Biology subjects

Fierke, C. A.

Publications and source records attributed to Fierke, C. A..

10 recordsLinked to original sources

bioRxiv2023

Phage-Assisted, Active Site-Directed Ligand Evolution with a Genetically Encoded Nε-Butyryl-ʟ-Lysine to Identify a Cellularly Potent and Selective Inhibitor for the ENL YEATS Domain as an Anti-Leukemia Agent

Eleven-nineteen leukemia protein (ENL) plays pivotal roles in the leukemogenesis. As a YEATS domain protein, ENL reads histone acylation marks and recruits key transcription factors to leukemic drivers such as HOXA9, MEIS1, and MYB and therefore promotes leukemia development. The histone-reading function of ENL has been proven essential in the onset and progression of several acute leukemias, suggesting a putative therapeutic window for ENL inhibition. In this study, we developed a phage-assisted, active site-directed ligand evolution (PADLE) approach for the identification of potent and selective ENL inhibitors, where N{varepsilon}-butyryl-O_SCPLOWLC_SCPLOW-lysine (BuK) that possesses known target-protein interactions with the ENL YEATS domain was genetically incorporated into a phage display library to serve as a warhead to direct displayed peptides to the active site of ENL YEATS for enrichment. Using this novel strategy in combination with structure-activity relationship that replaced BuK with other ncAAs for de novo {pi}-{pi}-{pi} stacking interactions with two aromatic residues in ENL YEATS, selective and potent ENL inhibitors with a Kd value as low as 2.0 nM were identified. One pentapeptide inhibitor tENL-S1f displayed selective inhibition of ENL over other YEATS domains as well as strong cellular target engagement and on-target effects in inhibiting leukemia cell growth and suppressing the expression of ENL target genes. As the first of its kind study, the current work opens a large avenue of research of using PADLE to develop selective and potent peptidyl inhibitors for a large variety of epigenetic reader proteins.

biochemistry↗

bioRxiv2022

Mode of inhibition of RNase P by gambogic acid and juglone

The first step in transfer RNA (tRNA) maturation is the cleavage of the 5 end of precursor transfer RNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein (RNP) complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of protein-only RNase P that can be used as tools for studying the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization (FP) cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 M range were identified and analyzed. These compounds similarly inhibit human mtRNase P, a multi-subunit protein enzyme, and are 50-fold less potent against bacterial RNA-dependent RNase P. Biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor that targets the PRORP1-substrate complex while juglone acts as time-dependent inhibitor of PRORP1. X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. A model consistent with the kinetic data is that juglone binds to PRORP1 rapidly to form an inactive enzyme-inhibitor (EI) complex, and then undergoes a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.

biochemistry↗

bioRxiv2022

Phage-Assisted, Active Site-Directed Ligand Evolution of a Potent and Selective Histone Deacetylase 8 Inhibitor

The phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn2+-dependent histone deacetylases (HDACs). Its ketone serves as an anchoring point to chelate the catalytic zinc ion in HDACs. Using a previously evolved N{varepsilon}-acetyl-lysyl-tRNA synthetase in combination with tRNAPyl, we showed that Aoda was efficiently incorporated into proteins in Escherichia coli by amber suppression. By propagating an amber codon-obligate phagemid library in E. coli encoding Aoda, we generated an Aoda-containing phage-displayed peptide library. Using this library to conduct PADLE against HDAC8 revealed a 7-mer peptide GH8P01F1 with Aoda-flanking amino acid residues that match existing peptide sequences in identified HDAC8 substrates. Switching Aoda in GH8P01F1 to a more Zn2+-chelating ncAA S-2-amino-8-hydroxyamino-8-oxooctanoic acid (Asuha) led to an extremely potent GH8HA01 that has an HDAC8-inhibition Ki value as 0.67 nM. GH8HA01 and its 5-mer truncation analogue Ac-GH8HA01{Delta}1{Delta}7 that has an HDAC8-inhibition Ki value as 0.31 nM represent two most potent HDAC8 inhibitors that have been developed so far and both are highly selective against HDAC8 over the other three tested HDACs, demonstrating the great potential of using PADLE to identify highly potent and selective ligands for targets.

biochemistry↗

bioRxiv2021

Structure-based prediction of KDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates

Histone deacetylases play important biological roles well beyond the deacetylation of histone tails. In particular, HDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as -tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of HDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of HDAC8 and other enzymes. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme. In particular, we propose novel regulatory roles of HDAC6 in tumorigenesis and cancer cell survival via the regulation of the EGFR/Akt pathway activation process. The calibration process and comparison of the results between HDAC6 and HDAC8 highlight structural differences that explain the established promiscuity of HDAC6.

