Antibiotic resistance remains a leading cause of severe infections worldwide. Small changes in protein sequence can impact antibiotic efficacy. Here, we report deposition of 58 X-ray crystal structures of bacterial proteins that are known targets for antibiotics, which expands knowledge of structural variation to support future antibiotic discovery or modifications.

ABSTRACT During infection, bacterial pathogens rely on secreted virulence factors to manipulate the host cell. However, in gram-positive bacteria, the molecular mechanisms underlying the folding and activity of these virulence factors after membrane translocation are not clear. Here, we solved the protein structures of two secreted parvulin and two secreted cyclophilin-like peptidyl-prolyl isomerase (PPIase) ATP-independent chaperones found in gram-positive streptococcal species. The extracellular parvulin-type PPIase, PrsA in Streptococcus pneumoniae and Streptococcus mutans maintain dimeric crystal structures reminiscent of folding catalysts that consist of two domains, a PPIase and foldase domain. Structural comparison of the two cyclophilin-like extracellular chaperones from S. pneumoniae and Streptococcus pyogenes with other cyclophilins demonstrates that this group of cyclophilin-like chaperones has novel structural appendages formed by 9- and 24-residue insertions. Furthermore, we demonstrate that deletion of prsA and slrA genes impairs the secretion of the cholesterol-dependent pore-forming toxin, pneumolysin in S. pneumoniae. Using protein pull-down and biophysical assays, we demonstrate a direct interaction between PrsA and SlrA with Ply. Then, we developed chaperone-assisted folding assays that show that the S. pneumoniae PrsA and SlrA extracellular chaperones accelerate pneumolysin folding. In addition, we demonstrate that SlrA and, for the first time, S. pyogenes PpiA exhibit PPIase activity and can bind the immunosuppressive drug, cyclosporine A. Altogether, these findings suggest a mechanistic role for streptococcal PPIase chaperones in the activity and folding of secreted virulence factors such as pneumolysin.IMPORTANCEStreptococcal species are a leading cause of lower respiratory infections that annually affect millions of people worldwide. During infection, streptococcal species secrete a medley of virulence factors that allow the bacteria to colonize and translocate to deeper tissues. In many gram-positive bacteria, virulence factors are secreted from the cytosol across the bacterial membrane in an unfolded state. The bacterial membrane-cell wall interface is exposed to the potentially harsh extracellular environment, making it difficult for native virulence factors to fold before being released into the host. ATP-independent PPIase-type chaperones, PrsA and SlrA, are thought to facilitate folding and stabilization of several unfolded proteins to promote the colonization and spread of streptococci. Here, we present crystal structures of the molecular chaperones of PrsA and SlrA homologs from streptococcal species. We provide evidence that the Streptococcus pyogenes SlrA homolog, PpiA, has PPIase activity and binds to cyclosporine A. In addition, we show that Streptococcus pneumoniae PrsA and SlrA directly interact and fold the cholesterol-dependent pore-forming toxin and critical virulence determinant, pneumolysin.

Drug discovery starts with known function, either of a compound or a protein, in-turn prompting investigations to probe 3D structure of the compound-protein interface. As protein structure determines function, we hypothesized that unique 3D structural motifs represent primary information denoting unique function that can drive discovery of novel agents. Using a physics-based protein structure analysis platform developed by us, designed to conduct computationally intensive analysis at supercomputing speeds, we probed a high-resolution protein x-ray crystallographic library developed by us. We selected 3D structural motifs whose function was not otherwise established, that offered environments supporting binding of drug-like chemicals and were present on proteins that were not established therapeutic targets. For each of eight potential binding pockets on six different proteins we accessed a 60 million compound library and used our analysis platform to evaluate binding. Using eight-day colony formation assays acquired compounds were screened for efficacy against human breast, prostate, colon and lung cancer cells and toxicity against human bone marrow stem cells. Compounds selectively inhibiting cancer growth segregated to two pockets on separate proteins. The compound, Dxr2-017, exhibited selective activity against human melanoma cells in the NCI-60 cell line screen, had an IC50 of 19 nM against human melanoma M14 cells in our eight-day assay, while over 2100-fold higher concentrations inhibited stem cells by less than 30%. We show that Dxr2-017 induces anoikis, a unique form of programmed cell death in need of targeted therapeutics. The predicted target protein for Dxr2-017 is expressed in bacteria, not in humans. This supports our strategy of focusing on unique 3D structural motifs. It is known that functionally important 3D structures are evolutionarily conserved. Here we demonstrate proof-of-concept that protein structure represents high value primary data to support discovery of novel therapeutics. This approach is widely applicable.

