scholarly journals BacPROTACs mediate targeted protein degradation in bacteria

2021 ◽  
Author(s):  
Francesca Ester Morreale ◽  
Stefan Kleine ◽  
Julia Leodolter ◽  
Stepan Ovchinnikov ◽  
Julilane Kley ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Degradation ◽  
Functional State ◽  
Small Molecule ◽  
Cellular Protein ◽  
Higher Order ◽  
Bacterial Degradation ◽  
Protein Substrate ◽  
Microbial Infections

Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis targeting chimeras (PROTACs). Despite their superior properties over classical inhibitors, it has so far not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, highlighting the potential of the technology to provide next generation antibiotics.

Quantitative Ranking of Ligand Binding Kinetics with a Multiscale Milestoning Simulation Approach

2018 ◽  
Author(s):  
Benjamin R. Jagger ◽  
Christoper T. Lee ◽  
Rommie Amaro
Keyword(s):  
Molecular Dynamics ◽  
Drug Discovery ◽  
Small Molecule ◽  
Brownian Dynamics ◽  
Model Simulation ◽  
Computational Cost ◽  
Lead Selection ◽  
Delta G ◽  
Dynamics Simulations

<p>The ranking of small molecule binders by their kinetic (kon and koff) and thermodynamic (delta G) properties can be a valuable metric for lead selection and optimization in a drug discovery campaign, as these quantities are often indicators of in vivo efficacy. Efficient and accurate predictions of these quantities can aid the in drug discovery effort, acting as a screening step. We have previously described a hybrid molecular dynamics, Brownian dynamics, and milestoning model, Simulation Enabled Estimation of Kinetic Rates (SEEKR), that can predict kon’s, koff’s, and G’s. Here we demonstrate the effectiveness of this approach for ranking a series of seven small molecule compounds for the model system, -cyclodextrin, based on predicted kon’s and koff’s. We compare our results using SEEKR to experimentally determined rates as well as rates calculated using long-timescale molecular dynamics simulations and show that SEEKR can effectively rank the compounds by koff and G with reduced computational cost. We also provide a discussion of convergence properties and sensitivities of calculations with SEEKR to establish “best practices” for its future use.</p>

Targeted Protein Degradation: "The Gold Rush is On!"

2020 ◽  
Vol 7 (1) ◽  
pp. 4-16
Author(s):  
Daria Kotlarek ◽  
Agata Pawlik ◽  
Maria Sagan ◽  
Marta Sowała ◽  
Alina Zawiślak-Architek ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Degradation ◽  
Small Molecule ◽  
Gold Rush ◽  
European Company ◽  
Drug Candidates ◽  
Protein Inhibition ◽  
Small Molecule Drug ◽  
Therapeutic Benefits ◽  
Talent Pool

Targeted Protein Degradation (TPD) is an emerging new modality of drug discovery that offers unprecedented therapeutic benefits over traditional protein inhibition. Most importantly, TPD unlocks the untapped pool of the proteome that to date has been considered undruggable. Captor Therapeutics (Captor) is the fourth global, and first European, company that develops small molecule drug candidates based on the principles of targeted protein degradation. Captor is located in Basel, Switzerland and Wroclaw, Poland and exploits the best opportunities of the two sites – experience and non-dilutive European grants, and talent pool, respectively. Through over $38 M of funding, Captor has been active in three areas of TPD: molecular glues, bi-specific degraders and direct degraders, ObteronsTM.

scholarly journals Substrate-Specific Effects of Natural Genetic Variation on Proteasome Activity

2021 ◽  
Author(s):  
Mahlon Collins ◽  
Randi R. Avery ◽  
Frank W Albert
Keyword(s):  
Genetic Variation ◽  
Protein Degradation ◽  
Dynamic Control ◽  
Cellular Protein ◽  
Proteasome Activity ◽  
Heritable Variation ◽  
Natural Genetic Variation ◽  
Specific Effects ◽  
Regulatory Particle

