A Prototype Assay Multiplexing SARS-CoV-2 3CL-Protease and Angiotensin-Converting Enzyme 2 for Saliva-Based Diagnostics in COVID-19.

With the current state of COVID-19 changing from a pandemic to being more endemic, the priorities of diagnostics will likely vary from rapid detection to stratification for the treatment of the most vulnerable patients. Such patient stratification can be facilitated using multiple markers, including SARS-CoV-2-specific viral enzymes, like the 3CL protease, and viral-life-cycle-associated host proteins, such as ACE2. To enable future explorations, we have developed a fluorescent and Raman spectroscopic SARS-CoV-2 3CL protease assay that can be run sequentially with a fluorescent ACE2 activity measurement within the same sample. Our prototype assay functions well in saliva, enabling non-invasive sampling. ACE2 and 3CL protease activity can be run with minimal sample volumes in 30 min. To test the prototype, a small initial cohort of eight clinical samples was used to check if the assay could differentiate COVID-19-positive and -negative samples. Though these small clinical cohort samples did not reach statistical significance, results trended as expected. The high sensitivity of the assay also allowed the detection of a low-activity 3CL protease mutant.

Translation of a Protease Turnover Assay for Clinical Discrimination of Mucinous Pancreatic Cysts.

The classification of pancreatic cyst fluids can provide a basis for the early detection of pancreatic cancer while eliminating unnecessary procedures. A candidate biomarker, gastricsin (pepsin C), was found to be present in potentially malignant mucinous pancreatic cyst fluids. A gastricsin activity assay using a magnetic bead-based platform has been developed using immobilized peptide substrates selective for gastricsin bearing a dimeric rhodamine dye. The unique dye structure allows quantitation of enzyme-cleaved product by both fluorescence and surface enhanced Raman spectroscopy (SERS). The performance of this assay was compared with ELISA assays of pepsinogen C and the standard of care, carcinoembryonic antigen (CEA), in the same clinical sample cohort. A retrospective cohort of mucinous (n = 40) and non-mucinous (n = 29) classes of pancreatic cyst fluid samples were analyzed using the new protease activity assay. For both assay detection modes, successful differentiation of mucinous and non-mucinous cyst fluid was achieved using 1 µL clinical samples. The activity-based assays in combination with CEA exhibit optimal sensitivity and specificity of 87% and 93%, respectively. The use of this gastricsin activity assay requires a minimal volume of clinical specimen, offers a rapid assay time, and shows improvements in the differentiation of mucinous and non-mucinous cysts using an accurate standardized readout of product formation, all without interfering with the clinical standard of care.

Inhibition of Band 3 tyrosine phosphorylation: a new mechanism for treatment of sickle cell disease.

Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso-occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin-actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane-derived microparticles (which initiate the coagulation cascade) and release of cell-free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso-occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell-free Hb, inhibit discharge of membrane-derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.

Natural Seminal Amyloids as Targets for Development of Synthetic Inhibitors of HIV Transmission.

Amyloids refer to a class of protein or peptide aggregates that are heterogeneous in size, morphology, and composition, and are implicated to play a central role in many neurodegenerative and systemic diseases. The strong correlation between biological activity and extent of aggregation of amyloidogenic proteins and peptides has led to an explosion of research efforts to target these materials with synthetic molecules or engineered antibodies to try to attenuate their function in disease pathology. Although many of these efforts to attenuate amyloid function have shown great promise in laboratory settings, the vast majority of work has been focused on targeting amyloids associated with neurologic diseases, which has been met with significant additional challenges that preclude clinical evaluation. Only recently have researchers started applying their efforts toward neutralizing the activity of amyloids associated with non-neurologic diseases. For instance, small peptides present in high abundance in human semen have been found to aggregate into amyloid-like fibrils, with in vitro experiments indicating that these amyloid fibrils could potentially increase the rate of infection of pathogens such as HIV by over 400 000-fold during sexual contact. Mechanistic investigations of naturally occurring seminal amyloid species such as Semen-derived Enhancer of Virus Infection (SEVI) and related natural peptide aggregates suggest that these materials interact strongly with virus particles and cell surfaces, facilitating viral attachment and internalization into cells and, thus, possibly promoting sexual transmission of disease. Such amyloid mediators in HIV transmission represent an attractive target for development of chemical approaches to attenuate their biological activity. For instance, the activity of seminal amyloids in genital fluids potentially allows for topical delivery of amyloid-targeting molecules, which could minimize common problems with systemic toxicity or permeability across biological barriers. In addition, molecules that target these amyloid mediators in viral attachment could potentially work synergistically with current antiviral agents to reduce the rate of HIV transmission. This Account will briefly summarize some of the key evidence in support of the capability of SEVI to enhance viral infection, and will highlight examples, many from our group, of recent efforts aimed at inhibiting its activity using synthetic small molecules, oligomeric peptides, and polymeric materials. We present various chemical strategies that have shown promise for neutralizing the role of SEVI in HIV transmission including the development of aggregation inhibitors of SEVI fibril formation, small molecule amyloid binders that modulate the charge or structure of SEVI, and synthetic molecules that form bioresistive coatings on SEVI and inhibit its interaction with the virus or cell surface. We discuss some unique challenges that hamper translation of these molecular strategies toward clinical evaluation, and propose several opportunities for researchers to address these challenges.