Abstract Introduction: Drug action is mediated by a small molecule therapeutic binding to a specific structural motif on a protein. As structure determines function, it follows that unique structural motifs are enriched for sites of unique function and high therapeutic target value. We hypothesized that protein structure constitutes high value primary information that can drive the discovery of novel therapeutic agents. Method: We developed a unique physics-based protein structure analysis platform scaled to run on a supercomputer, allowing us to consider dynamic protein flexibility and the influence of solvent upon protein structure, and to probe large protein libraries as a primary source of structure information that can support up-front structure-based screens. We developed and then probed a high resolution protein x-ray crystallographic library, created and curated by us. From a 60 million compound library, we conducted virtual screens for docking to identified sites. 8-day colony formation assays were conducted on a panel of 8 human breast, prostate, lung and colon cancer cell lines, as well as 8- and 14-day trilineage hematopoietic colony formation assays on human cord blood stem cells. Findings: From the first 180 deposited protein structures, we screened for unique protein surface structure. Structures were deemed unique if they were not otherwise known to be associated with any particular functional roles and were not the binding site of known therapeutics. Further selection included those that were clefts and possessed physical characteristics compatible with binding small chemicals whose properties have been linked to effective therapeutics. For each of 8 sites on 6 different proteins, a suite of analytics probed docking solutions from the compound library, generating a ranked list of 100 compounds for each site. Based on factors inclusive of availability and low potential for toxicity, compounds were acquired and used in in-parallel selection and de-selection functional assays. Cancer cell growth inhibition was a positive selection criterion, with additional ranking based on greater potency and broader efficacy. Bone marrow toxicity is limiting for most drugs, and inhibition of bone marrow stem cell growth was as a deselection criterion. Multiple compounds clustered around 2 sites on 2 different proteins. One compound, Dxr2-017, exhibited very high relative efficacy against colon and breast cancer cells. Evaluation of Dxr2-017 in the NCI-60 panel corroborated these findings, and showed even higher efficacy against melanoma. We demonstrated that Dxr2-017 inhibited melanoma growth with low nanomolar efficacy, while concentrations of over 2300-fold higher had only limited inhibitory effects on bone marrow stem cells. Conclusions: This study provides proof-of-principle that protein structure constitutes high value primary information that can support identification of novel therapeutics. This approach is widely applicable and expandable. Citation Format: Fangfang Qiao, T. Andrew Binkowski, Wayne Anderson, Weining Chen, Gray Schiltz, Karl Scheidt, Amarnath Natarajan, Raymond Bergan. Protein structure inspired drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4493.

Drug discovery historically starts with an established function, either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function, we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds, we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures, we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast, prostate, colon, or lung cancer, and for toxicity on normal human bone marrow stem cells, both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound, Dxr2-017, exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays, Dxr2-017 had an IC50 of 12 nM against melanoma cells, while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis, a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable.

Clostridioides difficile causes life-threatening gastrointestinal infections. It is a high-risk pathogen due to a lack of effective treatments, antimicrobial resistance, and a poorly conserved genomic core. Herein, we report 30 X-ray structures from a structure genomics pipeline spanning 13 years, representing 10.2% of the X-ray structures for this important pathogen.