The bulk of targeted cellular protein degradation is performed by the proteasome, a multi-subunit complex consisting of the 19S regulatory particle, which binds, unfolds, and translocates substrate proteins, and the 20S core particle, which degrades them. Protein homeostasis requires precise, dynamic control of proteasome activity. To what extent genetic variation creates differences in proteasome activity is almost entirely unknown. Using the ubiquitin-independent degrons of the ornithine decarboxylase and Rpn4 proteins, we developed reporters that provide high-throughput, quantitative measurements of proteasome activity in vivo in genetically diverse cell populations. We used these reporters to characterize the genetic basis of variation in proteasome activity in the yeast Saccharomyces cerevisiae. We found that proteasome activity is a complex, polygenic trait, shaped by variation throughout the genome. Genetic influences on proteasome activity were predominantly substrate-specific, suggesting that they primarily affect the function or activity of the 19S regulatory particle. Our results demonstrate that individual genetic differences create heritable variation in proteasome activity and suggest that genetic effects on proteasomal protein degradation may be an important source of variation in cellular and organismal traits.

scholarly journals Insulin and Analogue Effects on Protein Degradation in Different Cell Types

2000 ◽  
Vol 276 (15) ◽  
pp. 11552-11558 ◽  
Author(s):  
Janet Fawcett ◽  
Frederick G. Hamel ◽  
Robert G. Bennett ◽  
Zoltan Vajo ◽  
William C. Duckworth
Keyword(s):  
Protein Degradation ◽  
Hepg2 Cells ◽  
Cell Types ◽  
Major Effect ◽  
Cellular Protein ◽  
Experimental Conditions ◽  
L6 Cells ◽  
Different Cell Types

In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation. Cellular protein degradation is under the control of multiple systems, including lysosomes, proteasomes, calpains, and giant protease. Insulin has been shown to alter proteasome activityin vitroandin vivo. We examined the inhibition of protein degradation by insulin and insulin analogues (LysB28,ProB29-insulin (LysPro), AspB10-insulin (B10), and GluB4,GlnB16,PheB17-insulin (EQF)) in H4, HepG2, and L6 cells. These effects were compared with receptor binding. Protein degradation was examined by release of trichloroacetic acid-soluble radioactivity from cells previously labeled with [3H]leucine. Short- and intermediate-lived proteins were examined. H4 cells bound insulin with an EC50of 4.6 × 10−9m. LysPro was similar. The affinity of B10 was increased 2-fold; that of EQF decreased 15-fold. Protein degradation inhibition in H4 cells was highly sensitive to insulin (EC50= 4.2 × 10−11and 1.6 × 10−10m, short- and intermediate-lived protein degradation, respectively) and analogues. Despite similar binding, LysPro was 11- to 18-fold more potent than insulin at inhibiting protein degradation. Conversely, although EQF showed lower binding to H4 cells than insulin, its action was similar. The relative binding potencies of analogues in HepG2 cells were similar to those in H4 cells. Examination of protein degradation showed insulin, LysPro, and B10 were equivalent while EQF was less potent. L6 cells showed no difference in the binding of the analogues compared with insulin, but their effect on protein degradation was similar to that seen in HepG2 cells except B10 inhibited intermediate-lived protein degradation better than insulin. These studies illustrate the complexities of cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The differences seen in the action of the analogues cannot be attributed to binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.

scholarly journals Targeting the Ubiquitin-Proteasome System for Cancer Therapeutics by Small-Molecule Inhibitors

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3079
Author(s):  
Gabriel LaPlante ◽  
Wei Zhang
Keyword(s):  
Protein Degradation ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Proteasome Inhibitors ◽  
Cancer Therapeutics ◽  
Ubiquitin Proteasome System ◽  
Cellular Protein ◽  
E3 Ligases ◽  
Protein Levels ◽  
Ubiquitin Proteasome

The ubiquitin-proteasome system (UPS) is a critical regulator of cellular protein levels and activity. It is, therefore, not surprising that its dysregulation is implicated in numerous human diseases, including many types of cancer. Moreover, since cancer cells exhibit increased rates of protein turnover, their heightened dependence on the UPS makes it an attractive target for inhibition via targeted therapeutics. Indeed, the clinical application of proteasome inhibitors in treatment of multiple myeloma has been very successful, stimulating the development of small-molecule inhibitors targeting other UPS components. On the other hand, while the discovery of potent and selective chemical compounds can be both challenging and time consuming, the area of targeted protein degradation through utilization of the UPS machinery has seen promising developments in recent years. The repertoire of proteolysis-targeting chimeras (PROTACs), which employ E3 ligases for the degradation of cancer-related proteins via the proteasome, continues to grow. In this review, we will provide a thorough overview of small-molecule UPS inhibitors and highlight advancements in the development of targeted protein degradation strategies for cancer therapeutics.

