Structural and functional basis of low-affinity SAM/SAH-binding in the conserved MTase of the multi-segmented Alongshan virus distantly related to canonical unsegmented flaviviruses.

Alongshan virus (ALSV), a newly discovered member of unclassified Flaviviridae family, is able to infect humans. ALSV has a multi-segmented genome organization and is evolutionarily distant from canonical mono-segmented flaviviruses. The virus-encoded methyltransferase (MTase) plays an important role in viral replication. Here we show that ALSV MTase readily binds S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) but exhibits significantly lower affinities than canonical flaviviral MTases. Structures of ALSV MTase in the free and SAM/SAH-bound forms reveal that the viral enzyme possesses a unique loop-element lining side-wall of the SAM/SAH-binding pocket. While the equivalent loop in flaviviral MTases half-covers SAM/SAH, contributing multiple hydrogen-bond interactions; the pocket-lining loop of ALSV MTase is of short-length and high-flexibility, devoid of any physical contacts with SAM/SAH. Subsequent mutagenesis data further corroborate such structural difference affecting SAM/SAH-binding. Finally, we also report the structure of ALSV MTase bound with sinefungin, an SAM-analogue MTase inhibitor. These data have delineated the basis for the low-affinity interaction between ALSV MTase and SAM/SAH and should inform on antiviral drug design.

Development of a bispecific nanobody conjugate broadly neutralizes diverse SARS-CoV-2 variants and structural basis for its broad neutralization.

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and profound immune-escape capacity makes it an urgent need to develop broad-spectrum therapeutics. Nanobodies have recently attracted extensive attentions due to their excellent biochemical and binding properties. Here, we report two high-affinity nanobodies (Nb-015 and Nb-021) that target non-overlapping epitopes in SARS-CoV-2 S-RBD. Both nanobodies could efficiently neutralize diverse viruses of SARS-CoV-2. The neutralizing mechanisms for the two nanobodies are further delineated by high-resolution nanobody/S-RBD complex structures. In addition, an Fc-based tetravalent nanobody format is constructed by combining Nb-015 and Nb-021. The resultant nanobody conjugate, designated as Nb-X2-Fc, exhibits significantly enhanced breadth and potency against all-tested SARS-CoV-2 variants, including Omicron sub-lineages. These data demonstrate that Nb-X2-Fc could serve as an effective drug candidate for the treatment of SARS-CoV-2 infection, deserving further in-vivo evaluations in the future.

A Potent Neutralizing Nanobody Targeting the Spike Receptor-Binding Domain of SARS-CoV-2 and the Structural Basis of Its Intimate Binding.

The continuous spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) around the world has raised unprecedented challenges to the human society. Antibodies and nanobodies possessing neutralization activity represent promising drug candidates. In this study, we report the identification and characterization of a potent SARS-CoV-2 neutralizing nanobody that targets the viral spike receptor-binding domain (S-RBD). The nanobody, termed as Nb-007, engages SARS-CoV-2 S-RBD with the two-digit picomolar binding affinity and shows outstanding virus entry-inhibition activity. The complex structure of Nb-007 bound to SARS-CoV-2 S-RBD reveals an epitope that is partially overlapping with the binding site for the human receptor of angiotensin-converting enzyme 2 (ACE2). The nanobody therefore exerts neutralization by competing with ACE2 for S-RBD binding, which is further ascertained by our in-vitro biochemical analyses. Finally, we also show that Nb-007 reserves promising, though compromised, neutralization activity against the currently-circulating Delta variant and that fusion of the nanobody with Fc dramatically increases its entry-inhibition capacity. Taken together, these data have paved the way of developing Nb-007 as a drug-reserve for potential treatment of SARS-CoV-2 related diseases.

