Native Protein Research Articles

High-throughput structure determination of an intrinsically disordered protein using cell-free protein crystallization

Intrinsically disordered proteins (IDPs) play a crucial role in various biological phenomena, dynamically changing their conformations in response to external environmental cues. To gain a deeper understanding of these proteins, it is essential to identify the determinants that fix their structures at the atomic level. Here, we developed a pipeline for rapid crystal structure analysis of IDP using a cell-free protein crystallization (CFPC) method. Through this approach, we successfully demonstrated the determination of the structure of an IDP to uncover the key determinants that stabilize its conformation. Specifically, we focused on the 11-residue fragment of c-Myc, which forms an α-helix through dimerization with a binding partner protein. This fragment was strategically recombined with an in-cell crystallizing protein and was expressed in a cell-free system. The resulting crystal structures of the c-Myc fragment were successfully determined at a resolution of 1.92 Å and we confirmed that they are identical to the structures of the complex with the native binding partner protein. This indicates that the environment of the scaffold crystal can fix the structure of c-Myc. Significantly, these crystals were obtained directly from a small reaction mixture (30 µL) incubated for only 72 h. Analysis of eight crystal structures derived from 22 mutants revealed two hydrophobic residues as the key determinants responsible for stabilizing the α-helical structure. These findings underscore the power of our CFPC screening method as a valuable tool for determining the structures of challenging target proteins and elucidating the essential molecular interactions that govern their stability.

Virtual Discovery of Immune-Stimulating Epitopes in Chikungunya Virus for Vaccine Design

Epitope identification is a key step in vaccine development, and this can be achieved much faster and less expensively with in silico methods, compared to traditional methods for vaccine production. In silico methods applied in this research utilised both bioinformatics and immunoinformatics approaches for chikungunya virus vaccine design, which involved the retrieval of sequences from databases, and identification of conserved regions within the sequences by multiple sequence alignment on the MEGA X software (Pennsylvania State University, State College, USA). The epitopes in the conserved regions were selected, and various immunological predictions and screenings were carried out by employing immunological databases and tools. This process identifies epitopes such as conservation of cytotoxic T lymphocyte, helper T lymphocytes, and B cell epitopes. The primary, secondary, and tertiary structure of the vaccine was also predicted using structure predicting servers, and finally, the vaccine candidate was docked to toll-like receptor 4 to study its binding affinity and configuration. A total of 125 conserved antigenic epitopes were selected from capsid, 6K, and E1 proteins, which were found to be non-allergens and conform to acceptable physicochemical standards, as reported by other authors with similar work. The epitopes were predicted to be capable of inducing cytotoxic T lymphocytes, helper T lymphocytes, and B cell production. Construction of secondary structure was done using the Self-Optimized Prediction Method with Alignment (SOPMA), which predicted 17.96% α-helices, and 4.69% β-turns, among others. Predicting the tertiary structure provided five models, of which Model 1 was selected on the bases of its confidential score of 0.59, estimated TM-score of 0.79±0.09, and root mean square deviation of 8.0±4.4Å. Validity analysis revealed a Ramachandran plot where 97.2% of the vaccine residue was within the favoured region, and the peptide showed a Z-score of -1.52. The predicted peptide effectively docked with toll-like receptor 4 with a binding energy of -1,072.8. From the data obtained, it was revealed that the selected epitopes are highly immunogenic, non-allergenic, conform to native protein, and form a peptide capable of vaccine application. The authors can conclude this is a promising candidate for vaccine design and development.

