Biology subjects

Greco, F. A.

Publications and source records attributed to Greco, F. A..

5 recordsLinked to original sources

bioRxiv2025

Click. Screen. Degrade. A Miniaturized D2B Workflow for rapid PROTAC Discovery

Targeted protein degradation is one of the fastest developing fields in medicinal chemistry and chemical biology. Despite significant development in assay technologies and inhibitor discovery, the development of PROTACs remains a challenging endeavor since rational design approaches remain widely elusive. Our workflow eliminates the rate-limiting step of classic synthesis, namely compound purification, and pairs it with high-throughput, semi-automated plate-based synthesis, and direct cellular assay evaluation. We applied this direct-to-biology approach to four diverse targets demonstrating the general applicability of this technology. PROTAC synthesis was realized by using the highly efficient copper-catalyzed azide-alkyne cycloaddition reaction. This simplified reaction setup allowed synthesis in the nanomole scale with reaction volumes as low as 5 L. This high throughput approach enables the synthesis and testing of hundreds of PROTACs within a few days, allowing for a comprehensive assessment of target degradability and the identification of the most suitable E3 ligase for degrader development.

biochemistry↗

bioRxiv2025

Un-LOK-ing a new approach for conformational selective targeting of STK10 (LOK)

STK10 (serine/threonine kinase 10, LOK), is an important regulator of diverse cellular processes, such as cell cycle progression or lymphocyte migration. STK10 has emerged as a potential therapeutic target for diseases associated with impaired cell migration and cell division. Here we present a late-stage optimization of a macrocyclic pyrazolo[1,5-a]pyrimidine scaffold that led to a urea-based lead series targeting the back-pocket of STK10. Co-crystal structure analysis of 23 revealed that the optimized macrocycles adopted a unique binding mode that protrudes deep into the back pocket of STK10. Compound 23 exhibited potent on-target activity in biophysical and activity assays and displayed nanomolar activity for STK10 in cells. In addition, 23 shows good selectivity against the kinome and remarkably also against the closely related kinase SLK (STE20-like kinase). Therefore, we propose that targeting the unique and largely extended pocket in STK10 represents an opportunity to develop highly selective STK10 inhibitors. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/666149v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@36f039org.highwire.dtl.DTLVardef@d55025org.highwire.dtl.DTLVardef@80c007org.highwire.dtl.DTLVardef@bf2471_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Functional characterization of pathway inhibitors for the ubiquitin-proteasome system (UPS) as tool compounds for CRBN and VHL-mediated targeted protein degradation

Small molecule degraders such as PROteolysis TArgeting Chimeras (PROTACs) or molecular glues are new modalities for drug development and important tools for target validation. Both modalities recruit an E3 ubiquitin ligase to a protein of interest (POI) either via two independent, but linked ligands (PROTACs) or through binding of a small molecule that alters the E3 binding surface to recruit a neo-substrate (molecular glues). If optimized appropriately, both modalities result in the degradation of the POI. Due to the complexity of the induced multistep degradation process, controls for degrader evaluation are critical and they are commonly used in the literature. However, comparative studies and evaluation of cellular potencies of these control compounds and their appropriate uses have not been published so far. Additionally, the high diversity of mechanisms requires diverse small molecule controls to ensure appropriate inhibition of the investigated system while keeping potential cellular toxicity and unintended effects on cellular pathways as low as possible. Here, we scrutinized a diverse set of ubiquitin pathway inhibitors and evaluated their potency and utility within the CRBN and VHL mediated POI degradation pathway. We used the HiBiT system to measure the levels of target rescue after treatment with control compounds. Additionally, cell health was investigated using a multiplex high content assay. This assay panel allows us to determine non-toxic effective concentrations for control experiments and to perform rescue experiments in the absence of cellular toxicity, which has a profound effect on target degradation by ubiquitin-dependent and -independent pathways.

biochemistry↗

bioRxiv2024

Synthesis and evaluation of chemical linchpins for highly selective CK2α targeting

Casein kinase-2 (CK2) are serine/threonine kinases with dual co-factor (ATP and GTP) specificity, that are involved in the regulation of a wide variety of cellular functions. Small molecules targeting CK2 have been described in the literature targeting different binding pockets of the kinase with a focus on type I inhibitors such as the recently published chemical probe SGC-CK2-1. In this study, we investigated whether known allosteric inhibitors binding to a pocket adjacent to helix D could be combined with ATP mimetic moieties defining a novel class of ATP competitive compounds with a unique binding mode. Linking both binding sites requires a chemical linking moiety that would introduce a 90-degree angle between the ATP mimetic ring system and the D targeting moiety, which was realized using a sulfonamide. The synthesized inhibitors were highly selective for CK2 with binding constants in the nM range and low micromolar activity. While these inhibitors need to be further improved, the present work provides a structure-based design strategy for highly selective CK2 inhibitors.

biochemistry↗

bioRxiv2023

Targeting LC3/GABARAP for degrader development and autophagy modulation

Recent successes in developing small-molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the target recruitment to LC3/GABARAP, a family of membrane-bound proteins that tether autophagy receptors to the autophagosome. In order to validate the existing ligands, we rigorously tested target engagement of reported ATTEC ligands and handles. Surprisingly, using various biophysical methods, most available ligands did not interact with their designated target LC3. Intrigued by the idea of developing ATTECs, we evaluated the druggability of LC3/GABARAP by in silico docking and large scale crystallographic fragment screening. The data revealed that most fragments bound to the HP2, but not the HP1 pocket of the LC3-interacting region (LIR) docking site, suggesting favorable druggability of this binding pocket. Here, we present diverse comprehensively validated ligands for future ATTEC development.

biochemistry↗