Polyvalent Nanobody Structure Designed for Boosting SARS-CoV-2 Inhibition.

Coronavirus transmission and mutations have brought intensive challenges on pandemic control and disease treatment. Developing robust and versatile antiviral drugs for viral neutralization is highly desired. Here, we created a new polyvalent nanobody (Nb) structure that shows the effective inhibition of SARS-CoV-2 infections. Our polyvalent Nb structure, called "PNS", is achieved by first conjugating single-stranded DNA (ssDNA) and the receptor-binding domain (RBD)-targeting Nb with retained binding ability to SARS-CoV-2 spike protein and then coalescing the ssDNA-Nb conjugates around a gold nanoparticle (AuNP) via DNA hybridization with a desired Nb density that offers spatial pattern-matching with that of the Nb binding sites on the trimeric spike. The surface plasmon resonance (SPR) assays show that the PNS binds the SARS-CoV-2 trimeric spike proteins with a ∼1000-fold improvement in affinity than that of monomeric Nbs. Furthermore, our viral entry inhibition assays using the PNS against SARS-CoV-2 WA/2020 and two recent variants of interest (BQ1.1 and XBB) show an over 400-fold enhancement in viral inhibition compared to free Nbs. Our PNS strategy built on a new DNA-protein conjugation chemistry provides a facile approach to developing robust virus inhibitors by using a corresponding virus-targeting Nb with a desired Nb density.

APIPred: An XGBoost-Based Method for Predicting Aptamer-Protein Interactions.

Aptamers are single-stranded DNA or RNA oligos that can bind to a variety of targets with high specificity and selectivity and thus are widely used in the field of biosensing and disease therapies. Aptamers are generated by SELEX, which is a time-consuming procedure. In this study, using in silico and computational tools, we attempt to predict whether an aptamer can interact with a specific protein target. We present multiple data representations of protein and aptamer pairs and multiple machine-learning-based models to predict aptamer-protein interactions with a fair degree of selectivity. One of our models showed 96.5% accuracy and 97% precision, which are significantly better than those of the previously reported models. Additionally, we used molecular docking and SPR binding assays for two aptamers and the predicted targets as examples to exhibit the robustness of the APIPred algorithm. This reported model can be used for the high throughput screening of aptamer-protein pairs for targeting cancer and rapidly evolving viral epidemics.

Structural snapshots of Mycobacterium tuberculosis enolase reveal dual mode of 2PG binding and its implication in enzyme catalysis.

Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of MgB is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.

Designer DNA NanoGripper.

DNA has shown great biocompatibility, programmable mechanical properties, and structural addressability at the nanometer scale, making it a versatile material for building high precision nanorobotics for biomedical applications. Herein, we present design principle, synthesis, and characterization of a DNA nanorobotic hand, called the "NanoGripper", that contains a palm and four bendable fingers as inspired by human hands, bird claws, and bacteriophages evolved in nature. Each NanoGripper finger has three phalanges connected by two flexible and rotatable joints that are bendable in response to binding to other entities. Functions of the NanoGripper have been enabled and driven by the interactions between moieties attached to the fingers and their binding partners. We showcase that the NanoGripper can be engineered to interact with and capture various objects with different dimensions, including gold nanoparticles, gold NanoUrchins, and SARS-CoV-2 virions. When carrying multiple DNA aptamer nanoswitches programmed to generate fluorescent signal enhanced on a photonic crystal platform, the NanoGripper functions as a sensitive viral biosensor that detects intact SARS-CoV-2 virions in human saliva with a limit of detection of ~ 100 copies/mL, providing RT-PCR equivalent sensitivity. Additionally, we use confocal microscopy to visualize how the NanoGripper-aptamer complex can effectively block viral entry into the host cells, indicating the viral inhibition. In summary, we report the design, synthesis, and characterization of a complex nanomachine that can be readily tailored for specific applications. The study highlights a path toward novel, feasible, and efficient solutions for the diagnosis and therapy of other diseases such as HIV and influenza.

Net-Shaped DNA Nanostructures Designed for Rapid/Sensitive Detection and Potential Inhibition of the SARS-CoV-2 Virus.