molecular biology↗

bioRxiv2020

A Speedy Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro), we have designed and synthesized a series of SC2MPro inhibitors that contain {beta}-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active site cysteine C145. All inhibitors display high potency with IC50 values at or below 100 nM. The most potent compound MPI3 has as an IC50 value as 8.5 nM. Crystallographic analyses of SC2MPro bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 M and A549 cells at 0.16-0.31 M. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19.

biochemistry↗

bioRxiv2020

Disease associated mutations in mitochondrial precursor tRNAs affect binding, m1R9 methylation and tRNA processing by mtRNase P

Mitochondrial diseases linked to mutations in mitochondrial (mt)tRNA sequences are abundant. However, the contributions of these tRNA mutations to the development of diseases is mostly unknown. Mutations may affect interactions with (mt)tRNA maturation enzymes or protein synthesis machinery leading to mitochondrial dysfunction. In human mitochondria, the first step of tRNA processing is the removal of the 5'end of precursor tRNAs (pre-tRNA) catalyzed by the three-component enzyme, mtRNase P. Additionally, one sub-component of mtRNase P, mitochondrial RNase P protein 1 (MRPP1) catalyzes methylation of the R9 base in pre-tRNAs. Despite the central role of 5' end processing in mitochondrial tRNA maturation, the link between mtRNase P and diseases is mostly unexplored. Here we investigate how 11 different human disease-linked mutations in (mt)pre-tRNAs affect the activities of mtRNase P. We find that several mutations weaken the pre-tRNA binding affinity, while the majority of mutations decrease 5' end processing and methylation activity catalyzed by mtRNase P. Furthermore, all of the investigated mutations in (mt)pre-tRNALeu(UUR) alter the tRNA fold which contributes to the partial loss of function of mtRNase P. Overall, these results reveal an etiological link between early steps of (mt)tRNA-substrate processing and mitochondrial disease.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Unexpected Specificity within Dynamic Transcriptional Protein-Protein Complexes

A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator*coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator--not often considered as contributing to binding--play a key role in mediating conformational redistribution. The ETV/PEA3*Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators, and indicates that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity.

biochemistry↗

bioRxiv2019

Kinetic mechanism of human mitochondrial RNase P

A first step in processing mitochondrial precursor tRNA (pre-tRNA) is cleavage of the 5 leader catalyzed by ribonuclease P (RNase P). Human mitochondrial RNase P (mtRNase P) is composed of three protein subunits: mitochondrial RNase P protein (MRPP) 1, 2 and 3. Even though MRPP3 is the metallonuclease subunit responsible for catalysis, cleavage is observed only in the presence of the MRPP1/2 subcomplex. To understand the functional role of MRPP1/2, we reconstituted human mitochondrial RNase P in vitro and performed kinetic and thermodynamic analyses. MRPP1/2 significantly enhances both the catalytic activity and the apparent substrate affinity of mtRNase P. Additionally, pull-down and binding data demonstrate synergy between binding pre-tRNA and formation of a catalytically active MRPP1/2/3 complex. These data suggest that conformational changes in the MRPP1/2-pre-tRNA complex lead to protein-protein or protein-RNA interactions that increase both MRPP3 recognition and cleavage efficiency. This work presents the first kinetic model for human mtRNase P, providing a fundamental framework for the function of MRPP1/2 for recognition and processing of pre-tRNA.

biochemistry↗

bioRxiv2018

Alzheimer’s Amyloid-Beta Intermediates Generated by Polymer-Nanodiscs

Polymethacrylate-copolymer (PMA) encased lipid-nanodiscs (~10 nm) and macro-nanodiscs (>15 nm) are used to study A{beta}1-40 aggregation. We demonstrate that PMA-nanodiscs form a ternary association with A{beta} and regulate its aggregation kinetics by trapping intermediates. Results demonstrating reduced neurotoxicity of nanodisc-bound A{beta} oligomers are also reported.

biophysics↗

bioRxiv2018

Cationic polymethacrylate-copolymer acts as an agonist for beta-amyloid and antagonist for amylin fibrillation

In human, amyloid-beta (A{beta}) and islet amyloid polypeptide (hIAPP) aggregations are linked to Alzheimers disease and Type-2 Diabetes, respectively. There is significant interest in better understanding the aggregation process by using chemical tools. Here, we show the ability of a cationic polymethacrylate-copolymer (PMAQA) to quickly induce {beta}-hairpin structure and promote fibrillation in A{beta}40, and to constrain the conformational plasticity of hIAPP for several days and inhibit its aggregation at sub-micromolar concentrations. NMR experiments and atomistic molecular dynamics simulations reveal that PMAQA electrostatically interacts with A{beta}40s Glu22 and Asp23 followed by {beta}-sheet induction while it binds strongly to the closest proximity of amyloid core domain (NFGAIL) of hIAPP and restrain its structural rearrangement. This study provides a valuable approach to develop polymer-based anti-amyloid inhibitors that may diminish the population of intermediates of A{beta}40 or hIAPP.

biophysics↗