OpenFold: retraining AlphaFold2 yields new insights into its learning mechanisms and capacity for generalization.

AlphaFold2 revolutionized structural biology with the ability to predict protein structures with exceptionally high accuracy. Its implementation, however, lacks the code and data required to train new models. These are necessary to (1) tackle new tasks, like protein-ligand complex structure prediction, (2) investigate the process by which the model learns and (3) assess the model's capacity to generalize to unseen regions of fold space. Here we report OpenFold, a fast, memory efficient and trainable implementation of AlphaFold2. We train OpenFold from scratch, matching the accuracy of AlphaFold2. Having established parity, we find that OpenFold is remarkably robust at generalizing even when the size and diversity of its training set is deliberately limited, including near-complete elisions of classes of secondary structure elements. By analyzing intermediate structures produced during training, we also gain insights into the hierarchical manner in which OpenFold learns to fold. In sum, our studies demonstrate the power and utility of OpenFold, which we believe will prove to be a crucial resource for the protein modeling community.

Activation of Hepatocyte Growth Factor/MET Signaling as a Mechanism of Acquired Resistance to a Novel YAP1/TEAD Small Molecule Inhibitor.

Many tumor types harbor alterations in the Hippo pathway, including mesothelioma, where a high percentage of cases are considered YAP1/TEAD dependent. Identification of autopalmitoylation sites in the hydrophobic palmitate pocket of TEADs, which may be necessary for YAP1 protein interactions, has enabled modern drug discovery platforms to generate compounds that allosterically inhibit YAP1/TEAD complex formation and transcriptional activity. We report the discovery and characterization of a novel YAP1/TEAD inhibitor MRK-A from an aryl ether chemical series demonstrating potent and specific inhibition of YAP1/TEAD activity. In vivo, MRK-A showed a favorable tolerability profile in mice and demonstrated pharmacokinetics suitable for twice daily oral dosing in preclinical efficacy studies. Importantly, monotherapeutic targeting of YAP1/TEAD in preclinical models generated regressions in a mesothelioma CDX model; however, rapid resistance to therapy was observed. RNA-sequencing of resistant tumors revealed mRNA expression changes correlated with the resistance state and a marked increase of hepatocyte growth factor (HGF) expression. In vitro, exogenous HGF was able to fully rescue cytostasis induced by MRK-A in mesothelioma cell lines. In addition, co-administration of small molecule inhibitors of the MET receptor tyrosine kinase suppressed the resistance generating effect of HGF on MRK-A induced growth inhibition. In this work, we report the structure and characterization of MRK-A, demonstrating potent and specific inhibition of YAP1/TAZ-TEAD-mediated transcriptional responses, with potential implications for treating malignancies driven by altered Hippo signaling, including factors resulting in acquired drug resistance.

Rapid label-free cell-based Approach Membrane Permeability Assay using MALDI-hydrogen-deuterium exchange mass spectrometry for peptides.

Peptide therapeutics are a growing modality in the pharmaceutical industry and expanding these therapeutics to hit intracellular targets would require establishing cell permeability. Rapid measurement target-agnostic cell permeability of peptides is still analytically challenging. In this study, we demonstrate the development of a rapid high-throughput label-free methodology based on a MALDI-hydrogen-deuterium exchange mass spectrometry (MALDI-HDX-MS) approach to rank-order peptide cell membrane permeability using live THP-1 and AsPc-1 cells. Peptides were incubated in the presence of live cells and their permeability into the cells over time was measured by MALDI-HDX-MS. A differential hydrogen-deuterium exchange approach was used to distinguish the peptides outside of the cells from those inside. The peptides on the outside of the cells were labeled using sufficiently short exposure to deuterium oxide, while the peptides inside of the cells were protected from labeling as a result of permeation into the cells. The deuterium labeled and peak area ratios of unlabeled peptides were compared and plotted over time. The developed methodology, referred to as Cell-based Approach Membrane Permeability Assay (CAMPA), was applied to study an array of 24 diverse peptides including cell-penetrating peptides, stapled and macrocyclic peptides. The cell membrane permeability results observed by CAMPA were corroborated by previously reported in literature data. The CAMPA MALDI-MS analysis was fully automated including MS data processing using internally developed Python scripts. Moreover, CAMPA was demonstrated to be useful for differentiating passive and active cell transportation by using an endocytosis inhibitor in cell incubation media for selected peptides.

The Enzymatic Activity of Inosine 5'-Monophosphate Dehydrogenase May Not Be a Vulnerable Target for Staphylococcus aureus Infections.

Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 µM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. Five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

Mycophenolic anilides as broad specificity inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is a potential target for microorganisms. However, identifying inhibitor design determinants for IMPDH orthologs continues to evolve. Herein, a series of mycophenolic anilide inhibitors of Cryptosporidium parvum and human IMPDHs are reported. Furthermore, molecular docking of 12 (e.g. SH-19; CpIMPDH Ki,app = 0.042 ± 0.015 µM, HsIMPDH2 Ki,app = 0.13 ± 0.05 µM) supports different binding modes with the two enzymes. For CpIMPDH the inhibitor extends into a pocket in an adjacent subunit. In contrast, docking suggests the inhibitor interacts with Ser276 in the NAD binding site in HsIMPDH2, as well as an adjacent pocket within the same subunit. These results provide further guidance for generating IMPDH inhibitors for enzymes found in an array of pathogenic microorganisms, including Mycobacterium tuberculosis.

Numerical and experimental studies of gas-liquid flow and pressure drop in multiphase pump valves.

A numerical and experimental study was carried out to investigate the two-phase flow fields of the typical three valves used in the multiphase pumps. Under the gas volume fraction conditions in the range of 0%-100%, the three-dimensional steady and dynamic two-phase flow characteristics, pressure drops, and their multipliers of the ball valve, cone valve, and disk valve were studied, respectively, using Eulerian-Eulerian approach and dynamic grid technique in ANSYS FLUENT. In addition, a valve test system was built to verify the simulated results by the particle image velocimetry and pressure test. The flow coefficient CQ (about 0.989) of the disk valve is greater than those of the other valves (about 0.864) under the steady flow with a high Reynolds number. The two-phase pressure drops of the three valves fluctuate in different forms with the vibration of the cores during the dynamic opening. The two-phase multipliers of the fully opened ball valve are consistent with the predicted values of the Morris model, while those of the cone valve and disk valve had the smallest differences with the predicted values of the Chisholm model. Through the comprehensive analysis of the flow performance, pressure drop, and dynamic stability of the three pump valves, the disk valve is found to be more suitable for the multiphase pumps due to its smaller axial space, resistance loss, and better flow capacity.

Transgenerational transmission of neurodevelopmental disorders induced by maternal exposure to PM2.5.

The pathological traits or diseases susceptibility caused by maternal exposure to environmental adverse insults (infection, malnutrition, environmental toxicants) could be transmitted across generations. It remains uncertain, however, whether the neurodevelopmental disturbances of offspring induced by maternal exposure to PM2.5 during early life can be inherited by subsequent generations without further exposure. In the current study, using transgenerational animal models, we found that F1 female showed poorer performance in Morris Water Maze (MWM), and the deficits in spatial learning and memory similarly presented in F2-F3 female. The transgenerationally-transmitted neurobehavioral disorders were mediated both via maternal and paternal lineage. Since the epigenetic modifications have been reported to be involved in the disturbed neurodevelopment induced by maternal exposure to detrimental environmental factors during early life, we further explored the possible epigenetic mechanism of the transgenerational effects. Intriguingly, the results displayed the significant increase in expression of Dnmt3a in F1 female offspring. And the hypermethylation of Bdnf promoter Ⅳ and downregulated expression of Bdnf in hippocampus were stably transmitted across the generations until the third generation. There was another interesting finding that the transgenerational effects were sex-specific and only emerged in female offspring. Together, our study indicated for the first time that maternal exposure to PM2.5 during early life could detrimentally affect neurobehaviors in multiple generations, and the declined expression of Bdnf induced by hypermethylation of Bdnf promoter Ⅳ mediated by Dnmts might be the potential molecular mechanism.

Association between dietary inflammatory index and bone density in lactating women at 6 months postpartum: a longitudinal study.

BACKGROUND: Chronic inflammation contributes to the risk of osteoporosis and fracture. Dietary Inflammatory Index (DII), a novel method appraising the inflammatory potential of diet, has been utilized to examine the association between diet and bone health among postmenopausal women or the elderly. However, its relationship with bone density (BD) in lactating women has not been studied. METHODS: The prospective study was conducted to assess the possible association between DII and maternal BD during lactation. We enrolled 150 lactating women in the cohort. Participants were measured ultrasonic BD as baseline values at 1 month postpartum. After five-month follow up, the participants' BD were measured again. DII scores were calculated from semi-quantitative food frequency questionnaires (FFQ) and divided into tertiles. We compared the differences in the changes of BD at 6 months postpartum without or with adjustment for potential covariates across the tertiles. RESULTS: The women in Q1 of DII scores had less bone mass loss than those in Q2 and Q3 without adjustment for any covariates (p < 0.01); after adjusting demographic characteristics such as BMI (kg/m2) at 6 months postpartum, educational level, metabolic equivalent (MET), daily energy intake (kcal/d), we found that participants in the highest tertile of DII scores had much more bone loss than those in the lowest tertile (p = 0.038). However, in the test for trend, no significant association between DII and the changes of maternal BD at 6 months postpartum was observed. CONCLUSIONS: Chinese lactating women with higher DII scores have more bone mass loss; however significant differences and trends are attenuated and/or disappear depending on covariates and confounders that are taken into account in statistical analysis. The further study should be conducted in larger population to explore whether the significant association between DII and BD exists in Chinese lactating women.

