Biology subjects

Lehtio, L.

Publications and source records attributed to Lehtio, L..

6 recordsLinked to original sources

bioRxiv2026

Targeting collagen biosynthesis by small molecules inhibiting the function of the peptide-substrate-binding domain of collagen prolyl 4-hydroxylases

Collagen prolyl 4-hydroxylase (C-P4H) is an essential enzyme in collagen synthesis and known to be a potential target for drugs that prevent excess collagen formation. Currently known C-P4H inhibitors target the catalytic site of C-P4H, being analogues of 2-oxoglutarate (2OG). However, in mammalian cells there are many other 2OG-dependent dioxygenases with a highly similar catalytic domain, which limits the selective specificity of drugs targeting C-P4H activity. The peptide-substrate-binding (PSB) domain is unique for the C-P4H family and known to be important for the catalytic efficiency of C-P4H. Therefore, interfering with peptide binding to the PSB domain might allow more specific inhibition of the hydroxylation activity of C-P4Hs. We developed a robust FRET-based high-throughput screening assay (Z > 0.73) based on PSB-peptide interactions. This assay was used to screen a peptidomimetic library of 15614 compounds with the PSB domains of C-P4H isoforms I and II. A hit compound (OUL-PSBi-001) was identified with IC50s of 40 {micro}M and 62 {micro}M for PSB-I and PSB-II, respectively. We also showed that this compound indeed inhibits the catalytic activity of the full-length CP4H-I and -II. This FRET assay provides a new strategy for finding selective inhibitors for the treatment of fibrotic diseases and cancer.

molecular biology↗

bioRxiv2024

Identification of a series of pyrrolo-pyrimidine based SARS-CoV-2 Mac1 inhibitors that repress coronavirus replication

Coronaviruses (CoVs) can emerge from zoonotic sources and cause severe diseases in humans and animals. All CoVs encode for a macrodomain (Mac1) that binds to and removes ADP-ribose from target proteins. SARS-CoV-2 Mac1 promotes virus replication in the presence of interferon (IFN) and blocks the production of IFN, though the mechanisms by which it mediates these functions remain unknown. Mac1 inhibitors could help elucidate these mechanisms and serve as therapeutic agents against CoV-induced diseases. We previously identified compound 4a (a.k.a. MCD-628), a pyrrolo-pyrimidine that inhibited Mac1 activity in vitro at low micromolar levels. Here, we determined the binding mode of 4a by crystallography, further defining its interaction with Mac1. However, 4a did not reduce CoV replication, which we hypothesized was due to its acidic side chain limiting permeability. To test this hypothesis, we developed several hydrophobic derivatives of 4a. We identified four compounds that both inhibited Mac1 in vitro and inhibited murine hepatitis virus (MHV) replication: 5a, 5c, 6d, and 6e. Furthermore, 5c and 6e inhibited SARS-CoV-2 replication only in the presence of IFN{gamma}, similar to a Mac1 deletion virus. To confirm their specificity, we passaged MHV in the presence of 5a to identify drug-resistant mutations and identified an alanine-to-threonine and glycine-to-valine double mutation in Mac1. Recombinant virus with these mutations had enhanced replication compared to WT virus when treated with 5a, demonstrating the specificity of these compounds during infection. However, this virus is highly attenuated in vivo, indicating that drug-resistance emerged at the expense of viral fitness. IMPORTANCECoronaviruses (CoVs) present significant threats to human and animal health, as evidenced by recent outbreaks of MERS-CoV and SARS-CoV-2. All CoVs encode for a highly conserved macrodomain protein (Mac1) that binds to and removes ADP-ribose from proteins, which promotes virus replication and blocks IFN production, though the exact mechanisms remain unclear. Inhibiting Mac1 could provide valuable insights into these mechanisms and offer new therapeutic avenues for CoV-induced diseases. We have identified several unique pyrrolo-pyrimidine-based compounds as Mac1 inhibitors. Notably, at least two of these compounds inhibited both murine hepatitis virus (MHV) and SARS-CoV-2 replication. Furthermore, we identified a drug-resistant mutation in Mac1, confirming target specificity during infection. However, this mutant is highly attenuated in mice, indicating that drug-resistance appears to come at a fitness cost. These results emphasize the potential of Mac1 as a drug target and the promise of structure-based inhibitor design in combating coronavirus infections.

