Discovery of the First-in-Class Inhibitors of Hypoxia Up-Regulated Protein†1 (HYOU1) Suppressing Pathogenic Fibroblast Activation.

Fibroblasts are key regulators of inflammation, fibrosis, and cancer. Targeting their activation in these complex diseases has emerged as a novel strategy to restore tissue homeostasis. Here, we present a multidisciplinary lead discovery approach to identify and optimize small molecule inhibitors of pathogenic fibroblast activation. The study encompasses medicinal chemistry, molecular phenotyping assays, chemoproteomics, bulk RNA-sequencing analysis, target validation experiments, and chemical absorption, distribution, metabolism, excretion and toxicity (ADMET)/pharmacokinetic (PK)/in vivo evaluation. The parallel synthesis employed for the production of the new benzamide derivatives enabled us to a) pinpoint key structural elements of the scaffold that provide potent fibroblast-deactivating effects in cells, b) discriminate atoms or groups that favor or disfavor a desirable ADMET profile, and c) identify metabolic "hot spots". Furthermore, we report the discovery of the first-in-class inhibitor leads for hypoxia up-regulated protein 1 (HYOU1), a member of the heat shock protein 70 (HSP70) family often associated with cellular stress responses, particularly under hypoxic conditions. Targeting HYOU1 may therefore represent a potentially novel strategy to modulate fibroblast activation and treat chronic inflammatory and fibrotic disorders.

The Discovery of Peptide Macrocycle Rescuers of Pathogenic Protein Misfolding and Aggregation by Integrating SICLOPPS Technology and Ultrahigh-Throughput Screening in Bacteria.

The phenomenon of protein misfolding and aggregation has been widely associated with numerous human diseases, such as Alzheimer's disease, systemic amyloidosis and type 2 diabetes, the vast majority of which remain incurable. To advance early stage drug discovery against these diseases, investigation of molecular libraries with expanded diversities and ultrahigh-throughput screening methodologies that allow deeper investigation of chemical space are urgently required. Toward this, we describe how Escherichia coli can be engineered so as to enable (1) the production of expanded combinatorial libraries of short, drug-like, head-to-tail cyclic peptides and (2) their simultaneous functional screening for identifying effective inhibitors of protein misfolding and aggregation using a genetic assay that links protein folding and misfolding to cell fluorescence. In this manner, cyclic peptides with the ability to inhibit pathogenic protein misfolding and/or aggregation can be readily selected by flow cytometric cell sorting in an ultrahigh-throughput fashion. This biotechnological approach accelerates significantly the identification of hit/lead molecules with potentially therapeutic properties against devastating diseases.

Microbial Genetic Screens for Monitoring Protein Misfolding Associated with Neurodegeneration: Tools for Identifying Disease-Relevant Genes and for Screening Synthetic and Natural Compound Libraries for the Discovery of Potential Therapeutics.

Neurodegenerative Diseases (ND) are a major threat to the aging population and the lack of a single preventive or disease-modifying agent only serves to increase their impact. In the past few years, protein misfolding and the subsequent formation of neurotoxic oligomeric/aggregated protein species have emerged as a unifying theme underlying the pathology of these complex diseases. Recently developed microbial genetic screens and selection systems for monitoring ND-associated protein misfolding have allowed the establishment of highthroughput assays for the identification of cellular factors and processes that are important mediators of NDassociated proteotoxicities. In addition, such systems have facilitated the discovery of synthetic and natural compounds with the ability to rescue the misfolding and the associated pathogenic effects of aggregation-prone proteins associated with NDs. This review outlines such available systems in bacteria and yeast, whose usage will likely accelerate the pre-clinical discovery process for effective drugs against a variety of NDs with high socioeconomic impact.

An integrated bacterial system for the discovery of chemical rescuers of disease-associated protein misfolding.

Protein misfolding and aggregation are common pathological features of several human diseases, including Alzheimer's disease and type 2 diabetes. Here, we report an integrated and generalizable bacterial system for the facile discovery of chemical rescuers of disease-associated protein misfolding. In this system, large combinatorial libraries of macrocyclic molecules are biosynthesized in Escherichia coli cells and simultaneously screened for their ability to rescue pathogenic protein misfolding and aggregation using a flow cytometric assay. We demonstrate the effectiveness of this approach by identifying drug-like, head-to-tail cyclic peptides that modulate the aggregation of the Alzheimer's disease-associated amyloid ß peptide. Biochemical, biophysical and biological assays using isolated amyloid ß peptide, primary neurons and various established Alzheimer's disease nematode models showed that the selected macrocycles potently inhibit the formation of neurotoxic amyloid ß peptide aggregates. We also applied the system to the identification of misfolding rescuers of mutant Cu/Zn superoxide dismutase-an enzyme linked with inherited forms of amyotrophic lateral sclerosis. Overall, the system enables the identification of molecules with therapeutic potential for rescuing the misfolding of disease-associated polypeptides.

Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli.

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.

Engineered chimeric enzymes as tools for drug discovery: generating reliable bacterial screens for the detection, discovery, and assessment of estrogen receptor modulators.

Engineered protein-based sensors of ligand binding have emerged as attractive tools for the discovery of therapeutic compounds through simple screening systems. We have previously shown that engineered chimeric enzymes, which combine the ligand-binding domains of nuclear hormone receptors with a highly sensitive thymidylate synthase reporter, yield simple sensors that report the presence of hormone-like compounds through changes in bacterial growth. This work describes an optimized estrogen sensor in Escherichia coli with extraordinary reliability in identifying diverse estrogenic compounds and in differentiating between their agonistic/antagonistic pharmacological effects. The ability of this system to assist the discovery of new estrogen-mimicking compounds was validated by screening a small compound library, which led to the identification of two structurally novel estrogen receptor modulators and the accurate prediction of their agonistic/antagonistic biocharacter in human cells. Strong evidence is presented here that the ability of our sensor to detect ligand binding and recognize pharmacologically critical properties arises from allosteric communication between the artificially combined protein domains, where different ligand-induced conformational changes in the receptor are transmitted to the catalytic domain and translated to distinct levels of enzymic efficiency. To the best of our knowledge, this is one of the first examples of an engineered enzyme with the ability to sense multiple receptor conformations and to be either activated or inactivated depending on the nature of the bound effector molecule. Because the proposed mechanism of ligand dependence is not specific to nuclear hormone receptors, we anticipate that our protein engineering strategy will be applicable to the construction of simple sensors for different classes of (therapeutic) binding proteins.

Rapid detection of subtype-selective nuclear hormone receptor binding with bacterial genetic selection.

Subtype-selective nuclear hormone receptor modulators could potentially allow the development of valuable tissue-specific therapeutics. A simple biosensor that allows subtype-specific nuclear hormone receptor binding to be reflected by the growth phenotype of Escherichia coli cells has been constructed. This system will potentially enable the facile detection or evolution of subtype-selective hormone analogues.

A bacterial biosensor of endocrine modulators.

The nuclear hormone receptors comprise one of the largest classes of protein targets for drug discovery, as their function has been linked to a variety of serious diseases, including several forms of cancer. Identifying novel compounds with the ability to modulate the function of these targets could lead to the development of effective therapeutics. In vivo sensors of ligand binding have emerged as tools that can greatly accelerate the lead identification process, allowing new drugs to be discovered more rapidly and cheaply. In this work, a novel sensor of nuclear hormone binding has been developed in Escherichia coli by constructing a fusion of the ligand-binding domain of the human estrogen receptor with a thymidylate synthase enzyme (TS). Expression of this fusion protein in TS-deficient bacterial cells resulted in growth phenotypes that were dependent on the presence of estrogen. Subsequent replacement of the estrogen receptor with the ligand-binding domain of the human thyroid hormone receptor led to specific thyroid hormone-enhanced growth that was insensitive to estrogen. This biosensor was then challenged with a small library of estrogen and thyroid hormone analogues, and it was observed that levels of cell growth correlate well with ligand-binding affinity. Remarkably, this simple biosensor was able to discriminate between agonistic and antagonistic activities, as combinations of estrogen agonists had an additive impact on cell growth, whereas known estrogen antagonists were found to neutralize agonist effects. This system constitutes a technique for facile selection of lead compounds with potential medical applications.

Regulation of protein activity with small-molecule-controlled inteins.

Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts. Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision. These protein elements therefore have the potential to be used as general molecular "switches" for the control of arbitrary target proteins. Based on rational design, an intein-based protein switch has been constructed whose splicing activity is conditionally triggered in vivo by the presence of thyroid hormone or synthetic analogs. This modified intein was used in Escherichia coli to demonstrate that a number of different proteins can be inactivated by intein insertion and then reactivated by the addition of thyroid hormone via ligand-induced splicing. This conditional activation was also found to occur in a dose-dependent manner. Rational protein engineering was then combined with genetic selection to evolve an additional intein whose activity is controlled by the presence of synthetic estrogen ligands. The ability to regulate protein function post-translationally through the use of ligand-controlled intein splicing will most likely find applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, as well as many additional areas of biotechnology.