelivery systems focusing on the codelivery of drug-drug, drug-nucleic acid, nucleic acid-nucleic acid, and protein therapeutic-based combinations for targeted therapy in cancer and inflammatory diseases.

Topical ocular application of aggrelyte-2A reduces lens stiffness in mice.

Presbyopia is the progressive loss of the ability of the lens to focus on nearby objects due to its increased stiffness. It occurs in the mid-40s and continues to worsen until the mid-60s. The age-associated increase in protein cross-linking in the lens leads to protein aggregation and water insolubility, especially in the nuclear region, contributing to lens stiffness. This study reports the development of aggrelyte-2A (methyl S-acetyl-N-(3,3-dimethylbutanoyl) cysteinate, a derivative of our previously reported aggrelyte-2) for reversing the stiffness of aged lenses. Aggrelyte-2A showed minimal toxicity in cultured mouse lens epithelial cells (up to 2000 µM) and human lens epithelial cells (up to 250 µM). Lenses from aged mice (age: 24-25 months) treated with 1 mM aggrelyte-2A for 24 h, and human lenses (age: 47-67 years) treated with 250 µM aggrelyte-2A for 48 h showed 11-14% reductions in stiffness, accompanied by an increase in acetyllysine in lens proteins, and free-thiols in the lens. Topical application of aggrelyte-2A (40 mM, 5 µl twice daily for 4 weeks) on mouse eyes significantly reduced lens stiffness. The topical application showed no toxicity to the lens, cornea, or retina, as revealed by morphological examination, H&E staining, and optical coherence tomography. These data suggest that aggrelyte-2A could be developed as a presbyopia-reversing therapeutic.

Polymeric Chloroquine as an Effective Antimigration Agent in the Treatment of Pancreatic Cancer.

Hydroxychloroquine (HCQ) has been the subject of multiple recent preclinical and clinical studies for its beneficial use in the combination treatments of different types of cancers. Polymeric HCQ (PCQ), a macromolecular multivalent version of HCQ, has been shown to be effective in various cancer models both in vitro and in vivo as an inhibitor of cancer cell migration and experimental lung metastasis. Here, we present detailed in vitro studies that show that low concentrations of PCQ can efficiently inhibit cancer cell migration and colony formation orders of magnitude more effectively compared to HCQ. After intraperitoneal administration of PCQ in vivo, high levels of tumor accumulation and penetration are observed, combined with strong antimetastatic activity in an orthotopic pancreatic cancer model. These studies support the idea that PCQ may be effectively used at low doses as an adjuvant in the therapy of pancreatic cancer. In conjunction with previously published literature, these studies further undergird the potential of PCQ as an anticancer agent.

FAD-deficient P187S mutation of NAD(P)H:quinone oxidoreductase 1 (NQO1*2) binds and accelerates ß-amyloid aggregation.

Alzheimer's disease (AD) is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NAD(P)H quinone oxidoreductase 1 (NQO1) may accelerate the aggregation of Alzheimer's amyloid-ß peptide (Aß1-42). Here, we examined in vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Aß1-42 and modify its rate of aggregation. When monitored using the fluorescence of either noncovalent thioflavin T (ThT) or HiLyte Fluor 647 (HF647) dye covalently attached to the Aß1-42 peptide, the aggregation kinetics of Aß1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type (WT) NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor that binds near the FAD-binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Aß1-42. Imaging flow cytometry confirmed in-vitro coaggregation of NQO1 isoforms and Aß1-42. Aß1-42 alone forms rod-shaped fibril structures while in the presence of NQO1 isoforms, Aß1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry (ITC) analysis indicates that Aß1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of coaggregates of Aß1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.

Hyaluronate-coated perfluoroalkyl polyamine prodrugs as bioactive siRNA delivery systems for the treatment of peritoneal cancers.

RNA interference (RNAi) is an emerging therapeutic modality for cancer, which remains in critical need of effective delivery vectors due to the unfavorable biopharmaceutical properties of small RNAs. Polyamines are essential for functioning of mammalian cells. Dysregulated polyamine metabolism is found in many cancers and has been an attractive therapeutic target in combination therapies. Combination therapies based on drugs that affect polyamine metabolism and nucleic acids promise to enhance anticancer activity due to a cooperative effect on multiple oncogenic pathways. Here, we report bioactive polycationic prodrug (F-PaP) based on an anticancer polyamine analogue bisethylnorspermine (BENSpm) modified with perfluoroalkyl moieties. Following encapsulation of siRNA, F-PaP/siRNA nanoparticles were coated with hyaluronic acid (HA) to form ternary nanoparticles HA@F-PaP/siRNA. The presence of perfluoroalkyl moieties and HA reduced cell membrane toxicity and improved stability of the particles with cooperatively enhanced siRNA delivery in pancreatic and colon cancer cell lines. We then tested a therapeutic hypothesis that combining BENSpm with siRNA silencing of polo-like kinase 1 (PLK1) would result in cooperative cancer cell killing. HA@F-PaP/siPLK1 induced polyamine catabolism and cell cycle arrest, leading to enhanced apoptosis in the tested cell lines. The HA-coated nanoparticles facilitated tumor accumulation and contributed to strong tumor inhibition and favorable modulation of the immune tumor microenvironment in orthotopic pancreatic cancer model.

