Biology subjects

Hillyer, C. D.

Publications and source records attributed to Hillyer, C. D..

4 recordsLinked to original sources

bioRxiv2025

Mitofusin agonists enhances long-term engraftment and potency of cultured hematopoietic stem cells

Umbilical cord blood (CBU) is a valuable source of hematopoietic stem cells (HSCs) due to lower incidence of graft-versus-host disease. However, limited HSC counts restrict use in adults, necessitating improved methods for ex vivo expansion and improved HSC function. Mitofusin 2 (MFN2), a mitochondrial membrane fusion protein, is necessary for preserving HSC function and mitofusin agonists have been characterized. We report ex vivo culture of CBU HSCs with mitofusin agonists (MAs) enhances long-term repopulating activity by over five-fold in both primary and secondary transplantation assays without changes of total nucleated cells or phenotypic HSCs. Mechanistically, MA-treated HSCs show suppressed protein synthesis, increased autophagic flux, and elevated lysosomal mass. Transcriptomic analysis implicates downregulation of MTOR signaling, and immunoprecipitation studies suggest MFN2 interacts with MTOR. These data support a model in which fusion-competent MFN2 sequesters MTOR, promoting a catabolic state that preserves HSC potency. Our findings suggest a novel MFN2-MTOR regulatory axis that enhances the function of human HSCs cultured ex vivo for potential therapeutic application.

cell biology↗

bioRxiv2023

Discovery of highly potent small molecule pan-coronavirus fusion inhibitors

The unprecedented pandemic of COVID-19, caused by a novel coronavirus, SARS-CoV-2, has led to massive human suffering, death, and economic devastation worldwide. The virus is mutating fast to more transmissible and infectious variants. The Delta variant (B.1.617.2), initially identified in India, and the omicron variant (BA.4 and BA.5) have spread worldwide. In addition, recently alarming antibody evasive SARS-CoV-2 subvariants, BQ and XBB, have been reported. These new variants may pose a substantial challenge to controlling the spread of this virus. Therefore, the continued development of novel drugs having pan-coronavirus inhibition to treat and prevent infection of COVID-19 is urgently needed. These drugs will be critically important in dealing with new pandemics that will emerge in the future. We report the discovery of several highly potent small molecule pan-coronavirus inhibitors. One of which, NBCoV63, showed low nM potency against SARS-CoV-2 (IC50: 55 nM), SARS-CoV (IC50: 59 nM), and MERS-CoV (IC50: 75 nM) in pseudovirus-based assays with excellent selectivity indices (SI: as high as > 900) demonstrating its pan-coronavirus inhibition. NBCoV63 showed equally effective antiviral potency against SARS-CoV-2 mutant (D614G) and several variants of concerns (VOCs) such as B.1.617.2 (Delta), B.1.1.529/BA.1 and BA.4/BA.5 (Omicron) and K417T/E484K/N501Y (Gamma). NBCoV63 also showed similar efficacy profiles to Remdesivir against authentic SARS-CoV-2 (Hong Kong strain) and two of its variants (Delta and Omicron) by plaque reduction in Calu3 cells. Additionally, we show that NBCoV63 inhibits virus-mediated cell-to-cell fusion in a dose-dependent manner. Furthermore, the Absorption, distribution, metabolism, and excretion (ADME) data of NBCoV63 demonstrated drug-like properties.

pharmacology and toxicology↗

bioRxiv2020

Stapled peptides based on Human Angiotensin-Converting Enzyme 2 (ACE2) potently inhibit SARS-CoV-2 infection in vitro

SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as the primary receptor to enter host cells and initiate the infection. The critical binding region of ACE2 is a [~]30 aa long helix. Here we report the design of four stapled peptides based on the ACE2 helix, which is expected to bind to SARS-CoV-2 and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50-94% helicity). On the contrary, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus based single-cycle assay in HT1080/ACE2 and human lung cells A549/ACE2, overexpressing ACE2. Three of the four stapled peptides showed potent antiviral activity in HT1080/ACE2 (IC50: 1.9 - 4.1 {micro}M) and A549/ACE2 cells (IC50: 2.2 - 2.8 {micro}M). The linear peptides NYBSP-C and the double-stapled peptide StRIP16, used as controls, showed no antiviral activity. Most significantly, none of the stapled peptides show any cytotoxicity at the highest dose tested. We also evaluated the antiviral activity of the peptides by infecting Vero E6 cells with the replication-competent authentic SARS-CoV-2 (US_WA-1/2020). NYBSP-1 was the most efficient preventing the complete formation of cytopathic effects (CPEs) at an IC100 17.2 {micro}M. NYBSP-2 and NYBSP-4 also prevented the formation of the virus-induced CPE with an IC100 of about 33 {micro}M. We determined the proteolytic stability of one of the most active stapled peptides, NYBSP-4, in human plasma, which showed a half-life (T1/2) of >289 min.

pharmacology and toxicology↗

bioRxiv2020

Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.

microbiology↗