The discovery of drug-like molecules that bind pockets in proteins that are not present in crystallographic structures yet exert allosteric control over activity has generated great interest in ...designing pharmaceuticals that exploit allosteric effects. However, there have only been a small number of successes, so the therapeutic potential of these pockets—called hidden allosteric sites—remains unclear. One challenge for assessing their utility is that rational drug design approaches require foreknowledge of the target site, but most hidden allosteric sites are only discovered when a small molecule is found to stabilize them. We present a means of decoupling the identification of hidden allosteric sites from the discovery of drugs that bind them by drawing on new developments in Markov state modeling that provide unprecedented access to microsecond- to millisecond-timescale fluctuations of a protein’s structure. Visualizing these fluctuations allows us to identify potential hidden allosteric sites, which we then test via thiol labeling experiments. Application of these methods reveals multiple hidden allosteric sites in an important antibiotic target—TEM-1 β-lactamase. This result supports the hypothesis that there are many as yet undiscovered hidden allosteric sites and suggests our methodology can identify such sites, providing a starting point for future drug design efforts. More generally, our results demonstrate the power of using Markov state models to guide experiments.
Significance Rational drug design efforts typically focus on identifying inhibitors that bind to protein active sites. Pockets that are not present in crystallographic structures yet can exert allosteric (i.e., long-range) control over distant active sites present an exciting alternative. However, identifying these hidden allosteric sites is extremely challenging because one usually has to simultaneously find a small molecule that binds to and stabilizes the open conformation of the pocket. Here, we present a means of combining advances in computer modeling—using Markov state models to capture long timescale dynamics—with biophysical experiments to identify hidden allosteric sites without requiring the simultaneous discovery of drug-like compounds that bind them. Using this technology, we discover multiple hidden allosteric sites in a single protein.
Proteins from thermophiles are generally more thermostable than their mesophilic homologs, but little is known about the evolutionary process driving these differences. Here we attempt to understand ...how the diverse thermostabilities of bacterial ribonuclease H1 (RNH) proteins evolved. RNH proteins from Thermus thermophilus (ttRNH) and Escherichia coli (ecRNH) share similar structures but differ in melting temperature (T(m)) by 20 °C. ttRNH's greater stability is caused in part by the presence of residual structure in the unfolded state, which results in a low heat capacity of unfolding (ΔCp) relative to ecRNH. We first characterized RNH proteins from a variety of extant bacteria and found that Tm correlates with the species' growth temperatures, consistent with environmental selection for stability. We then used ancestral sequence reconstruction to statistically infer evolutionary intermediates along lineages leading to ecRNH and ttRNH from their common ancestor, which existed approximately 3 billion years ago. Finally, we synthesized and experimentally characterized these intermediates. The shared ancestor has a melting temperature between those of ttRNH and ecRNH; the T(m)s of intermediate ancestors along the ttRNH lineage increased gradually over time, while the ecRNH lineage exhibited an abrupt drop in Tm followed by relatively little change. To determine whether the underlying mechanisms for thermostability correlate with the changes in T(m), we measured the thermodynamic basis for stabilization--ΔCp and other thermodynamic parameters--for each of the ancestors. We observed that, while the T(m) changes smoothly, the mechanistic basis for stability fluctuates over evolutionary time. Thus, even while overall stability appears to be strongly driven by selection, the proteins explored a wide variety of mechanisms of stabilization, a phenomenon we call "thermodynamic system drift." This suggests that even on lineages with strong selection to increase stability, proteins have wide latitude to explore sequence space, generating biophysical diversity and potentially opening new evolutionary pathways.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
TEM β-lactamase confers bacteria with resistance to many antibiotics and rapidly evolves activity against new drugs. However, functional changes are not easily explained by differences in crystal ...structures. We employ Markov state models to identify hidden conformations and explore their role in determining TEM's specificity. We integrate these models with existing drug-design tools to create a new technique, called Boltzmann docking, which better predicts TEM specificity by accounting for conformational heterogeneity. Using our MSMs, we identify hidden states whose populations correlate with activity against cefotaxime. To experimentally detect our predicted hidden states, we use rapid mass spectrometric footprinting and confirm our models' prediction that increased cefotaxime activity correlates with reduced Ω-loop flexibility. Finally, we design novel variants to stabilize the hidden cefotaximase states, and find their populations predict activity against cefotaxime in vitro and in vivo. Therefore, we expect this framework to have numerous applications in drug and protein design.
