Biology subjects

Plach, M. G.

Publications and source records attributed to Plach, M. G..

6 recordsLinked to original sources

bioRxiv2024

SAXS/MC studies of the mixed-folded protein Cdt1 reveal monomeric, folded over conformations

Cdt1 is a protein critical for DNA replication licensing and is well-established to be a binding partner of the minichromosome maintenance (MCM) complex. Cdt1 has also been demonstrated to have an emerging, "moonlighting" role at the kinetochore via direct binding to microtubules and to the Ndc80 complex. However, it is not known how the structure and conformations of Cdt1 could allow for these multiple, completely unique sets of protein complexes. And while there exist multiple robust methods to study entirely folded or entirely unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. It this work, we employ multiple orthogonal biophysical and computational techniques to provide a detailed structural characterization of human Cdt1 92-546. DSF and DSCD show both folded winged helix (WH) domains of Cdt1 are relatively unstable. CD and NMR show the N-terminal and the linker regions are intrinsically disordered. Using DLS and SEC-MALS, we show that Cdt1 is polydisperse, monomeric at high concentrations, and without any apparent inter-molecular self-association. SEC-SAXS of the monomer in solution enabled computational modeling of the protein in silico. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble. Using experimental SAXS data, we filtered for conformations which did and did not fit our data. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with our SAXS data. The best fit models have the N-terminal and linker regions extended into solution and the two folded domains close to each other in apparent "folded over" conformations. The best fit Cdt1 conformations are consistent with a function as a scaffold protein which may be sterically blocked without the presence of binding partners. Our studies also provide a template for combining experimental and computational biophysical techniques to study mixed-folded proteins.

biophysics↗

bioRxiv2022

Impact of single mutations on binding kinetics oftriplex forming oligos revealed by fluorescence proximitysensing in heliX(R) biosensor

The sequence specific association of RNA with DNA via formation of RNA/DNA triple helices is relevant for regulation of gene expression, repair mechanisms, and chromatin organization. Formation of such RNA/DNA triplexes requires poly-purine sequences, which allow for Hoogsteen base-paring in addition to Watson-Crick pairing in the major groove of DNA. Binding of triplex forming oligos (TFOs) is sequence-specific and understanding sequence dependencies is key for the development of targeted tools for gene therapy. Here, we highlight a direct approach for determining binding kinetics and binding constants for TFOs using the state-of-the-art heliX(R)biosensor tool. With this, we provide key insights into the binding kinetics of RNA and DNA TFOs to a triplex targeting site (TTS)-containing DNA double helix measured in real-time. Dependent on the introduced base, point mutations in one position of a triplex forming oligo (TFO) can change the dissociation constant (KD) by several orders of magnitude or just by one log, affecting primarily the dissociation rate. Furthermore, we demonstrate that the heliX(R)biosensor assay is also well-suited for detection of rather weak triplex formation. The weakest binding we could identify was 140 M, for a TFO, which other studies considered as non-binding.

biophysics↗

bioRxiv2020

Multi-parameter formulation development for an HIV-vaccine protein with direct validation of epitope binding integrity and stoichiometry

Vaccines are on the front line-of-defense against infectious diseases, ranging from threats we are familiar with, including polio, tuberculosis, or HIV, to novel and emerging threats such as SARS-CoV-2. Successful development of new protein-based vaccines requires sophisticated and efficient development of storage and formulation conditions. Here we demonstrate the combined power of 2binds sophisticated buffer matrix FORMOscreen(R) and NanoTemper Technologies novel Prometheus Panta high-throughput Dynamic Light Scattering/Nano Differential Scanning Fluorimetry instrument. We show that this combination can comprehensively improve critical biophysical parameters for the HIV-1 vaccine BG505-SOSIP and find the optimal formulation condition with unmatched efficiency.

biophysics↗

bioRxiv2020

GNS561, a clinical-stage PPT1 inhibitor, has powerful antitumor activity against hepatocellular carcinoma via modulation of lysosomal functions