scholarly journals Targeted Protein Degradation via a Covalent Reversible Degrader Based on Bardoxolone

2020 ◽  
Author(s):  
Bingqi Tong ◽  
Mai Luo ◽  
Yi Xie ◽  
Jessica Spradlin ◽  
John A. Tallarico ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Binding ◽  
Protein Degradation ◽  
Small Molecules ◽  
Small Molecule ◽  
E3 Ligase ◽  
E3 Ligases ◽  
Protein Levels ◽  
Nrf2 Activator

<p>Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules (i.e. PROTACs). E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the >500 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-NRF2 activator bardoxolone to a BRD4 inhibitor JQ1. Notably, this work reports the first covalent, reversible E3 ligase recruiter for TPD applications. </p>

scholarly journals Targeted Protein Degradation via a Covalent Reversible Degrader Based on Bardoxolone

2020 ◽  
Author(s):  
Bingqi Tong ◽  
Mai Luo ◽  
Yi Xie ◽  
Jessica Spradlin ◽  
John A. Tallarico ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Binding ◽  
Protein Degradation ◽  
Small Molecules ◽  
Small Molecule ◽  
E3 Ligase ◽  
E3 Ligases ◽  
Protein Levels ◽  
Nrf2 Activator

<p>Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules (i.e. PROTACs). E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the >500 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-NRF2 activator bardoxolone to a BRD4 inhibitor JQ1. Notably, this work reports the first covalent, reversible E3 ligase recruiter for TPD applications. </p>

scholarly journals Tubular cell protein degradation in early diabetic renal hypertrophy.

1994 ◽  
Vol 4 (8) ◽  
pp. 1582-1587
Author(s):  
P Shechter ◽  
G Boner ◽  
R Rabkin
Keyword(s):  
Protein Degradation ◽  
Protein Content ◽  
Cellular Protein ◽  
Diabetic Rats ◽  
Tubular Cell ◽  
Cell Protein ◽  
Renal Hypertrophy ◽  
Cathepsin Activity ◽  
Wet Weight

Renal hypertrophy in diabetes is accompanied by an increase in kidney protein content, which reflects an imbalance between protein synthesis and degradation. This study determines whether altered cellular protein degradation contributes to the imbalance. Diabetes was induced in rats with streptozotocin (55 mg/kg/ip). After 2 or 4 days of diabetes, kidney weight and protein content were measured. Over the 4 days, despite a loss in body weight, kidney wet weight increased by 35% and protein content by 37% in the diabetic rats. Treatment with insulin prevented this increase. Long-lived protein degradation was measured in isolated proximal tubules prelabeled with (14C)valine in vivo. Two days after streptozotocin, protein degradation was depressed by 19% (P < 0.05) and by the fourth day by 27% compared with that in nondiabetic controls (2.6% +/- 0.2 versus 1.9 +/- 0.1% degraded/h; P < 0.01). This was accompanied by a similar diabetes-induced decrease in proximal tubule cathepsin B and L activity. Accordingly, this study provides direct evidence that, in diabetes, tubular cell protein breakdown is depressed and suggests that altered lysosomal cathepsin activity may contribute to this effect. Depressed proteolysis likely contributes to the increase in kidney protein content and hence to diabetic renal hypertrophy.

scholarly journals Bardoxolone conjugation enables targeted protein degradation of BRD4

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bingqi Tong ◽  
Mai Luo ◽  
Yi Xie ◽  
Jessica N. Spradlin ◽  
John A. Tallarico ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Binding ◽  
Protein Degradation ◽  
Small Molecules ◽  
Small Molecule ◽  
E3 Ligase ◽  
E3 Ligases ◽  
Protein Levels ◽  
Nrf2 Activator

Abstract Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.

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