Association between total water intake and dietary intake of pregnant and breastfeeding women in China: a cross-sectional survey.

BACKGROUND: Pregnant and lactating women are at high risk of insufficient water intake. The cross-sectional study was mainly designed to evaluate the water intake, including total water intake (TWI), plain water intake, and water intake from beverages and foods of 200 pregnant women and 150 breastfeeding women in Beijing. METHODS: A semi-quantitative Food Frequency Questionnaire (FFQ) was employed to assess their dietary intake, TWI, plain water, and water intake from beverages and foods. Multivariate regression analysis was conducted for evaluating the association between water intake and dietary variables. RESULTS: On average, the TWI of pregnant and breastfeeding women was 2638 mL/day and 3218 mL/day, respectively. Only 28% of pregnant women and 27% of breastfeeding women were complied with the adequate intake (AI). Water from foods was the greatest contributor to TWI both in pregnant and breastfeeding women. TWI was positively related to some dietary variables (P < 0.001). For pregnant women, with each 100 kcal/day increase in energy intake, the TWI increased by 67 mL. With each 5 g increase in daily intake of dietary protein, fat, carbohydrate and fiber, TWI increased by 72 mL, 66 mL, 22 mL, 353 mL, respectively. When the energy contribution of protein increased by 5%, TWI increased by 210 mL. The each 100 mg increase in daily sodium intake was accompanied with 52 mL increase in TWI. For breastfeeding women, with each 100 kcal/day increase in energy intake, the TWI increased by 54 mL. With each 5 g increase in daily intake of dietary protein, fat, carbohydrate and fiber, TWI increased by 53 mL, 58 mL, 16 mL, 212 mL, respectively. The each 100 mg increase in daily sodium intake was accompanied with 54 mL increase in TWI. CONCLUSIONS: A large proportion of pregnant and breastfeeding women in Beijing were not adherent to AI for TWI set by Chinese Nutrition Society. Water intake from foods was the greatest contributor to TWI both in pregnant and breastfeeding women, and maternal dietary intake posed impacts on water intake during pregnancy and lactation. More researches are required to assess the water intake and hydration status of the populations.

Neurodevelopmental toxicity induced by maternal PM2.5 exposure and protective effects of quercetin and Vitamin C.

Epidemiological studies show that maternal exposure to PM2.5 affects the neurodevelopment of the offspring, especially the neurocognitive function. However, no relevant experimental researches have been published on toxic mechanism and diet intervention. We evaluated the effects of exposure to different doses of PM2.5 on the behavioral development of offspring via a PM2.5 exposure model established by intratracheal instillation, explored its mechanism and the protective effects of quercetin and VC intervention, and focused on the protein expression of CREB/BDNF signaling pathway. Specifically, Exposure to PM2.5 during gestation and lactation period caused maternal oxidative stress. Maternal exposure to PM2.5 changed postnatal open-field behaviors in both gender, impaired spatial learning and memory in the female offspring, increased the level of IL-1ß, IL-6, down-regulated p-CREB/CREB, BDNF, TrkB, p-CaMKII/CaMKII, p-CaMKIV/CaMKIV, up-regulated p-Akt/Akt and p-ERK1/2/ERK1/2 in the offspring. In addition, maternal supplementation with quercetin ameliorate the maternal oxidative stress, improved progeny inflammatory response, regulated BDNF, TrkB, p-Akt/Akt, p-ERK1/2/ERK1/2 in female offspring, regulated TrkB, p-CREB/CREB and p-Akt/Akt in male offspring. Maternal supplementation with VC increased the levels of CAT in maternal mice, up-regulated BDNF in female offspring, regulated p-CREB/CREB and p-ERK1/2/ERK1/2 in male offspring. Our findings indicate that PM2.5 exposure during pregnancy and lactation could impair behavioral development of offspring. Quercetin shows more protective effects than VC. The mechanism of neurodevelopmental toxicity induced by PM2.5 may be related to oxidative stress, inflammatory response and modulation of the CREB/BDNF signaling pathway.