Hyaluronate-coated perfluoroalkyl polyamine prodrugs as bioactive siRNA delivery systems for the treatment of peritoneal cancers.

RNA interference (RNAi) is an emerging therapeutic modality for cancer, which remains in critical need of effective delivery vectors due to the unfavorable biopharmaceutical properties of small RNAs. Polyamines are essential for functioning of mammalian cells. Dysregulated polyamine metabolism is found in many cancers and has been an attractive therapeutic target in combination therapies. Combination therapies based on drugs that affect polyamine metabolism and nucleic acids promise to enhance anticancer activity due to a cooperative effect on multiple oncogenic pathways. Here, we report bioactive polycationic prodrug (F-PaP) based on an anticancer polyamine analog bisethylnorspermine (BENSpm) modified with perfluoroalkyl moieties. Following encapsulation of siRNA, F-PaP/siRNA nanoparticles were coated with hyaluronic acid (HA) to form ternary nanoparticles HA@F-PaP/siRNA. The presence of perfluoroalkyl moieties and HA reduced cell membrane toxicity and improved stability of the particles with cooperatively enhanced siRNA delivery in pancreatic and colon cancer cell lines. We then tested a therapeutic hypothesis that combining BENSpm with siRNA silencing of polo-like kinase 1 (PLK1) would result in cooperative cancer cell killing. HA@F-PaP/siPLK1 induced polyamine catabolism and cell cycle arrest, leading to enhanced apoptosis in the tested cell lines. The HA-coated nanoparticles facilitated tumor accumulation and contributed to strong tumor inhibition and favorable modulation of the immune tumor microenvironment in orthotopic pancreatic cancer model. Combination anticancer therapy with polyamine prodrug-mediated delivery of siRNA. Hyaluronate coating of the siRNA nanoparticles facilitates selective accumulation in orthotopic pancreatic tumors. Perfluoroalkyl conjugation reduces toxicity and improves gene silencing effect. Nanoparticle treatment induces polyamine catabolism and cell cycle arrest leading to strong tumor inhibition and favorable modulation of immune tumor microenvironment.

Modified chitosan for effective renal delivery of siRNA to treat acute kidney injury.

Acute kidney injury (AKI) is characterized by a sudden decrease in renal function and impacts growing number of people worldwide. RNA interference (RNAi) showed potential to treat diseases with no or limited conventional therapies, including AKI. Suitable carriers are needed to protect and selectively deliver RNAi to target cells to fully explore this therapeutic modality. Here, we report on the synthesis of chitosan modified with α-cyclam-p-toluic acid (C-CS) as a novel siRNA carrier for targeted delivery to injured kidneys. We demonstrate that conjugation of the α-cyclam-p-toluic acid to chitosan imparts the C-CS polymer with targeting and antagonistic properties to cells overexpressing chemokine receptor CXCR4. In contrast, the parent α-cyclam-p-toluic acid showed no such properties. Self-assembled C-CS/siRNA nanoparticles rapidly accumulate in the injured kidneys and show long retention in renal tubules. Apoptosis and metabolic and inflammatory pathways induced by p53 are important pathological mechanisms in the development of AKI. Nanoparticles with siRNA against p53 (sip53) were formulated and intravenously injected for attenuation of IRI-AKI. Due to the favorable accumulation in injured kidneys, the treatment with C-CS/sip53 decreased renal injury, extent of renal apoptosis, macrophage and neutrophil infiltration, and improved renal function. Overall, our study suggests that C-CS/siRNA nanoparticles have the potential to effectively accumulate and deliver therapeutic siRNAs to injured kidneys through CXCR4 binding, providing a novel way for AKI therapy.

Self-Assembled Alkylated Polyamine Analogs as Supramolecular Anticancer Agents.

Conformationally restrained polyamine analog PG11047 is a well-known drug candidate that modulates polyamine metabolism and inhibits cancer cell growth in a broad spectrum of cancers. Here, we report a structure-activity relationship study of the PG11047 analogs (HPGs) containing alkyl chains of varying length, while keeping the unsaturated spermine backbone unchanged. Synthesis of higher symmetrical homologues was achieved through a synthetic route with fewer steps than the previous route to PG11047. The amphiphilic HPG analogs underwent self-assembly and formed spherically shaped nanoparticles whose size increased with the hydrophobic alkyl group's increasing chain length. Assessment of the in vitro anticancer activity showed more than an eight-fold increase in the cancer cell inhibition activity of the analogs with longer alkyl chains compared to PG11047 in human colon cancer cell line HCT116, and a more than ten-fold increase in human lung cancer cell line A549. Evaluation of the inhibition of spermine oxidase (SMOX) showed no activity for PG11047, but activity was observed for its higher symmetrical homologues. Comparison with a reference SMOX inhibitor MDL72527 showed nine-fold better activity for the best performing HPG analog.

