Controlled Proteolysis of an Essential Virulence Determinant Dictates Infectivity of Lyme Disease Pathogens.

The Borrelia burgdorferi BB0323 protein undergoes a complex yet poorly defined proteolytic maturation event that generates N-terminal and C-terminal proteins with essential functions in cell growth and infection. Here, we report that a borrelial protease, B. burgdorferi high temperature requirement A protease (BbHtrA), cleaves BB0323 between asparagine (N) and leucine (L) at positions 236 and 237, while the replacement of these residues with alanine in the mutant protein prevents its cleavage, despite preserving its normal secondary structure. The N-terminal BB0323 protein binds BbHtrA, but its cleavage site mutant displays deficiency in such interaction. An isogenic borrelial mutant with NL-to-AA substitution in BB0323 (referred to as Bbbb0323NL) maintains normal growth yet is impaired for infection of mice or transmission from infected ticks. Notably, the BB0323 protein is still processed in Bbbb0323NL, albeit with lower levels of mature N-terminal BB0323 protein and multiple aberrantly processed polypeptides, which could result from nonspecific cleavages at other asparagine and leucine residues in the protein. The lack of infectivity of Bbbb0323NL is likely due to the impaired abundance or stoichiometry of a protein complex involving BB0238, another spirochete protein. Together, these studies highlight that a precise proteolytic event and a particular protein-protein interaction, involving multiple borrelial virulence determinants, are mutually inclusive and interconnected, playing essential roles in the infectivity of Lyme disease pathogens.

Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors.

Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. (2016).

Systematic mapping of WNT-FZD protein interactions reveals functional selectivity by distinct WNT-FZD pairs.

The seven-transmembrane-spanning receptors of the FZD1-10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD2, FZD4, or FZD5, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-ß-catenin pathway through FZD2/4/5 as measured by phosphorylation of LRP6 and ß-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.

Functional consequences of Wnt-induced dishevelled 2 phosphorylation in canonical and noncanonical Wnt signaling.

Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594-609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/ß-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ε as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser(594), Thr(595), and Ser(597) of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates ß-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.

Advances in luminescence-based technologies for drug discovery.

INTRODUCTION: Luminescence-based technologies, specifically bioluminescence and chemiluminescence, are powerful tools with extensive use in drug discovery. Production of light during chemiluminescence and bioluminescence, unlike fluorescence, doesn't require an excitation light source, resulting in high signal-to-noise ratio, less background interference, and no issues from phototoxicity and photobleaching. These characteristics of luminescence technologies offer unique advantages for experimental designs, allowing for greater flexibility to target a wide range of proteins and biological processes for drug discovery at different stages. AREAS COVERED: This review provides a basic overview of luciferase-based technologies and details recent advances and use cases of luciferase and luciferin variations and their applicability in the drug discovery toolset. The authors expand upon specific applications of luciferase technologies, including chemiluminescent and bioluminescent-based microscopy. Finally, the authors lay out forward-looking statements on the field of luminescence and how it may shape the translational scientists' work moving forward. EXPERT OPINION: The demand for improved luciferase and luciferin pairs correlates strongly with efforts to improve the sensitivity and robustness of high-throughput assays. As luminescent reporter systems improve, so will the expansion of use cases for luminescence-based technologies in early-stage drug discovery. With the synthesis of novel, non-enzymatic chemiluminescence-based probes, which previously were restrained to only basic research applications, they may now be readily implemented in drug discovery campaigns.

Auranofin targets UBA1 and enhances UBA1 activity by facilitating ubiquitin trans-thioesterification to E2 ubiquitin-conjugating enzymes.

UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. Decreased or insufficient ubiquitination can cause or drive aging and many diseases. Therefore, a small-molecule enhancing UBA1 activity could have broad therapeutic potential. Here we report that auranofin, a drug approved for the treatment of rheumatoid arthritis, is a potent UBA1 activity enhancer. Auranofin binds to the UBA1's ubiquitin fold domain and conjugates to Cys1039 residue. The binding enhances UBA1 interactions with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest that auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration.

A unique borrelial protein facilitates microbial immune evasion.

IMPORTANCE: Lyme disease is a major tick-borne infection caused by a bacterial pathogen called Borrelia burgdorferi, which is transmitted by ticks and affects hundreds of thousands of people every year. These bacterial pathogens are distinct from other genera of microbes because of their distinct features and ability to transmit a multi-system infection to a range of vertebrates, including humans. Progress in understanding the infection biology of Lyme disease, and thus advancements towards its prevention, are hindered by an incomplete understanding of the microbiology of B. burgdorferi, partly due to the occurrence of many unique borrelial proteins that are structurally unrelated to proteins of known functions yet are indispensable for pathogen survival. We herein report the use of diverse technologies to examine the structure and function of a unique B. burgdorferi protein, annotated as BB0238-an essential virulence determinant. We show that the protein is structurally organized into two distinct domains, is involved in multiplex protein-protein interactions, and facilitates tick-to-mouse pathogen transmission by aiding microbial evasion of early host cellular immunity. We believe that our findings will further enrich our understanding of the microbiology of B. burgdorferi, potentially impacting the future development of novel prevention strategies against a widespread tick-transmitted infection.

Dome1-JAK-STAT signaling between parasite and host integrates vector immunity and development.

Ancestral signaling pathways serve critical roles in metazoan development, physiology, and immunity. We report an evolutionary interspecies communication pathway involving a central Ixodes scapularis tick receptor termed Dome1, which acquired a mammalian cytokine receptor motif exhibiting high affinity for interferon-gamma (IFN-γ). Host-derived IFN-γ facilitates Dome1-mediated activation of the Ixodes JAK-STAT pathway. This accelerates tick blood meal acquisition and development while upregulating antimicrobial components. The Dome1-JAK-STAT pathway, which exists in most Ixodid tick genomes, regulates the regeneration and proliferation of gut cells-including stem cells-and dictates metamorphosis through the Hedgehog and Notch-Delta networks, ultimately affecting Ixodes vectorial competence. We highlight the evolutionary dependence of I. scapularis on mammalian hosts through cross-species signaling mechanisms that dually influence arthropod immunity and development.

Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells.

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co-expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial-mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co-transfectants exhibited invasive properties in three-dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf-1, a specific antagonist of the Wnt/ß-catenin pathway, or short hairpin RNA targeting ß-catenin expression demonstrated that the invasive activity was not mediated by ß-catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context-dependent. While Rspo2 and Wnt1 act synergistically in the ß-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures.

Application of biophysical methods for improved protein production and characterization: A case study on an high-temperature requirement A-family bacterial protease.

The high-temperature requirement A (HtrA) serine protease family presents an attractive target class for antibacterial therapeutics development. These proteins possess dual protease and chaperone functions and contain numerous binding sites and regulatory loops, displaying diverse oligomerization patterns dependent on substrate type and occupancy. HtrA proteins that are natively purified coelute with contaminating peptides and activating species, shifting oligomerization and protein structure to differently activated populations. Here, a redesigned HtrA production results in cleaner preparations with high yields by overexpressing and purifying target protein from inclusion bodies under denaturing conditions, followed by a high-throughput screen for optimal refolding buffer composition using function-agnostic biophysical techniques that do not rely on target-specific measurements. We use Borrelia burgdorferi HtrA to demonstrate the effectiveness of our function-agnostic approach, while characterization with both new and established biophysical methods shows the retention of proteolytic and chaperone activity of the refolded protein. This systematic workflow and toolset will translate to the production of HtrA-family proteins in higher quantities of pure and monodisperse composition than the current literature standard, with applicability to a broad array of protein purification strategies.

Application of temperature-responsive HIS-tag fluorophores to differential scanning fluorimetry screening of small molecule libraries.

Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient, most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in histidine-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this HIS-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of Tm values with an average coefficient of variation of 0.51% and median coefficient of variation of compound Tm of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.

Discovery of small molecule inhibitors that effectively disrupt IQGAP1-Cdc42 interaction in breast cancer cells.

The small GTPase Cdc42 is an integral component of the cytoskeleton, and its dysregulation leads to pathophysiological conditions, such as cancer. Binding of Cdc42 to the scaffold protein IQGAP1 stabilizes Cdc42 in its active form. The interaction between Cdc42 and IQGAP1 enhances migration and invasion of cancer cells. Disrupting this association could impair neoplastic progression and metastasis; however, no effective means to achieve this has been described. Here, we screened 78,500 compounds using a homogeneous time resolved fluorescence-based assay to identify small molecules that disrupt the binding of Cdc42 to IQGAP1. From the combined results of the validation assay and counter-screens, we selected 44 potent compounds for cell-based experiments. Immunoprecipitation and cell viability analysis rendered four lead compounds, namely NCGC00131308, NCGC00098561, MLS000332963 and NCGC00138812, three of which inhibited proliferation and migration of breast carcinoma cells. Microscale thermophoresis revealed that two compounds bind directly to Cdc42. One compound reduced the amount of active Cdc42 in cells and effectively impaired filopodia formation. Docking analysis provided plausible models of the compounds binding to the hydrophobic pocket adjacent to the GTP binding site of Cdc42. In conclusion, we identified small molecules that inhibit binding between Cdc42 and IQGAP1, which could potentially yield chemotherapeutic agents.

Allosteric Binders of ACE2 Are Promising Anti-SARS-CoV-2 Agents.

The COVID-19 pandemic has had enormous health, economic, and social consequences. Vaccines have been successful in reducing rates of infection and hospitalization, but there is still a need for acute treatment of the disease. We investigate whether compounds that bind the human angiotensin-converting enzyme 2 (ACE2) protein can decrease SARS-CoV-2 replication without impacting ACE2's natural enzymatic function. Initial screening of a diversity library resulted in hit compounds active in an ACE2-binding assay, which showed little inhibition of ACE2 enzymatic activity (116 actives, success rate ∼4%), suggesting they were allosteric binders. Subsequent application of in silico techniques boosted success rates to ∼14% and resulted in 73 novel confirmed ACE2 binders with K d values as low as 6 nM. A subsequent SARS-CoV-2 assay revealed that five of these compounds inhibit the viral life cycle in human cells. Further effort is required to completely elucidate the antiviral mechanism of these ACE2-binders, but they present a valuable starting point for both the development of acute treatments for COVID-19 and research into the host-directed therapy.

Allosteric binders of ACE2 are promising anti-SARS-CoV-2 agents.

The COVID-19 pandemic has had enormous health, economic, and social consequences. Vaccines have been successful in reducing rates of infection and hospitalization, but there is still a need for an acute treatment for the disease. We investigate whether compounds that bind the human ACE2 protein can interrupt SARS-CoV-2 replication without damaging ACE2’s natural enzymatic function. Initial compounds were screened for binding to ACE2 but little interruption of ACE2 enzymatic activity. This set of compounds was extended by application of quantitative structure-activity analysis, which resulted in 512 virtual hits for further confirmatory screening. A subsequent SARS-CoV-2 replication assay revealed that five of these compounds inhibit SARS-CoV-2 replication in human cells. Further effort is required to completely determine the antiviral mechanism of these compounds, but they serve as a strong starting point for both development of acute treatments for COVID-19 and research into the mechanism of infection.

Real-Time Cellular Thermal Shift Assay to Monitor Target Engagement.

Determining a molecule's mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a protein's unfolding and aggregation properties in response to thermal challenge. In traditional CETSA experiments, each temperature requires an individual sample, which restricts throughput and requires substantial optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform by coupling a real-time PCR instrument with a CCD camera to detect luminescence. A thermally stable Nanoluciferase variant (ThermLuc) was bioengineered to withstand unfolding at temperatures greater than 90 °C and was compatible with monitoring target engagement events when fused to diverse targets. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA to detect on-target binding. RT-CETSA technology advances capabilities of the CETSA method and facilitates the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule.

Discovery and Optimization of Pyrrolopyrimidine Derivatives as Selective Disruptors of the Perinucleolar Compartment, a Marker of Tumor Progression toward Metastasis.

The perinucleolar compartment (PNC) is a dynamic subnuclear body found at the periphery of the nucleolus. The PNC is enriched with RNA transcripts and RNA-binding proteins, reflecting different states of genome organization. PNC prevalence positively correlates with cancer progression and metastatic capacity, making it a useful marker for metastatic cancer progression. A high-throughput, high-content assay was developed to identify novel small molecules that selectively reduce PNC prevalence in cancer cells. We identified and further optimized a pyrrolopyrimidine series able to reduce PNC prevalence in PC3M cancer cells at submicromolar concentrations without affecting cell viability. Structure-activity relationship exploration of the structural elements necessary for activity resulted in the discovery of several potent compounds. Analysis of in vitro drug-like properties led to the discovery of the bioavailable analogue, metarrestin, which has shown potent antimetastatic activity with improved survival in rodent models and is currently being evaluated in a first-in-human phase 1 clinical trial.

Hybrid In Silico Approach Reveals Novel Inhibitors of Multiple SARS-CoV-2 Variants.

The National Center for Advancing Translational Sciences (NCATS) has been actively generating SARS-CoV-2 high-throughput screening data and disseminates it through the OpenData Portal (https://opendata.ncats.nih.gov/covid19/). Here, we provide a hybrid approach that utilizes NCATS screening data from the SARS-CoV-2 cytopathic effect reduction assay to build predictive models, using both machine learning and pharmacophore-based modeling. Optimized models were used to perform two iterative rounds of virtual screening to predict small molecules active against SARS-CoV-2. Experimental testing with live virus provided 100 (∼16% of predicted hits) active compounds (efficacy > 30%, IC50 ≤ 15 µM). Systematic clustering analysis of active compounds revealed three promising chemotypes which have not been previously identified as inhibitors of SARS-CoV-2 infection. Further investigation resulted in the identification of allosteric binders to host receptor angiotensin-converting enzyme 2; these compounds were then shown to inhibit the entry of pseudoparticles bearing spike protein of wild-type SARS-CoV-2, as well as South African B.1.351 and UK B.1.1.7 variants.

Applications of Differential Scanning Fluorometry and Related Technologies in Characterization of Protein-Ligand Interactions.

Differential scanning fluorometry (DSF) is an efficient and high-throughput method to analyze protein stability, as well as detect ligand interactions through perturbations of the protein's melting temperature. The method monitors protein unfolding by observing the fluorescence changes of a sample, whether through an environmentally sensitive fluorophore or by intrinsic protein fluorescence, while a temperature gradient is applied. Here, we describe in detail how to develop and optimize DSF assays to identify protein-ligand interactions while exploring different buffer and additive conditions. Analysis of the data and further applications of the method are also discussed.

An OpenData portal to share COVID-19 drug repurposing data in real time.

The National Center for Advancing Translational Sciences (NCATS) has developed an online open science data portal for its COVID-19 drug repurposing campaign - named OpenData - with the goal of making data across a range of SARS-CoV-2 related assays available in real-time. The assays developed cover a wide spectrum of the SARS-CoV-2 life cycle, including both viral and human (host) targets. In total, over 10,000 compounds are being tested in full concentration-response ranges from across multiple annotated small molecule libraries, including approved drug, repurposing candidates and experimental therapeutics designed to modulate a wide range of cellular targets. The goal is to support research scientists, clinical investigators and public health officials through open data sharing and analysis tools to expedite the development of SARS-CoV-2 interventions, and to prioritize promising compounds and repurposed drugs for further development in treating COVID-19.

Mannose receptor (CD206) activation in tumor-associated macrophages enhances adaptive and innate antitumor immune responses.

Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206high patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.