Background & AimsHepatocellular carcinoma (HCC) is the most frequent primary liver cancer. Autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. Approach & ResultsTo explore the antitumor effects of GNS561, a new autophagy inhibitor, we first achieved in vitro assays using various human cancer cell lines. Having demonstrated that GNS561 displayed high liver tropism using mass spectrometry imaging, the potency of GNS561 on tumor was evaluated in vivo in two HCC models (human orthotopic patient-derived xenograft mouse model and diethylnitrosanime-induced cirrhotic immunocompetent rat model). Oral administration of GNS561 was well tolerated and decreased tumor growth in these two models. GNS561 mechanism of action was assessed in an HCC cell line, HepG2. We showed that due to its lysosomotropic properties, GNS561 could reach and inhibited its enzyme target, palmitoyl-protein thioesterase 1, resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of mTOR, lysosomal membrane permeabilization, caspase activation and cell death. ConclusionsGNS561, currently tested in a global Phase 1b/2a clinical trial against primary liver cancer, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment. With an estimated 782,000 deaths in 2018, hepatocellular carcinoma (HCC) stands as the most common primary liver cancer and constitutes the fourth leading cause of cancer-related death worldwide (1). The rising incidence of HCC, the high worldwide mortality rate, and limited therapeutic options at advanced stages, make HCC a significant unmet medical need. Autophagy-related lysosomal cell death, either alone or in connection with several other cell death pathways, has been recognized as a major target for cancer therapy (2). Dysregulated autophagic-lysosomal activity and mTOR signaling were shown to allow cancer cells to become resistant to the cellular stress induced by chemotherapy and targeted therapy (3). Recently, several lysosome-specific inhibitors were shown to target palmitoyl-protein thioesterase 1 (PPT1), resulting in the modulation of protein palmitoylation and autophagy, and antitumor activity in melanoma and colon cancer models (4, 5). Chloroquine (CQ) and hydroxychloroquine (HCQ) have been used for more than 50 years to prevent and treat malarial infections and autoimmune diseases. Based on the lysosomotropic properties and the capacity for autophagy inhibition, these molecules have been proposed as active drugs in cancer (6-9). Over 40 clinical trials have been reported to evaluate the activity of both CQ or HCQ as single agent or in combination with chemotherapy in several tumor types (6-8. However, the required drug concentrations to inhibit autophagy were not achieved in humans, leading to inconsistent results in cancer clinical trials (5, 10). This prompted research to identify novel compounds with potent inhibitory properties against autophagy for cancer therapy. We previously reported that GNS561 was efficient in intrahepatic cholangiocarcinoma (iCCA) by inhibiting late-stage autophagy (11). In this study, we investigated the mechanism of action of GNS561. We identified lysosomal PPT1 as a target of GNS561. Exposure to GNS561 induced lysosomal accumulation of unbound zinc ion (Zn2+), inhibition of PPT1 and cathepsin activity, blockage of autophagic flux and mTOR displacement. Interestingly, these effects resulted in lysosomal membrane permeabilization (LMP) and caspase activation that led to cancer cell death. This mechanism was associated with dose-dependent inhibition of cancer cell proliferation and tumor growth inhibition in two HCC in vivo models. These data establish PPT1 and lysosomes as major targets for cancer cells and led to the development of a clinical program investigating the effects of GNS561 in patients with advanced HCC.

cancer biology↗

bioRxiv2020

In Silico Driven Prediction of MAPK14 Off-Targets Reveals Unrelated Proteins with High Accuracy

During the discovery and development of new drugs, candidates with undesired and potentially harmful side-effects can arise at all stages, which poses significant scientific and economic risks. Most of such phenotypic side-effects can be attributed to binding of the drug candidate to unintended proteins, so-called off-targets. The early identification of potential off-targets is therefore of utmost importance to mitigate any downstream risks. We showcase how the combination of knowledge-based in silico off-target screening and state-of-the-art biophysics can be applied to rapidly identify off-targets for a MAPK14 inhibitor. Out of 13 predicted off-targets, six proteins were confirmed to interact with the inhibitor in vitro, which translates to an exceptional hit rate of 46%. For two proteins, affinities in the lower micromolar range were obtained: The kinase IRE1 and the Hematopoietic Prostaglandin D Synthase, which is entirely unrelated to MAPK14 and is involved in different cell-regulatory processes. The whole off-target identification/validation pipeline can be completed as fast as within two months, excluding delivery times of proteins. These results emphasize how computational off-target screening in combination with MicroScale Thermophoresis can effectively reduce downstream development risks in a very competitive time frame and at low cost.

bioinformatics↗

bioRxiv2020

Focus Your Screening Library: Rapid Identification of Novel PDE2 Inhibitors with in silico Driven Library Prioritization and MicroScale Thermophoresis

Accelerated development of lead structures is of high interest to the pharmaceutical industry in order to decrease development times and costs. We showcase how an intelligent combination of AI-based drug screening with state-of-the-art biophysics drives the rapid identification of novel inhibitor structures with high chemical diversity for cGMP-dependent 3,5-cyclic phosphodiesterase (PDE2). The starting point was an off-the-shelve chemical library of two million drug-like compounds. In a single in silico reduction step, we short-listed 125 compounds - the focused library - as potential binders to PDE2 and tested their binding behavior in vitro using MicroScale Thermophoresis (MST). Of this focused library, seven compounds indicated binding to PDE2, translating to a hit rate of 6%. Three of these compounds have affinities in the lower micromolar range. The compound with the highest affinity showed a KD of 10 {micro}M and is thus an excellent starting point for further medicinal chemistry optimization. The results show how innovative and structure-driven in silico approaches and biophysics can be used to accelerate drug discovery and to obtain new molecular scaffolds at a fraction of the costs and time - compared with standard high-throughput screening.

bioinformatics↗