Klebsiella pneumoniae is a leading cause of antibiotic-resistant-associated deaths in the world. Here, we report the deposition of 14 structures of enzymes from both the core and accessory genomes of sequence type 23 (ST23) K1 hypervirulent K. pneumoniae.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Chronic pain involves sensitization of nociceptors and synaptic transmission of painful signals in nociceptive circuits in the dorsal horn of the spinal cord. We investigated the contribution of clathrin-dependent endocytosis to sensitization of nociceptors by G protein-coupled receptors (GPCRs) and to synaptic transmission in spinal nociceptive circuits. We determined whether therapeutic targeting of endocytosis could ameliorate pain. mRNA encoding dynamin (Dnm) 1 to 3 and adaptor-associated protein kinase 1 (AAK1), which mediate clathrin-dependent endocytosis, were localized to primary sensory neurons of dorsal root ganglia of mouse and human and to spinal neurons in the dorsal horn of the mouse spinal cord by RNAScope. When injected intrathecally to mice, Dnm and AAK1 siRNA or shRNA knocked down Dnm and AAK1 mRNA in dorsal root ganglia neurons, reversed mechanical and thermal allodynia and hyperalgesia, and normalized nonevoked behavior in preclinical models of inflammatory and neuropathic pain. Intrathecally administered inhibitors of clathrin, Dnm, and AAK1 also reversed allodynia and hyperalgesia. Disruption of clathrin, Dnm, and AAK1 did not affect normal motor functions of behaviors. Patch clamp recordings of dorsal horn neurons revealed that Dnm1 and AAK1 disruption inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing release of synaptic vesicles from presynaptic primary afferent neurons. Patch clamp recordings from dorsal root ganglion nociceptors indicated that Dnm siRNA prevented sustained GPCR-mediated sensitization of nociceptors. By disrupting synaptic transmission in the spinal cord and blunting sensitization of nociceptors, endocytosis inhibitors offer a therapeutic approach for pain treatment.

The infectious disease human monkeypox is spreading rapidly in 2022, causing a global health crisis. The genomics of Monkeypox virus (MPXV) have been extensively analyzed and reported, although little is known about the virus-encoded proteome. In particular, there are no reported experimental MPXV protein structures other than computational models. Here, a 1.52 Å resolution X-ray structure of the MPXV protein A42R, the first MPXV-encoded protein with a known structure, is reported. A42R shows structural similarity to profilins, which are cellular proteins that are known to function in the regulation of actin cytoskeletal assembly. However, structural comparison of A42R with known members of the profilin family reveals critical differences that support prior biochemical findings that A42R only weakly binds actin and does not bind poly(L-proline). In addition, the analysis suggests that A42R may make distinct interactions with phosphatidylinositol lipids. Overall, the data suggest that the role of A42R in the replication of orthopoxviruses may not be readily determined by comparison to cellular profilins. Furthermore, these findings support the need for increased efforts to determine high-resolution structures of other MPXV proteins to inform physiological studies of the poxvirus infection cycle and to reveal potential new strategies to combat human monkeypox should this emerging infectious disease with pandemic potential become more common in the future.

Pathogenic bacteria attack their host by secreting virulence factors that in various ways interrupt host defenses and damage their cells. Functions of many virulence factors, even from well-studied pathogens, are still unknown. Francisella tularensis is a class A pathogen and a causative agent of tularemia, a disease that is lethal without proper treatment. Here we report the three-dimensional structure and preliminary analysis of the potential virulence factor identified by the transcriptomic analysis of the F. tularensis disease models that is encoded by the FTT_1539 gene. The structure of the FTT_1539 protein contains two sets of three stranded antiparallel beta sheets, with a helix placed between the first and the second beta strand in each sheet. This structural motif, previously seen in virulence factors from other pathogens, was named the SHS2 motif and identified to play a role in protein-protein interactions and small molecule recognition. Sequence and structure analysis identified FTT_1539 as a member of a large family of secreted proteins from a broad range of pathogenic bacteria, such as Helicobacter pylori and Mycobacterium tuberculosis. While the specific function of the proteins from this class is still unknown, their similarity to the H. pylori Tip-α protein that induces TNF-a and other chemokines through NF-kB activation suggests the existence of a common pathogen-host interference mechanism shared by multiple human pathogens.

