No abstract

Aromatic aldehydes that bind to sickle hemoglobin (HbS) to increase the protein oxygen affinity and/or directly inhibit HbS polymer formation to prevent the pathological hypoxia-induced HbS polymerization and the subsequent erythrocyte sickling have for several years been studied for the treatment of sickle cell disease (SCD). With the exception of Voxelotor, which was recently approved by the U.S. Food and Drug Administration (FDA) to treat the disease, several other promising antisickling aromatic aldehydes have not fared well in the clinic because of metabolic instability of the aldehyde moiety, which is critical for the pharmacologic activity of these compounds. Over the years, our group has rationally developed analogs of aromatic aldehydes that incorporate a stable Michael addition reactive center that we hypothesized would form covalent interactions with Hb to increase the protein affinity for oxygen and prevent erythrocyte sickling. Although, these compounds have proven to be metabolically stable, unfortunately they showed weak to no antisickling activity. In this study, through additional targeted modifications of our lead Michael addition compounds, we have discovered other novel antisickling agents. These compounds, designated MMA, bind to the α-globin and/or β-globin to increase Hb affinity for oxygen and concomitantly inhibit erythrocyte sickling with significantly enhanced and sustained pharmacologic activities in vitro

The pyridoxal 5′‐phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP‐dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP‐dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function.

Piscidin 1 (P1) and piscidin 3 (P3), isolated from the mast cells of hybrid striped bass, are host defense peptides (HDPs) that are active against several types of pathogens, including viruses and bacteria, as well as some tumor cells. They contain an amino‐terminal copper and nickel binding (ATCUN) motif, and have enhanced antimicrobial functionality when metal‐bound. Studies suggest that P1’s preferred method of attack is disruption of cell membranes and P3 is more damaging to DNA, but their mechanisms of action, particularly in the metallated state, are not yet well characterized. Resilience of cell membranes under adverse conditions is known to be greater when a portion of their constituent lipids are polyunsaturated fatty acids (PUFAs). It has also been shown that both peptide activity and structure respond to changes in lipid environment. Here, we posit that P1 and P3 retain their permeabilizing function in PUFA‐containing membranes by chelating a redox metal ion that enhances their ability to disrupt membranes physically as well as chemically by oxidizing double bonds. We take a multi‐faceted approach utilizing both functional assays (dye leakage, antimicrobial assays) and experimental and computational structural studies including circular dichroism, solid‐state NMR, neutron diffraction, and molecular dynamics simulations, to examine the complex interplay between P1 and P3 and cell membrane mimics with different lipid compositions. One model mimics bacterial cells with incorporated PUFAs and the other mimics mammalian cells under oxidative stress conditions. We show that in the presence of PUFAs, membrane lytic activity of both P1 and P3 decreases, but increases as much as five fold upon metallation and twofold when membranes contain oxidized lipids. We investigate how changing peptide‐lipid interactions in these different membranes could explain these results. The combined findings give insight into protein lipid‐interactions and valuable information for the design of novel therapeutics for combating drug‐resistant infections.