Expanding Benzoxazole-Based Inosine 5'-Monophosphate Dehydrogenase (IMPDH) Inhibitor Structure-Activity As Potential Antituberculosis Agents.

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. Mycobacterium tuberculosis ( Mtb) inosine 5'-monophosphate dehydrogenase 2 ( MtbIMPDH2) is a promising yet controversial potential target. The inhibition of MtbIMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure-activity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 µM can be achieved. The antibacterial activity of the most promising compound, 17b (Q151), is derived from the inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of 17b, alleviating the concern that guanine salvage can protect Mtb in vivo. These findings suggest that MtbIMPDH2 is a vulnerable target for tuberculosis.

The relationship among serum lipocalin 2, bone turnover markers, and bone mineral density in outpatient women.

PURPOSE: We aimed to investigate associations among serum levels of LCN2, bone resorption marker carboxy-terminal cross-linking telopeptide of type-1 collagen (CTx), bone formation marker osteocalcin (OCN), and bone mineral densities (BMDs) in ambulatory healthy women. METHODS: This cross-sectional study analyzed 1012 previously enrolled outpatient Han Chinese women. BMDs of the lumbar spine and femoral neck were measured using dual energy X-ray absorptiometry. Serum levels of LCN2, CTx, OCN, and creatinine (Scr) were measured. RESULTS: Circulating LCN2 was inversely correlated with BMDs at the lumbar spine and femoral neck (Spearman's r = -0.08, P = 0.010 and r = -0.14, P < 0.001; respectively). A significant positive correlation between LCN2 and CTx (r = 0.11, P < 0.001), OCN (r = 0.06, P = 0.047), age (r = 0.21, P < 0.001), and Scr (r = 0.24, P < 0.001) was also observed. After adjusting for age and Scr, the correlation among LCN2, BMDs and OCN disappeared, but LCN2 was still positively associated with CTx (r = 0.08, P = 0.010). The circulating concentration of LCN2 showed no significant difference between subjects with and without osteoporotic fractures (43.63 (35.29, 53.66) vs. 42.25 (34.43, 51.46) ng/ml, respectively, P = 0.111). Serum CTx concentrations rose with serum LCN2 increasing from the lowest to the highest quartile (P for trend = 0.005), even after adjusting for age and Scr (P for trend = 0.040). In multivariate regression analysis, LCN2 was one of the main determinants for changes in serum CTx (standard ß = 0.061, P = 0.005). CONCLUSIONS: In ambulatory healthy women, the relationships among serum LCN2 level, BMDs, and OCN were confounded by age and Scr. Although LCN2 was positively related with CTx, the correlation was very weak and may not be physiologically relevant.

Benzoxazoles, Phthalazinones, and Arylurea-Based Compounds with IMP Dehydrogenase-Independent Antibacterial Activity against Francisella tularensis.

Francisella tularensis is the causative agent of tularemia and a potential biowarfare agent. The virulence of F. tularensis is decreased by deletion of guaB, the gene encoding IMP dehydrogenase (IMPDH), suggesting that this enzyme is a target for antibacterial design. Here we report that F. tularensis growth is blocked by inhibitors of bacterial IMPDHs. Seventeen compounds from two different frameworks, designated the D and Q series, display antibacterial activities with MICs of <1 µM. These compounds are also active against intracellular infections. Surprisingly, antibacterial activity does not correlate with IMPDH inhibition. In addition, the presence of guanine does not affect the antibacterial activity of most compounds, nor does the deletion of guaB These observations suggest that antibacterial activity derives from inhibition of another target(s). Moreover, D compounds display antibacterial activity only against F. tularensis, suggesting the presence of a unique target or uptake mechanism. A ΔguaB mutant resistant to compound D73 contained a missense mutation (Gly45Cys) in nuoB, which encodes a subunit of bacterial complex I. Overexpression of the nuoB mutant conferred resistance to D73 in both wild-type and ΔguaB strains. This strain was not resistant to Q compounds, suggesting that a different off-target mechanism operates for these compounds. Several Q compounds are also effective against Mycobacterium tuberculosis, in which a second target has also been implicated, in addition to IMPDH. The fortuitous presence of multiple targets with overlapping structure-activity relationships presents an intriguing opportunity for the development of robust antibiotics that may avoid the emergence of resistance.

The Influence of Quercetin on Maternal Immunity, Oxidative Stress, and Inflammation in Mice with Exposure of Fine Particulate Matter during Gestation.