We present a net-shaped DNA nanostructure (called "DNA Net" herein) design strategy for selective recognition and high-affinity capture of intact SARS-CoV-2 virions through spatial pattern-matching and multivalent interactions between the aptamers (targeting wild-type spike-RBD) positioned on the DNA Net and the trimeric spike glycoproteins displayed on the viral outer surface. Carrying a designer nanoswitch, the DNA Net-aptamers release fluorescence signals upon virus binding that are easily read with a handheld fluorimeter for a rapid (in 10 min), simple (mix-and-read), sensitive (PCR equivalent), room temperature compatible, and inexpensive (∼$1.26/test) COVID-19 test assay. The DNA Net-aptamers also impede authentic wild-type SARS-CoV-2 infection in cell culture with a near 1 × 103-fold enhancement of the monomeric aptamer. Furthermore, our DNA Net design principle and strategy can be customized to tackle other life-threatening and economically influential viruses like influenza and HIV, whose surfaces carry class-I viral envelope glycoproteins like the SARS-CoV-2 spikes in trimeric forms.

Malaria parasites utilize two essential plasma membrane fusogens for gamete fertilization.

Cell fusion of female and male gametes is the climax of sexual reproduction. In many organisms, the Hapless 2 (HAP2) family of proteins play a critical role in gamete fusion. We find that Plasmodium falciparum, the causative agent of human malaria, expresses two HAP2 proteins: PfHAP2 and PfHAP2p. These proteins are present in stage V gametocytes and localize throughout the flagellum of male gametes. Gene deletion analysis and genetic crosses show that PfHAP2 and PfHAP2p individually are essential for male fertility and thereby, parasite transmission to the mosquito. Using a cell fusion assay, we demonstrate that PfHAP2 and PfHAP2p are both authentic plasma membrane fusogens. Our results establish nonredundant essential roles for PfHAP2 and PfHAP2p in mediating gamete fusion in Plasmodium and suggest avenues in the design of novel strategies to prevent malaria parasite transmission from humans to mosquitoes.

Characterization of a triazole scaffold compound as an inhibitor of Mycobacterium tuberculosis imidazoleglycerol-phosphate dehydratase.

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), employs ten enzymes including imidazoleglycerol-phosphate dehydratase (IGPD) for de novo biosynthesis of histidine. The absence of histidine-biosynthesis in humans combined with its essentiality for Mtb makes the enzymes of this pathway major anti-TB drug targets. We explored the inhibitory potential of a small molecule ß-(1,2,4-Triazole-3-yl)-DL-alanine (DLA) against Mtb IGPD. DLA exhibits an in vitro inhibitory efficacy in the lower micromolar range. Higher-resolution crystal structures of native and substrate-bound Mtb IGPD provided additional structural features of this important drug target. Crystal structure of IGPD-DLA complex at a resolution of 1.75 Å, confirmed that DLA locks down the function of the enzyme by binding in the active site pocket of the IGPD mimicking the substrate-binding mode to a high degree. In our biochemical study, DLA showed an efficient inhibition of Mtb IGPD. Furthermore, DLA also showed bactericidal activity against Mtb and Mycobacterium smegmatis and inhibited their growth in respective culture medium. Importantly, owing to the favorable ADME and physicochemical properties, it serves as an important lead molecule for further derivatizations.

Characterization of the NiRAN domain from RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV-2.

Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, a well-known kinase inhibitor- Sorafenib showed a significant inhibition and dampened viral load in SARS-CoV-2 infected cells. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.

Characterization of putative transcriptional regulator (PH0140) and its distal homologue.

In this study, a phylogenetic tree was constructed using 1854 sequences of various Lrp/AnsC (FFRPs) and ArsR proteins from pathogenic and non-pathogenic organisms. Despite having sequence similarities, FFRPs and ArsR proteins functioning differently as a transcriptional regulator and de-repressor in the presence of exogenous amino acids and metal ions, respectively. To understand these functional differences, the structures of various FFRPs and ArsR proteins (134 sequences) were modeled. Several ArsR proteins exhibited high similarity to the FFRPs while in few proteins, unusual structural folds were observed. However, the Helix-turn-Helix (HTH) domains are common among them and the ligand-binding domains are structurally dissimilar suggest the differences in their binding preferences. Despite low sequence conservation, most of these proteins revealed negatively charged surfaces in the active site pockets. Representative structures (PH0140 and TtArsR protein) from FFRPs and ArsR protein families were considered and evaluated for their functional differences using molecular modeling studies. Our earlier study has explained the binding preference of exogenous Tryptophan and the related transcriptional regulatory mechanism of PH0140 protein. In this study, a Cu2+ ion-induced de-repression mechanism of the TtArsR-DNA complex was characterized through docking and molecular dynamics. Further, the proteins were purified and their efficiency for sensing Tryptophan and Cu2+ ions were analyzed using cyclic voltammetry. Overall, the study explores the structural evolution and functional difference of FFRPs and ArsR proteins that present the possibilities of PH0140 and TtArsR as potential bio-sensory molecules.