An engineered 5-helix bundle derived from SARS-CoV-2 S2 pre-binds sarbecoviral spike at both serological- and endosomal-pH to inhibit virus entry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related sarbecoviruses enter host cells by receptor-recognition and membrane-fusion. An indispensable step in fusion is the formation of 6-helix bundle by viral spike heptad repeats 1 and 2 (HR1 and HR2). Here, we report the construction of 5-helix bundle (5HB) proteins for virus infection inhibition. The optimal construct inhibits SARS-CoV-2 pseudovirus entry with sub-micromolar IC50. Unlike HR2-based peptides that cannot bind spike in the pre-fusion conformation, 5HB features with the capability of binding to pre-fusion spike. Furthermore, 5HB binds viral HR2 at both serological- and endosomal-pH, highlighting its entry-inhibition capacity when SARS-CoV-2 enters via either cell membrane fusion or endosomal route. Finally, we show that 5HB could neutralize S-mediated entry of the predominant SARS-CoV-2 variants and a wide spectrum of sarbecoviruses. These data provide proof-of-concept evidence that 5HB might be developed for the prevention and treatment of SARS-CoV-2 and other emerging sarbecovirus infections.

S19W, T27W, and N330Y mutations in ACE2 enhance SARS-CoV-2 S-RBD binding toward both wild-type and antibody-resistant viruses and its molecular basis.

SARS-CoV-2 recognizes, via its spike receptor-binding domain (S-RBD), human angiotensin-converting enzyme 2 (ACE2) to initiate infection. Ecto-domain protein of ACE2 can therefore function as a decoy. Here we show that mutations of S19W, T27W, and N330Y in ACE2 could individually enhance SARS-CoV-2 S-RBD binding. Y330 could be synergistically combined with either W19 or W27, whereas W19 and W27 are mutually unbeneficial. The structures of SARS-CoV-2 S-RBD bound to the ACE2 mutants reveal that the enhanced binding is mainly contributed by the van der Waals interactions mediated by the aromatic side-chains from W19, W27, and Y330. While Y330 and W19/W27 are distantly located and devoid of any steric interference, W19 and W27 are shown to orient their side-chains toward each other and to cause steric conflicts, explaining their incompatibility. Finally, using pseudotyped SARS-CoV-2 viruses, we demonstrate that these residue substitutions are associated with dramatically improved entry-inhibition efficacy toward both wild-type and antibody-resistant viruses. Taken together, our biochemical and structural data have delineated the basis for the elevated S-RBD binding associated with S19W, T27W, and N330Y mutations in ACE2, paving the way for potential application of these mutants in clinical treatment of COVID-19.

Reduction of monocyte chemoattractant protein-1 and interleukin-8 levels by ticlopidine in TNF-alpha stimulated human umbilical vein endothelial cells.

Atherosclerosis and its associated complications represent major causes of morbidity and mortality in the industrialized or Western countries. Monocyte chemoattractant protein-1 (MCP-1) is critical for the initiating and developing of atherosclerotic lesions. Interleukin-8 (IL-8), a CXC chemokine, stimulates neutrophil chemotaxis. Ticlopidine is one of the antiplatelet drugs used to prevent thrombus formation relevant to the pathophysiology of atherothrombosis. In this study, we found that ticlopidine dose-dependently decreased the mRNA and protein levels of TNF-alpha-stimulated MCP-1, IL-8, and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Ticlopidine declined U937 cells adhesion and chemotaxis as compared to TNF-alpha stimulated alone. Furthermore, the inhibitory effects were neither due to decreased HUVEC viability, nor through NF-kB inhibition. These results suggest that ticlopidine decreased TNF-alpha induced MCP-1, IL-8, and VCAM-1 levels in HUVECs, and monocyte adhesion. Therefore, the data provide additional therapeutic machinery of ticlopidine in treatment and prevention of atherosclerosis.

Performance characteristics of DRI, CEDIA, and REMEDi systems for preliminary tests of amphetamines and opiates in human urine.

Arrestee urine specimens (930) were tested with DRI, CEDIA, and REMEDi; those that tested positive for amphetamines and opiates (616 and 414, respectively) were then confirmed by gas chromatography-mass spectrometry. The performance characteristics of these three preliminary systems were evaluated using the following commonly used parameters: true positive, true negative, false positive, and false negative. The sensitivity, specificity, and efficiency of these methods were also calculated. Data derived from this study indicated DRI and CEDIA adapted by this study generated acceptable preliminary test results for amphetamine/methamphetamine and morphine/codeine, but not for MDA/MDMA and REMEDi has lower sensitivity than DRI and CEDIA, but with better specificity and efficiency, supporting its use under emergency room settings where drug concentrations in overdose cases are expectedly at high levels.