microbiology↗

bioRxiv2023

DTX3L and USP28 fine-tune DNA double strand repair through mutual regulation of their protein levels

The DNA damage response (DDR) relies on a complex protein network to maintain genomic integrity, yet the interplay between post-translational modifiers remains poorly understood. Here, we uncover a novel regulatory axis between the E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 at DNA double-strand breaks (DSBs). Our results reveal a sophisticated feedback mechanism in which DTX3L ubiquitinates USP28, leading to its proteasomal degradation, while USP28 counteracts by deubiquitinating both itself and DTX3L. This cross-regulation fine-tunes DSB repair in multiple pathways, including non-homologous end joining (NHEJ), homologous recombination (HR), single-strand annealing (SSA), and microhomology-mediated end joining (MMEJ). Strikingly, the detrimental effects of USP28 depletion on these repair pathways were rescued by concurrent DTX3L knockdown. Collectively, our work uncovers a novel layer of DDR regulation in which DTX3L and USP28s antagonistic activities calibrate cellular responses to genotoxic stress, thus identifying promising therapeutic targets to combat diseases associated with genomic instability. HighlightsO_LIDTX3L and USP28 physically interact and colocalize in cellular sub-compartments, with the N-terminal D1-D3 domains of DTX3L primarily mediating the interaction C_LIO_LIDTX3L ubiquitinates USP28 for degradation, while USP28 deubiquitinates itself and DTX3L, creating a sophisticated feedback mechanism. C_LIO_LIThe DTX3L-USP28 circuit influences levels of key proteins like HIF-1, p53, and c-MYC, suggesting broader impacts on cellular stress responses. C_LIO_LIDTX3L and USP28 cooperatively regulate multiple DSB repair pathways, including NHEJ, HR, SSA, and MMEJ, with USP28 depletion effects rescued by DTX3L silencing. C_LI

biochemistry↗

bioRxiv2022

Design, Synthesis and Evaluation of Inhibitors of the SARS-CoV-2 nsp3 Macrodomain

A series of amino acid based 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low M Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/482176v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@167aceorg.highwire.dtl.DTLVardef@1d88c47org.highwire.dtl.DTLVardef@1e1b34borg.highwire.dtl.DTLVardef@c2321a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

A molecular toolbox for ADP-ribosyl binding proteins

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or in viral infections, which makes them attractive targets for the development of inhibitors. Our goal was to develop a robust and accessible assay technology that is suitable for high-throughput screening and applicable to a wide range of proteins acting as either hydrolysing or non-hydrolysing binders of mono- and poly-ADP-ribosyl groups. As a foundation of our work, we developed a C-terminal protein fusion tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups as well as chemical ADP-ribosyl analogs. By fusion of the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we were able to generate robust FRET signals and the interaction with 22 previously described ADP-ribosyl-binders was confirmed. To demonstrate the applicability of this binding assay for high-throughput screening, we utilized it to screen for inhibitors of the SARS-CoV-2 nsp3 macrodomain and identified the drug suramin as a moderate yet unspecific inhibitor of this protein. To complement the binding technology, we prepared high-affinity ADP-ribosyl binders fused to a nanoluciferase, which enabled simple blot-based detection of mono- and poly-ADP-ribosylated proteins. These tools can be expressed recombinantly in E. coli using commonly available agents and will help to investigate ADP-ribosylation systems and aid in drug discovery.

biochemistry↗

bioRxiv2020

Activation of ARTD2/PARP2 by DNA damage induces conformational changes relieving enzyme autoinhibition

Human ARTD2/PARP2 is an ADP-ribosyltransferase which, when activated by 5- phosphorylated DNA ends, catalyzes poly-ADP-ribosylation of itself, other proteins and DNA. A crystal structure of ARTD2 in complex with an activating 5-phosphorylated DNA shows that the WGR domain bridges the dsDNA gap and joins the DNA ends. This DNA binding results in major conformational changes, reorganization of helical fragments, in the ARTD2 regulatory domain. Comparison of ARTD1-3 crystal structures reveal how binding to a DNA damage site leads to formation of a catalytically competent conformation capable of binding substrate NAD+ and histone PARylation factor 1 changing the ARTD2 residue specificity from glutamate to serine when initiating DNA repair processes. The structure also reveals how the conformational changes in the autoinhibitory regulatory domain would promote the flexibility needed by the enzyme to reach the target macromolecule for ADP-ribosylation.

biochemistry↗