In situ interaction of pea peptides and bile salts under in vitro digestion: Potential impact on lipolysis

The present work evaluated how a native pea protein isolate (PPI) affects the key roles carried out by bile salts (BS) in lipid digestion by means of the in vitro static INFOGEST protocol. Two gastric residence times were evaluated (10 and 60 min), and then the peptides obtained (GPPP) were mixed with BS at physiological concentration in simulated intestinal fluid to understand how they interact with BS both at the bulk and at the interface. Both GPPP give rise to a film with a predominant viscous character that does not constitute a barrier to the penetration of BS, but interact with BS in the bulk duodenal fluid. When the peptides flushing from the stomach after the different gastric residence times undergo duodenal digestion, it was found that for the longer gastric residence time the percentage of soluble fraction in the duodenal phase, that perform synergistically with BS micelles, was twice that of the lower residence time, leading to an increase in the solubilization of oleic acid. These results finally lead to a greater extent of lipolysis of olive oil emulsions.This work demonstrates the usefulness of in vitro models as a starting point to study the influence of gastric residence time of pea protein on its interaction with BS, affecting lipolysis. Pea proteins were shown to be effective emulsifiers that synergistically perform with BS improving the release and bioaccessibility of bioactive lipids as olive oil.

Exploring the binding mechanism and esterase-like activity of human serum albumin with levofloxacin and its choline based conjugates: A biophysical approach

Human serum albumin (HSA) effectively binds to compounds having different molecular weight and thus facilitates their distribution in the living organisms. Thus, the binding interactions between a potential antibacterial drug (levofloxacin) and synthesized choline based levofloxacinate conjugates with HSA have been explored. The binding efficacy and mechanism were explored by utilizing different spectroscopic techniques; UV–Visible, steady state fluorescence, time resolved fluorescence and esterase-like activity. The interactions between the ligands and protein were electrostatic as well as hydrophobic in nature. The influence of different ligands having different alkyl chain shows quenching of the fluorescence emission of HSA. The spontaneous binding/quenching of HSA with ligands was static in nature, validated by steady state and time resolved fluorescence spectroscopy. Also, the impact of these ligands on the conformation of the native HSA structure was evaluated by using circular dichroism spectroscopy. In combination to the structural change study, the native protein functionality was observed (in terms of ‘esterase-like activity’) which has been found to be on lower side due to ligand binding. Further, we have performed the reverse study to check the impact of HSA on the fluorescent fluoroquinolone drug. The current study may prove helpful in elucidating the chemico-biological interactions which may prove useful in the pharmaceuticals, pharmacology, and different biochemistry fields.

Surpassing the natural limits of serological diagnostic tests

Serological tests have an important and irreplaceable role in the diagnosis of infectious diseases, both for individual patients and society. They indirectly identify the causative pathogen through the capture of antibodies, which contrasts with the direct detection by antigen and molecular biology tests that are also limited to active infections. Within point-of-care platforms, serodiagnostic assays can support immediate decisions in patient care and contain the spread of disease as they do not require highly trained personnel or dedicated infrastructure. By employing serodiagnosis, health officials can proactively respond and eliminate emerging health risks. Larger sample numbers can be screened in other formats for surveillance as well as securing donated blood supplies. Furthermore, an assay can be designed to detect any immunoglobulin from IgM to IgG and its subclasses, to IgA and IgE. However, most serological assays employ natural proteins as the defining antibody-capturing reagent, which compromises their performance by limiting the two critical parameters of a serodiagnostic test: specificity and sensitivity. To surpass this natural limitation, we have repurposed the β-barrel of fluorescent proteins to receive epitope sequences that dependably produce high-performing designer immunological reagents. Consequently, serodiagnosis can be conducted more accurately at a lower cost.

Analyzing noncovalent interactions between notoginseng saponins and lysozyme by deposition scanning intensity fading MALDI-TOF mass spectrometry.