Listeria monocytogenes ( Lm ) is a widespread environmental Gram‐positive bacterium that can transition into a pathogen following ingestion by a susceptible host. To cross host barriers and establish infection, Lm is dependent upon the regulated secretion and activity of many proteins including PrsA2, a peptidyl‐prolyl cis‐trans isomerase with foldase activity. PrsA2 contributes to the stability and activity of a number of secreted virulence factors that are required for Lm invasion, replication, and cell‐to‐cell spread within the infected host. In contrast, a second related secretion chaperone, PrsA1, has thus far no identified contributions to Lm pathogenesis. Here we describe the characterization of a two‐component signal transduction system PieRS that regulates the expression of a regulon that includes the secretion chaperones PrsA1 and PrsA2. PieRS regulated gene products are required for bacterial resistance to ethanol exposure and are important for bacterial survival during transit through the gastrointestinal tract. PrsA1 was also found to make a unique contribution to Lm survival in the GI tract, revealing for the first time a non‐overlapping requirement for both secretion chaperones PrsA1 and PrsA2 during the process of intra‐gastric infection.

The unexpected findings that are described by Czub et al. [IUCrJ (2022), 9, 551-561] provide a very interesting study demonstrating how small differences in structure can result in significant changes in the relative affinities of the seemingly promiscuous binding sites that are seen with serum albumins.

V. cholerae has a dynamic life cycle, which requires rapid adaptation to changing external conditions. Two-component signal transduction (TCS) systems allow V. cholerae to sense and respond to these environmental changes.

BACKGROUND The relative roles of mucus plugs and emphysema in mechanisms of airflow limitation and hypoxemia in smokers with chronic obstructive pulmonary disease (COPD) are uncertain. METHODS We analyzed computed tomography (CT) lung images and lung function in participants in the Subpopulations and Intermediate Outcome Measures in COPD Study. Radiologists scored mucus plugs on CT lung images and imaging software automatically quantified percent emphysema. Unadjusted and adjusted relationships between mucus plug score, percent emphysema, and lung function were determined using regression. RESULTS Among 400 smokers, 229 (57%) had mucus plugs and 207 (52%) had emphysema and subgroups could be identified with mucus dominant and emphysema dominant disease. Only 33% of smokers with high mucus plug scores had mucus symptoms. Mucus plug score and percent emphysema were independently associated with lower values for forced expiratory volume in one second and peripheral oxygen saturation (p values < 0.001). The relationships between mucus plug score and lung function outcomes were strongest in smokers with limited emphysema (p values <0.001). Compared to smokers with low mucus plug scores, those with high scores had worse COPD Assessment Test scores (17.4 ± 7.7 vs. 14.4 ± 13.3), more frequent annual exacerbations (0.75 ± 1.1 vs. 0.43 ± 0.85), and shorter 6-minute walk distance (329 ± 115 vs. 392 ± 117 meters)(p values < 0.001). CONCLUSION Symptomatically silent mucus plugs are highly prevalent in smokers and independently associate with lung function outcomes. These data provide rationale for targeting mucus-high/emphysema-low COPD patients in clinical trials of muco-active treatments.