Study of Renal Accumulation of Targeted Polycations in Acute Kidney Injury.

Acute kidney injury (AKI) is a global healthcare burden characterized by rapid loss of renal function and high morbidity and mortality. Chemokine receptor CXCR4 participates in the renal infiltration of immune cells following injury and in local inflammatory enhancement. Injured renal tubule cells overexpress CXCR4, which could be used as a target for improved drug delivery in AKI. Plerixafor is a small-molecule CXCR4 antagonist that has shown beneficial effects against AKI and has been previously developed into a polymeric analog (polymeric plerixafor, PP). With the goal of gaining a better understanding of how overall charge and hydrophilicity affect renal accumulation of PP, we have synthesized PP copolymers containing hydroxyl, carboxyl, primary amine, and alkyl moieties using Michael-type addition copolymerization. All synthesized copolymers showed excellent CXCR4-binding and inhibiting ability in vitro and improved cellular uptake in hypoxia-reoxygenation stimulated mouse tubule cells. Analysis of serum protein binding revealed that polymers with hydroxyl group modification showed the least amount of protein binding. Biodistribution of the polymers was tested in a unilateral ischemia reperfusion-induced AKI mouse model. The results showed significant differences in accumulation in the injured kidneys depending on the net charge and hydrophilicity of the polymers. The findings of this study will guide the development of polymeric drug carriers for targeted delivery to injured kidneys for better AKI therapy.

Biodegradable Elastomers Enabling Thermoprocessing Below 100 °C.

Biodegradable and biocompatible elastomers are highly desirable for many biomedical applications. Here, we report synthesis and characterization of poly(ε-caprolactone)-co-poly(ß-methyl-δ-valerolactone)-co-poly(ε-caprolactone) (PCL-PßMδVL-PCL) elastomers. These materials have strain to failure values greater than 1000%. Tensile set measurements according to an ASTM standard revealed a 98.24% strain recovery 10 min after the force was removed and complete strain recovery 40 min after the force was removed. The PßMδVL midblock is amorphous with a glass-transition temperature of -51 °C, and PCL end blocks are semicrystalline and have a melting temperature in the range of 52-55 °C. Due to their thermoplastic nature and the low melting temperature, these elastomers can be readily processed by printing, extrusion, or hot-pressing at 60 °C. Lysozyme, a model bioactive agent, was incorporated into a PCL-PßMδVL-PCL elastomer through melt blending in an extruder, and the blend was further hot-pressed into films; both processing steps were performed at 60 °C. No loss of lysozyme bioactivity was observed. PCL-PßMδVL-PCL elastomers are as cytocompatible as tissue culture polystyrene in supporting cell viability and cell growth, and they are degradable in aqueous environments through hydrolysis. The degradable, cytocompatible, elastomeric, and thermoplastic properties of PCL-PßMδVL-PCL polymers collectively render them potentially valuable for many applications in the biomedical field, such as medical devices and tissue engineering scaffolds.

Receptor binding, immune escape, and protein stability direct the natural selection of SARS-CoV-2 variants.

Emergence of new severe acute respiratory syndrome coronavirus 2 variants has raised concerns related to the effectiveness of vaccines and antibody therapeutics developed against the unmutated wildtype virus. Here, we examined the effect of the 12 most commonly occurring mutations in the receptor-binding domain of the spike protein on its expression, stability, activity, and antibody escape potential. Stability was measured using thermal denaturation, and the activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to the human angiotensin-converting enzyme 2 and to neutralizing human antibody CC12.1, respectively. Our results show that mutants differ in their expression levels. Of the eight best-expressed mutants, two (N501Y and K417T/E484K/N501Y) showed stronger affinity to angiotensin-converting enzyme 2 compared with the wildtype, whereas four (Y453F, S477N, T478I, and S494P) had similar affinity and two (K417N and E484K) had weaker affinity than the wildtype. Compared with the wildtype, four mutants (K417N, Y453F, N501Y, and K417T/E484K/N501Y) had weaker affinity for the CC12.1 antibody, whereas two (S477N and S494P) had similar affinity, and two (T478I and E484K) had stronger affinity than the wildtype. Mutants also differ in their thermal stability, with the two least stable mutants showing reduced expression. Taken together, these results indicate that multiple factors contribute toward the natural selection of variants, and all these factors need to be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.