Analysis of noncovalent interactions between natural products and proteins is important for rapid screening of active ingredients and understanding their pharmacological activities. In this work, the intensity fading MALDI-TOF mass spectrometry (IF-MALDI-MS) method with improved reproducibility was implemented to investigate the binding interactions between saponins from Panax notoginseng and lysozyme. The benchmark IF-MALDI-MS experiment was established using N,N',N″-triacetylchitotriose-lysozyme as a model system. The reproducibility of ion intensities in IF-MALDI-MS was improved by scanning the whole sample deposition with a focused laser beam. The relative standard deviation (RSD) of deposition scanning IF-MALDI-MS is 5.7%. Similar decay trends of the relative intensities of notoginseng saponins against increasing amounts of lysozyme were observed for all six notoginseng saponins. The half-maximal fading concentration (FC50) was calculated to quantitatively characterize the binding affinity of each ligand based on the decay curve. According to the FC50 values obtained, the binding affinities of the six notoginseng saponins were evaluated in the following order: notoginsenoside S > notoginsenoside Fc > ginsenoside Rb1 > ginsenoside Rd > notoginsenoside Ft1 > ginsenoside Rg1. The binding order was in accordance with molecular docking studies, which showed hydrogen bonding might play a key role in stabilizing the binding interaction. Our results demonstrated that deposition scanning IF-MALDI-MS can provide valuable information on the noncovalent interactions between ligands and proteins.

Construction and application of H1R ligand screening materials based on SMA stabilization and his-tag covalent immobilization of membrane proteins

The histamine H1 receptor (H1R) plays a pivotal role in allergy initiation and undergoes the necessity of devising a high-throughput screening approach centered on H1R to screen novel ligands effectively. This study suggests a method employing styrene maleic acid (SMA) extraction and His-tag covalent bonding to immobilize H1R membrane proteins, minimizing the interference of nonspecific proteins interference while preserving native protein structure and maximizing target exposure. This approach was utilized to develop a novel material for high-throughput ligand screening and implemented in cell membrane chromatography (CMC). An H1R-His-SMALPs/CMC model was established and its chromatographic performance (selectivity, specificity and lifespan) validated, demonstrating a significant enhancement in lifespan compared to previous CMC models. Subsequently, this model facilitated high-throughput screening of H1R ligands in the compound library and preliminary activity verification of potential H1R antagonists. Identification of a novel H1R antagonist laid the foundation for further development in this area.

Inflammatory caspase substrate specificities.

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. Thousands of putative substrates have been identified for caspases that regulate an immunologically silent type of cell death known as apoptosis, but less is known about substrates of the inflammatory caspases that regulate an immunostimulatory type of cell death called pyroptosis. Furthermore, much of our understanding of caspase substrate specificities is derived from work done with peptide substrates, which do not often translate to native protein substrates. Our knowledge of inflammatory caspase biology and substrates has recently expanded and here, we discuss the recent advances in our understanding of caspase substrate specificities, with a focus on inflammatory caspases. We highlight new substrates that have been discovered and discuss the factors that engender specificity. Recent evidence suggests that inflammatory caspases likely utilize two binding interfaces to recognize and process substrates, the active site and a conserved exosite.

Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens

Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein’s specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.

Novel Approach to Exploring Protease Activity and Targets in HIV-associated Obstructive Lung Disease using Combined Proteomic-Peptidomic Analysis.

Obstructive lung disease (OLD) is increasingly prevalent among persons living with HIV (PLWH). However, the role of proteases in HIV-associated OLD remains unclear. We combined proteomics and peptidomics to comprehensively characterize protease activities. We combined mass spectrometry (MS) analysis on bronchoalveolar lavage fluid (BALF) peptides and proteins from PLWH with OLD (n=25) and without OLD (n=26) with a targeted Somascan aptamer-based proteomic approach to quantify individual proteases and assess their correlation with lung function. Endogenous peptidomics mapped peptides to native proteins to identify substrates of protease activity. Using the MEROPS database, we identified candidate proteases linked to peptide generation based on binding site affinities which were assessed via z-scores. We used t-tests to compare average forced expiratory volume in 1 second per predicted value (FEV1pp) between samples with and without detection of each cleaved protein and adjusted for multiple comparisons by controlling the false discovery rate (FDR). We identified 101 proteases, of which 95 had functional network associations and 22 correlated with FEV1pp. These included cathepsins, metalloproteinases (MMP), caspases and neutrophil elastase. We discovered 31 proteins subject to proteolytic cleavage that associate with FEV1pp, with the top pathways involved in small ubiquitin-like modifier mediated modification (SUMOylation). Proteases linked to protein cleavage included neutrophil elastase, granzyme, and cathepsin D. In HIV-associated OLD, a significant number of proteases are up-regulated, many of which are involved in protein degradation. These proteases degrade proteins involved in cell cycle and protein stability, thereby disrupting critical biological functions.