The objective is to investigate the influence of PM2.5 exposure on peripheral blood lymphocyte subsets in pregnant mice and the antagonism of quercetin on adverse effects induced by PM2.5 exposure. Pregnant mice were randomly divided into control group, PM2.5 model group and 3 quercetin intervention groups. Dams in all groups except the control group were exposed to PM2.5 suspension by intratracheal instillation on gestational day (GD) 3, 6, 9, 12 and 15. Meanwhile, each dam was given 0.15% carboxymethylcellulose sodium (CMCS) (control group & PM2.5 model group) and different doses of quercetin (quercetin intervention groups) by gavage once a day from GD0 to GD17. The percentage of lymphocyte subsets, Biomarkers of systemic inflammation injuries (IL-2, IL-6, IL-8 & TNF-α) and oxidative stress indicators (CAT, GSH & HO-1) in peripheral blood of the dams were analyzed. The number of T cells increased, accompanied by increased level of IL-2, IL-6, IL-8 and HO-1 due to PM2.5 exposure. Less CD4+ and CD8+ T cells were counted in 100 mg/kg quercetin intervention group, compared with PM2.5 model group. Quercetin may inhibit cytokine production, especially in IL-6 and IL-8 and may upgrade the level of HO-1. Our findings indicate that PM2.5 could significantly influence the distribution of T-lymphocyte subsets, activate inflammatory reaction and elevate oxidative stress level in peripheral blood of pregnant mice. Certain dose of quercetin administration during pregnancy may protect the dams against the adverse effects through various ways.

Identification of two novel mutations in the GALNT3 gene in a Chinese family with hyperphosphatemic familial tumoral calcinosis.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare, autosomal recessive genetic disease. This disease is characterized by the progressive calcification of soft tissues leading to symptoms of pressure and hyperphosphatemia but normal concentrations of serum calcium with or without an elevation of 1,25-dihydroxyvitamin D3 levels.HFTC is caused by loss-of-function mutations in the GALNT3, FGF23 or KL genes. Here, we identified two novel mutations in the GALNT3 gene in a Chinese family with HFTC. Identification of a novel genotype in HFTC provides clues for understanding the phenotype-genotype relationships in HFTC and may assist not only in the clinical diagnosis of HFTC but also in the interpretation of the genetic information used for prenatal diagnosis and genetic counseling.

Paragonimiasis mimicking chest cancer and abdominal wall metastaisis: A case report.

Typical human paragonimiasis demonstrates an elevated eosinophil count, positive immunoblot, nodular shadows of the lung and pleural thickening with pleural effusion, and these symptoms may be confused with chest cancer. In the present case, a rare case of human paragonimiasis mimicking chest cancer and abdominal wall metastasis is described, the 39-year-old male patient was admitted in our hospital for cough, weight loss 5 kg and a firm mass in right upper abdominal wall. The laboratory test showed unremarkable hematology and biochemistry results. Chest X-ray, Plain computed tomography of the chest and abdomen showed right pleural effusion, several nodules in right lower lung and a mass in the right upper abdominal wall. The initial diagnosis was lung or chest cancer with abdominal wall metastasis, and the abdominal wall mass was resected for the final diagnosis. The biopsy revealed eosinophilic granuloma with Charcot-Leyden crystal formation infiltrated in the muscular fibers. Subsequent to assessment of the antibodies against parasites, the final diagnosis of paragonimiasis was made.

Double primary hepatic cancer (hepatocellular carcinoma and intrahepatic cholangiocarcinoma) in a single patient: A case report.

Double primary hepatic cancer, consisting of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) located separately within a single liver simultaneously, is extremely rare. The present study reports a case of double hepatic nodules, in which HCC and ICC occurred simultaneously in the right hepatic lobe. The 47-year-old male patient, who was a carrier of hepatitis B virus, was admitted to our hospital for physical examination, which revealed two liver masses. The results of initial laboratory tests, including liver function tests, were within normal limits, with the exception of mildly elevated aspartate aminotransferase and alanine aminotransferase, and decreased albumin levels. α-fetoprotein was in the normal range, while carbohydrate antigen 19-9 was marginally elevated. Abdominal ultrasonography and enhanced computed tomography revealed two tumors located in segments (S) VI and VII of the liver, respectively, with malignant behavior. Examination of the two masses following resection of S VI and VII confirmed a diagnosis of combined HCC and ICC. After 8 months of follow-up, no signs of recurrence have been observed with chemical therapy.

Mycobacterium tuberculosis IMPDH in Complexes with Substrates, Products and Antitubercular Compounds.

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5'-monophosphate into xanthosine 5'-monophosphate with the concomitant reduction of NAD+ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD+ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.

A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity.

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization.