Biology subjects

Beld, J.

Publications and source records attributed to Beld, J..

6 recordsLinked to original sources

bioRxiv2023

Discovery of novel brain permeable human ACSS2 inhibitors for blocking breast cancer brain metastatic growth

Breast-cancer brain metastasis (BCBM) poses a significant clinical challenge, resulting in an end-stage diagnosis and hindered by limited therapeutic options. The blood-brain barrier (BBB) acts as an anatomical and physiological hurdle for therapeutic compounds, restricting the effective delivery of therapies to the brain. In order to grow and survive in a nutrient-poor environment, tumors in the brain must adapt to their metabolic needs, becoming highly dependent on acetate. These tumors rely on the conversion of acetate to acetyl-CoA by the enzyme Acetyl-CoA synthetase 2 (ACSS2), a key metabolic enzyme involved in regulating fatty acid synthesis and protein acetylation in tumor cells. ACSS2 has emerged as a crucial enzyme required for the growth of tumors in the brain. Here, we utilized a computational pipeline, combining pharmacophore-based shape screen methodology with ADME property predictions to identify novel brain-permeable ACSS2 inhibitors. From a small molecule library, this approach identified 30 potential ACSS2 binders, from which two candidates, AD-5584 and AD-8007, were validated for their binding affinity, predicted metabolic stability, and, notably, their ability to traverse the BBB. We show that treatment of BCBM cells, MDA-MB-231BR, with AD-5584 and AD-8007 leads to a significant reduction in lipid storage, reduction in colony formation, and increase in cell death in vitro. Utilizing an ex vivo orthotopic brain-slice tumor model, we show that treatment with AD-8007 and AD-5584 significantly reduces tumor size and synergizes with radiation in blocking BCBM tumor growth ex vivo. Importantly, we show that following intraperitoneal injections with AD-5584 and AD-8007, we can detect these compounds in the brain, confirming their BBB permeability. Thus, we have identified and validated novel ACSS2 inhibitor candidates for further drug development and optimization as agents for treating patients with breast cancer brain metastasis.

cancer biology↗

bioRxiv2023

Strategic engineering unlocks in vitro type II polyketide biosynthesis

Microbial polyketides represent a structurally diverse class of secondary metabolites with medicinally relevant properties and are synthesized by polyketide synthases (PKS). While Type I PKSs are large biosynthetic machineries composed of a single polypeptide chain, type II PKSs are minimally composed of a ketosynthase-chain length factor (KS-CLF) and a phosphopantetheinylated acyl carrier protein (holo-ACP) expressed separately. Although type II PKSs are found throughout the bacterial kingdom, and despite their importance to strategic bioengineering, type II PKSs have not been well-studied in vitro: In cases where the KS-CLF can be accessed via E. coli heterologous expression, the cognate ACPs are not activatable by the broad specificity Bacillus subtilis surfactin-producing phosphopantetheinyl transferase (PPTase) Sfp and, conversely, in systems where the ACP can be activated by Sfp, the corresponding KS-CLF is typically not readily obtained. Here, we report the high-yield heterologous expression of both cyanobacterial Gloeocapsa sp. PCC 7428 minimal type II PKS (gloPKS) components in E. coli, which allowed us to functionally reconstitute this minimal type II PKS in vitro. Initially, neither the cognate PPTase nor Sfp converted gloACP to its active holo state. However, by examining sequence differences between Sfp-compatible and -incompatible ACPs, we identified two conserved residues in gloACP that, when mutated, enabled high-yield phosphopantetheinylation of gloACP by Sfp. Using analogous mutations, other previously Sfp-incompatible type II PKS ACPs from different bacterial phyla were also rendered activatable by Sfp. This demonstrates the generalizability of our approach and breaks down a longstanding barrier to type II PKS studies and the exploration of complex biosynthetic pathways.