De novo design of proteins housing excitonically coupled chlorophyll special pairs

Natural photosystems couple light harvesting to charge separation using a ‘special pair’ of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.

The conformational landscape of fold-switcher KaiB is tuned to the circadian rhythm timescale.

How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from Rhodobacter sphaeroides. Unique among known natural metamorphic proteins, this variant of KaiB spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) state. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and KaiC binding-competent FS state, underscoring the ability of a single protein sequence to encode the slow process needed for its biological function. We reveal that the rate-limiting step between the Ground and FS state is the cis-trans isomerization of three prolines in the C-terminal fold-switching region by demonstrating acceleration of interconversion by the prolyl cis/trans isomerase CypA. The interconversion proceeds through a "partially disordered" (PD) state, where the N-terminal half remains stably folded while the C-terminal half becomes disordered. We discovered two additional properties of KaiB's landscape: firstly, the Ground state experiences cold denaturation: at 4°C, the PD state becomes the majorly populated state. Secondly, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to suggest that this "Enigma" state represents a beta-strand register shift that eases buried charged residues in the Ground state. These results provide mechanistic insight in how evolution can design a single sequence that achieves specific timing needed for its function.

Native and tagged CENP-A histones are functionally inequivalent

BackgroundOver the past several decades, the use of biochemical and fluorescent tags has elucidated mechanistic and cytological processes that would otherwise be impossible. The challenging nature of certain nuclear proteins includes low abundancy, poor antibody recognition, and transient dynamics. One approach to get around those issues is the addition of a peptide or larger protein tag to the target protein to improve enrichment, purification, and visualization. However, many of these studies were done under the assumption that tagged proteins can fully recapitulate native protein function.ResultsWe report that when C-terminally TAP-tagged CENP-A histone variant is introduced, it undergoes altered kinetochore protein binding, differs in post-translational modifications (PTMs), utilizes histone chaperones that differ from that of native CENP-A, and can partially displace native CENP-A in human cells. Additionally, these tagged CENP-A-containing nucleosomes have reduced centromeric incorporation at early G1 phase and poorly associates with linker histone H1.5 compared to native CENP-A nucleosomes.ConclusionsThese data suggest expressing tagged versions of histone variant CENP-A may result in unexpected utilization of non-native pathways, thereby altering the biological function of the histone variant.

A phase I/II open label study to assess safety and preliminary evidence of a therapeutic effect of azeliragon in patients refractory to first-line treatment of metastatic pancreatic cancer.

TPS4212 Background: Azeliragon is an orally administered small molecule, administered once daily, that inhibits RAGE interactions with its natural ligands, including HMGB1 and S100 proteins. Azeliragon was previously under development for Alzheimer’s disease and failed to show efficacy in phase 3 trials. Clinical safety data from these trials, with over 1500 individuals dosed for periods up to 18 months, indicated that azeliragon has a high level of safety and tolerability, virtually indistinguishable from placebo. RAGE, the receptor for advanced glycation end products, is highly expressed in pancreatic ductal adenocarcinoma (PDAC). RAGE interaction with its ligands, including S100 proteins and HMGB1 released from PDAC cells, promotes PDAC invasion, metastasis, and resistance to 5 FU and Gemcitabine. PDAC-secreted S100 proteins can also stimulate fibroblasts surrounding PDAC cells since RAGE is positively expressed in fibroblasts, promoting the desmoplastic response. RAGE has also been implicated in the muscle wasting associated with cancer and with aging, as well as in neuropathic pain, suggesting a potential supportive role in PDAC treatment. Methods: This is an open label study to determine the safety and preliminary evidence of a therapeutic effect of azeliragon in patients refractory to the standard of care treatment of metastatic pancreatic cancer. Azeliragon is administered at three dose levels in a rolling dose escalation strategy with 6 patients at each dose level: 5 mg/day (level 1), 10 mg/day (level 2) and 20 mg/day (level 3). These dose levels have an initial loading dose for 6 days at 15 mg daily, 15 mg twice a day and 30 mg twice a day, respectively. The primary objective is to determine the RP2D, defined as the dose for which <33% patients experience a dose limiting toxicity (DLT) within 28 days from initiation of dosing. Secondary endpoints include disease control, overall survival, changes in pain as determined by the Brief Pain Inventory, and changes in ECOG performance status, weight, and serum albumin. The study received regulatory approval and accrual started in June 2023. Clinical trial information: NCT05766748 .