Hydrophobic Nanoparticles Reduce the ß-Sheet Content of SEVI Amyloid Fibrils and Inhibit SEVI-Enhanced HIV Infectivity.

Semen-derived enhancer of virus infection (SEVI) fibrils are naturally abundant amyloid aggregates found in semen that facilitate viral attachment and internalization of human immunodeficiency virus (HIV) in cells, thereby increasing the probability of infection. Mature SEVI fibrils are composed of aggregated peptides exhibiting high ß-sheet secondary structural characteristics. Herein, we show that polymers containing hydrophobic side chains can interact with SEVI and reduce its ß-sheet content by ∼45% compared with the ß-sheet content of SEVI in the presence of polymers with hydrophilic side chains, as estimated by polarization modulation-infrared reflectance absorption spectroscopy measurements. A nanoparticle (NP) formulation of this hydrophobic polymer reduced SEVI-mediated HIV infection in TMZ-bl cells by 60% compared with the control treatment. Although these NPs lacked specific amyloid-targeting groups, thus requiring high concentrations to observe biological activity, the use of hydrophobic interactions to alter the secondary structure of amyloids represents a useful approach to neutralizing the SEVI function. These results could, therefore, have general implications in the design of novel materials that can modify the activity of amyloids associated with a variety of other neurological and systemic diseases.

Inhibition of the enhancement of infection of human immunodeficiency virus by semen-derived enhancer of virus infection using amyloid-targeting polymeric nanoparticles.

The semen-derived enhancer of virus infection (SEVI) is a natural amyloid material that has been shown to substantially increase viral attachment and infectivity of HIV in cells. We previously reported that synthetic monomeric and oligomeric amyloid-targeting molecules could form protein-resistive coatings on SEVI and inhibit SEVI- and semen-mediated enhancement of HIV infectivity. While oligomeric amyloid-binding compounds showed substantial improvement in apparent binding to SEVI compared to monomeric compounds, we observed only a modest correlation between apparent binding to SEVI and activity for reducing SEVI-mediated HIV infection. Here, we synthesized amyloid-binding polyacrylate-based polymers and polymeric nanoparticles of comparable size to HIV virus particles (∼150 nm) to assess the effect of sterics on the inhibition of SEVI-mediated enhancement of HIV infectivity. We show that these polymeric materials exhibit excellent capability to reduce SEVI-mediated enhancement of HIV infection, with the nanoparticles exhibiting the greatest activity (IC50 value of ∼4 µg/mL, or 59 nM based on polymer) of any SEVI-neutralizing agent reported to date. The results support that the improved activity of these nanomaterials is likely due to their increased size (diameters = 80-200 nm) compared to amyloid-targeting small molecules and that steric interactions may play as important a role as binding affinity in inhibiting viral infection mediated by SEVI amyloids. In contrast to the previously reported SEVI-neutralizing, amyloid-targeting molecules (which required concentrations at least 100-fold above the Kd to observe activity), the approximate 1:1 ratio of apparent Kd to IC50 for activity of these polymeric materials suggests the majority of polymer molecules that are bound to SEVI contribute to the inhibition of HIV infectivity enhanced by SEVI. Such size-related effects on physical inhibition of protein-protein interactions may open further opportunities for the use of targeted nanomaterials in disease intervention.