biochemistry↗

bioRxiv2023

The VanS sensor histidine kinase from type-B VRE recognizes vancomycin directly

Vancomycin-resistant enterococci (VRE) are among the most common causes of nosocomial infections and have been prioritized as targets for new therapeutic development. Many genetically distinct types of VRE have been identified; however, they all share a common suite of resistance genes that function together to confer resistance to vancomycin. Expression of the resistance phenotype is controlled by the VanRS two-component system. This system senses the presence of the antibiotic, and responds by initiating transcription of resistance genes. VanS is a transmembrane sensor histidine kinase, and plays a fundamental role in antibiotic resistance by detecting vancomycin or its effects; it then transduces this signal to the VanR transcription factor, thereby alerting the organism to the presence of the antibiotic. Despite the critical role played by VanS, fundamental questions remain about its function, and in particular about how it senses vancomycin. Here, we focus on a purified VanRS system from one of the most clinically prevalent forms of VRE, type B. We show that in a native-like membrane environment, the autokinase activity of type-B VanS is strongly stimulated by vancomycin. We additionally demonstrate that this effect is mediated by a direct physical interaction between the antibiotic and the type-B VanS protein, and localize the interacting region to the proteins periplasmic domain. This represents the first time that a direct sensing mechanism has been confirmed for any VanS protein. Significance StatementWhen vancomycin-resistant enterococci (VRE) sense the presence of vancomycin, they remodel their cell walls to block antibiotic binding. This resistance phenotype is controlled by the VanS protein, a histidine kinase that senses the antibiotic or its effects and signals for transcription of resistance genes. However, the mechanism by which VanS detects the antibiotic has remained unclear, with no consensus emerging as to whether the protein interacts directly with vancomycin, or instead detects some downstream consequence of vancomycins action. Here, we show that for one of the most clinically relevant types of VRE, type B, VanS is activated by direct binding of the antibiotic. Such mechanistic insights will likely prove useful in circumventing vancomycin resistance.

biochemistry↗

bioRxiv2023

Acyl-CoA thioesterase-2 facilitates beta-oxidation in glycolytic skeletal muscle in a lipid supply dependent manner

Acyl-Coenzyme A (acyl-CoA) thioesters are compartmentalized intermediates that participate in in multiple metabolic reactions within the mitochondrial matrix. The limited availability of free CoA (CoASH) in the matrix raises the question of how the local acyl-CoA concentration is regulated to prevent trapping of CoASH from overload of any specific substrate. Acyl-CoA thioesterase-2 (ACOT2) hydrolyzes long-chain acyl-CoAs to their constituent fatty acids and CoASH, and is the only mitochondrial matrix ACOT refractory to inhibition by CoASH. Thus, we reasoned that ACOT2 may constitutively regulate matrix acyl-CoA levels. Acot2 deletion in murine skeletal muscle (SM) resulted in acyl-CoA build-up when lipid supply and energy demands were modest. When energy demand and pyruvate availability were elevated, lack of ACOT2 activity promoted glucose oxidation. This preference for glucose over fatty acid oxidation was recapitulated in C2C12 myotubes with acute depletion of Acot2, and overt inhibition of {beta}-oxidation was demonstrated in isolated mitochondria from Acot2-depleted glycolytic SM. In mice fed a high fat diet, ACOT2 enabled the accretion of acyl-CoAs and ceramide derivatives in glycolytic SM, and this was associated with worse glucose homeostasis compared to when ACOT2 was absent. These observations suggest that ACOT2 supports CoASH availability to facilitate {beta}-oxidation in glycolytic SM when lipid supply is modest. However, when lipid supply is high, ACOT2 enables acyl-CoA and lipid accumulation, CoASH sequestration, and poor glucose homeostasis. Thus, ACOT2 regulates matrix acyl-CoA concentration in glycolytic muscle, and its impact depends on lipid supply.

biochemistry↗

bioRxiv2023

Novel Diazirine Photoprobes for the Identification of Vancomycin-Binding Proteins

Vancomycins interactions with cellular targets drive its antimicrobial activity, and also trigger expression of resistance against the antibiotic. Interaction partners for vancomycin have previously been identified using photoaffinity probes, which have proven to be useful tools for exploring vancomycins interactome. This work seeks to develop diazirine-based vancomycin photoprobes that display enhanced specificity and bear fewer chemical modifications, as compared to previous photoprobes. Using proteins fused to vancomycins main cell-wall target, D-alanyl-D-alanine, we use mass spectrometry to show that these photoprobes specifically label known vancomycin-binding partners within minutes. In a complementary approach, we developed a Western-blot strategy targeting the vancomycin adduct of the photoprobes, eliminating the need for affinity tags and simplifying the analysis of photolabeling reactions. Together, the probes and identification strategy provide a novel and streamlined pipeline for identifying novel vancomycin-binding proteins. For Table of Contents Only. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/545140v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1d21b8forg.highwire.dtl.DTLVardef@10f4d4borg.highwire.dtl.DTLVardef@19bb1f0org.highwire.dtl.DTLVardef@bf3dd5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Structure of VanS from Vancomycin-Resistant Enterococci: A Sensor Kinase with Weak ATP Binding

The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci (VRE). VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from VRE types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with KD values in the low millimolar range. Since these KD values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.

biochemistry↗