Identification of a norovirus GII-specific antigenic epitope.

Noroviruses (NoVs) are the chief cause of acute viral gastroenteritis worldwide. By employing the major capsid protein VP1 of a GII.6 NoV strain as an immunogen, we generated two monoclonal antibodies (mAbs) with wide-spectrum binding activities against NoV genogroup II (GII) VP1 proteins. One mAb (10G7) could bind to native and denatured GII-specific VP1 proteins. The other mAb (10F2) could bind to all tested native GII VP1 proteins, but not to denatured GII.3, GII.4, GII.7, or GII.17 VP1 proteins. Using GII.6/GII.4 fusion proteins, the mAb 10F2 binding region was confirmed to be located in the C-terminal P1 domain. An enzyme-linked immunosorbent assay based on peptides covering the P domain did not detect any binding. Using a panel of VP1 proteins with swapped regions, deletions, and mutations, the mAb 10F2 binding region was determined to be located between residues 496 and 513. However, the residue(s) responsible for its varied binding affinity for different denatured GII VP1 proteins remain to be identified. In summary, two NoV GII-specific cross-reactive mAbs were generated, and their binding regions were determined. Our results might facilitate the detection and immunogenic study of NoVs.

Soluble and insoluble expression of recombinant human interleukin-2 protein using pET expression vector in Escherichia coli

Interleukin-2 has emerged as a potent protein-based drug to treat various cancers, AIDS, and autoimmune diseases. Despite its immense requirement, the production procedures are inefficient to meet the demand. Therefore, efficient production procedures must be adopted to improve protein yield and decrease procedural loss. This study analyzed cytoplasmic and periplasmic IL-2 expression for increased protein yield and significant biological activity. The study is focused on cloning IL-2 into a pET-SUMO and pET-28a vector that expresses IL-2 in soluble form and inclusion bodies, respectively. Both constructs were expressed into different E. coli expression strains, but the periplasmic and cytoplasmic expression of IL-2 was highest in overnight culture in Rosetta 2 (DE3). Therefore, E. coli Rosetta 2 (DE3) was selected for large-scale production and purification. Purified IL-2 was characterized by SDS-PAGE and western blotting, while its biological activity was determined using MTT bioassay. The results depict that the periplasmic and cytoplasmic IL-2 achieved adequate purification, yielding 0.86 and 0.51 mg/mL, respectively, with significant cytotoxic activity of periplasmic and cytoplasmic IL-2. Periplasmic IL-2 has shown better yield and significant biological activity in vitro which describes its attainment of native protein structure and function.

AsIII Selectively Induces a Disorder-to-Order Transition in the Metalloid Binding Region of the AfArsR Protein.

Arsenic is highly toxic and a significant threat to human health, but certain bacteria have developed defense mechanisms initiated by AsIII binding to AsIII-sensing proteins of the ArsR family. The transcriptional regulator AfArsR responds to AsIII and SbIII by coordinating the metalloids with three cysteines, located in a short sequence of the same monomer chain. Here, we characterize the binding of AsIII and HgII to a model peptide encompassing this fragment of the protein via solution equilibrium and spectroscopic/spectrometric techniques (pH potentiometry, UV, CD, NMR, PAC, EXAFS, and ESI-MS) combined with DFT calculations and MD simulations. Coordination of AsIII changes the peptide structure from a random-coil to a well-defined structure of the complex. A trigonal pyramidal AsS3 binding site is formed with almost exactly the same structure as observed in the crystal structure of the native protein, implying that the peptide possesses all of the features required to mimic the AsIII recognition and response selectivity of AfArsR. Contrary to this, binding of HgII to the peptide does not lead to a well-defined structure of the peptide, and the atoms near the metal binding site are displaced and reoriented in the HgII model. Our model study suggests that structural organization of the metal site by the inducer ion is a key element in the mechanism of the metalloid-selective recognition of this protein.