Background: Chronic cough and phlegm are frequently reported chronic obstructive pulmonary disease (COPD) symptoms. Prior research classified chronic mucus hypersecretion (CMH) based on the presence of these symptoms for >= 3 months, called chronic bronchitis (CB) if respiratory infection symptoms were present for 1-2 years (Medical Research Council [MRC] definition). We explored whether the COPD Assessment Test (CAT), a simple measure developed for routine clinical use, captures CMH populations and outcomes similarly to MRC and St. George's Respiratory Questionnaire (SGRQ) definitions. Methods: We identified CMH in the SPIROMICS COPD cohort using (a) MRC definitions, (b) SGRQ questions for cough and phlegm (both as most/several days a week), and (c) CAT cough and phlegm questions. We determined optimal cut-points for CAT items and described exacerbation frequencies for different CMH definitions. Moderate exacerbations required a new prescription for antibiotics/oral corticosteroids or emergency department visit; severe exacerbations required hospitalization. Results were stratified by smoking status. Results: In a population of 1431 participants (57% male; mean FEV1% predicted 61%), 47% and 49% of evaluable participants had SGRQ- or CAT-defined CMH, respectively. A cut-point of >= 2 for cough and phlegm items defined CMH in CAT. Among SGRQ-CMH+ participants, 80% were also defined as CMH+ by the CAT. CMH+ participants were more likely to be current smokers. A higher exacerbation frequency was observed for presence of CMH+ versus CMH- in the year prior to baseline for all CMH definitions; this trend continued across 3 years of follow-up, regardless of smoking status. Conclusion: Items from the CAT identified SGRQ-defined CMH, a frequent COPD trait that correlated with exacerbation frequency. The CAT is a short, simple questionnaire and a potentially valuable tool for telemedicine or real-world trials. CAT-based CMH is a novel approach for identifying clinically important characteristics in COPD that can be ascertained in these settings.

Emerging evidence suggests the Pseudomonas aeruginosa accessory genome is enriched with uncharacterized virulence genes. Identification and characterization of such genes may reveal novel pathogenic mechanisms used by particularly virulent isolates. Here, we utilized a mouse bacteremia model to quantify the virulence of 100 individual P. aeruginosa bloodstream isolates and performed whole-genome sequencing to identify accessory genomic elements correlated with increased bacterial virulence. From this work, we identified a specific contact-dependent growth inhibition (CDI) system enriched among highly virulent P. aeruginosa isolates. CDI systems contain a large exoprotein (CdiA) with a C-terminal toxin (CT) domain that can vary between different isolates within a species. Prior work has revealed that delivery of a CdiA-CT domain upon direct cell-to-cell contact can inhibit replication of a susceptible target bacterium. Aside from mediating interbacterial competition, we observed our virulence-associated CdiA-CT domain to promote toxicity against mammalian cells in culture and lethality during mouse bacteremia. Structural and functional studies revealed this CdiA-CT domain to have in vitro tRNase activity, and mutations that abrogated this tRNAse activity in vitro also attenuated virulence. Furthermore, CdiA contributed to virulence in mice even in the absence of contact-dependent signaling. Overall, our findings indicate that this P. aeruginosa CDI system functions as both an interbacterial inhibition system and a bacterial virulence factor against a mammalian host. These findings provide an impetus for continued studies into the complex role of CDI systems in P. aeruginosa pathogenesis.

The p38αMAPK is a serine/threonine protein kinase and a key node in the intracellular signaling networks that transduce and amplify stress signals into physiological changes. A preponderance of preclinical data and clinical observations established p38αMAPK as a brain drug discovery target involved in neuroinflammatory responses and synaptic dysfunction in multiple degenerative and neuropsychiatric brain disorders. We summarize the discovery of highly selective, brain-penetrant, small molecule p38αMAPK inhibitors that are efficacious in diverse animal models of neurologic disorders. A crystallography and pharmacoinformatic approach to fragment expansion enabled the discovery of an efficacious hit. The addition of secondary pharmacology screens to refinement delivered lead compounds with improved selectivity, appropriate pharmacodynamics, and efficacy. Safety considerations and additional secondary pharmacology screens drove optimization that delivered the drug candidate MW01-18-150SRM (MW150), currently in early stage clinical trials.

As SCUBA diving continues to rapidly grow in the United States and worldwide, physicians should have a fundamental working knowledge to provide care for an injured diver. SCUBA divers are faced with many hazards at depths that are normally well compensated for at sea level. Pressure gradients, changes in the partial pressure of inhaled gases and gas solubility can have disastrous effects to the diver if not managed properly. Many safety measures in SCUBA diving are governed by the laws of physics, but some have come under scrutiny. This has prompted increased research concerning in water recompression and flying after diving. This article will give physicians an understanding of the dangers divers encounter and the current treatment recommendations. We will also explore some controversies in diving medicine.