Immune Response to the Recombinant Apa Protein from Mycobacterium tuberculosis Expressed in Streptomyces lividans After Intranasal Administration in Mice. Induction of Protective Response to Tubercle Bacillus Aerosols Exposure

Identifying and evaluating potential vaccine candidates has become one of the main objectives to combat tuberculosis. Among them, mannosylated Apa antigen from Mycobacterium tuberculosis and the non-mannosylated protein expressed in Escherichia coli, have been studied. Although both proteins can induce a protective response in mice, it has been considered that native protein can be dispensed. In this work, we study the protective response induced by Apa expressed in E. coli and in Streptomyces lividans. The latter, like native is secreted as a double band of 45/47 kDa, however, only its 47 kDa band is mannosylated. Both antigens and BCG were intranasal administrated in mice, and animals were then challenged by aerosol with M. tuberculosis H37Rv. The results showed that both, Apa from S. lividans and E. coli conferred statistically significantly protection to animals compared to controls. The cytokine immune response was studied by an immunoassay after animals’ immunization, revealing that Apa from S. lividans induced a statistically significant proliferation of T cell, as well as the expression of IFN-γ, IL-1β, IL-17 and IL-10. In contrast, non-proliferation was obtained with non-mannosylated protein, but induction of IL-12 and IL-17 was observed. Together, these results demonstrate that both proteins were able to modulate a specific immune response against M. tuberculosis, that could be driven by different mechanisms possibly associated with the presence or not of mannosylation. Furthermore, stimulation of cells from BCG-vaccinated animals with the proteins could be an important tool, to help define the use of a given subunit-vaccine after BCG vaccination.

De novo design of buttressed loops for sculpting protein functions

In natural proteins, structured loops have central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that designs tandem repeat proteins with structured loops (9–14 residues) buttressed by extensive hydrogen bonding interactions. Experimental characterization shows that the designs are monodisperse, highly soluble, folded and thermally stable. Crystal structures are in close agreement with the design models, with the loops structured and buttressed as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute generally to efforts to design new protein functions.

A high content imaging assay for identification of specific inhibitors of native Plasmodium liver stage protein synthesis.

Plasmodium parasite resistance to antimalarial drugs is a serious threat to public health in malaria-endemic areas. Compounds that target core cellular processes like translation are highly desirable, as they should be multistage actives, capable of killing parasites in the liver and blood, regardless of molecular target or mechanism. Assays that can identify these compounds are thus needed. Recently, specific quantification of native Plasmodium berghei liver stage protein synthesis as well as that of the hepatoma cells supporting parasite growth, was achieved via automated confocal feedback microscopy of the o-propargyl puromycin (OPP)-labeled nascent proteome, but this imaging modality is limited in throughput. Here, we developed and validated a miniaturized high content imaging (HCI) version of the OPP assay that increases throughput, before deploying this approach to screen the Pathogen Box. We identified only two hits, both of which are parasite-specific quinoline-4-carboxamides, and analogues of the clinical candidate and known inhibitor of blood and liver stage protein synthesis, DDD107498/cabamiquine. We further show that these compounds have strikingly distinct relationships between their antiplasmodial and translation inhibition efficacies. These results demonstrate the utility and reliability of the P. berghei liver stage OPP HCI assay for specific, single-well quantification of Plasmodium and human protein synthesis in the native cellular context, allowing identification of selective Plasmodium translation inhibitors with the highest potential for multistage activity.