Allosteric drugs, which bind to proteins in regions other than their main ligand-binding or active sites, make it possible to target proteins considered "undruggable" and to develop new therapies ...that circumvent existing resistance. Despite growing interest in allosteric drug discovery, rational design is limited by a lack of sufficient structural information about alternative binding sites in proteins. Previously, we used Markov State Models (MSMs) to identify such "cryptic pockets," and here we describe a method for identifying compounds that bind in these cryptic pockets and modulate enzyme activity. Experimental tests validate our approach by revealing both an inhibitor and two activators of TEM β-lactamase (TEM). To identify hits, a library of compounds is first virtually screened against either the crystal structure of a known cryptic pocket or an ensemble of structures containing the same cryptic pocket that is extracted from an MSM. Hit compounds are then screened experimentally and characterized kinetically in individual assays. We identify three hits, one inhibitor and two activators, demonstrating that screening for binding to allosteric sites can result in both positive and negative modulation. The hit compounds have modest effects on TEM activity, but all have higher affinities than previously identified inhibitors, which bind the same cryptic pocket but were found, by chance, via a computational screen targeting the active site. Site-directed mutagenesis of key contact residues predicted by the docking models is used to confirm that the compounds bind in the cryptic pocket as intended. Because hit compounds are identified from docking against both the crystal structure and structures from the MSM, this platform should prove suitable for many proteins, particularly targets whose crystal structures lack obvious druggable pockets, and for identifying both inhibitory and activating small-molecule modulators.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Increased respiration during physical activity may increase air pollution dose, which may attenuate the benefits of physical activity on cardiovascular disease (CVD) risk and overall mortality.
We ...aimed to examine the multiplicative interaction between long-term ambient residential exposure to fine particulate matter
(
) and physical activity in the association with CVD risk and overall mortality.
We followed 104,990 female participants of the U.S.-based prospective Nurses' Health Study from 1988 to 2008. We used Cox proportional hazards models to assess the independent associations of 24-months moving average residential
exposure and physical activity updated every 4 y and the multiplicative interaction of the two on CVD (myocardial infarction and stroke) risk and overall mortality, after adjusting for demographics and CVD risk factors.
During 20 years of follow-up, we documented 6,074 incident CVD cases and 9,827 deaths. In fully adjusted models,
exposure was associated with modest increased risks of CVD hazard ratio (HR) for fifth quintile
compared to first quintile
: 1.09, 95% confidence interval (CI): 0.99, 1.20;
and overall mortality (HR fifth compared to first quintile: 1.10, 95% CI: 1.02, 1.19;
). Higher overall physical activity was associated with substantially lower risk of CVD HR fourth quartile, which was
equivalent of task (MET)-h/wk, compared to first quartile (
): 0.61, 95% CI: 0.57, 0.66;
and overall mortality (HR fourth compared to first quartile: 0.40, 95% CI: 0.37, 0.42;
). We observed no statistically significant interactions between
exposure and physical activity (overall, walking, vigorous activity) in association with CVD risk and overall mortality.
In this study of U.S. women, we observed no multiplicative interaction between long-term
exposure and physical activity; higher physical activity was strongly associated with lower CVD risk and overall mortality at all levels of
exposure. https://doi.org/10.1289/EHP7402.
Celotno besedilo
Dostopno za:
CEKLJ, DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Ambient air pollution exposures have been frequently linked to cardiovascular disease (CVD) morbidity and mortality. However, less is known about the populations most susceptible to these adverse ...effects.
We assessed the associations of long-term particulate matter (PM) exposures with incident CVD in a nationwide cohort of 114 537 women in the Nurses' Health Study, and performed analyses to identify subpopulations at the greatest risk. Residential address level time-varying monthly exposures to PM2.5, PM10, and PM2.5 to 10 microns in diameter were estimated from spatio-temporal prediction models. In multivariable models, increases in all size fractions of PM were associated with small, but not statistically significant, increased risks of total CVD, coronary heart disease, and stroke. PM-associated CVD risks were statistically significantly higher among women with diabetes as compared to those without (P-for-interaction <0.0001 for PM10 and PM2.5 and 0.007 for PM2.5 to 10). For each 10 μg/m(3) increase in 12-month average PM2.5, PM2.5 to 10, and PM10, the multivariable adjusted hazard ratios were 1.44 (95% CI: 1.23 to 1.68), 1.17 (95% CI: 1.05 to 1.30), and 1.19 (95% CI: 1.10 to 1.28) among women with diabetes. There were also suggestions of higher risks among older (≥70 years) women, the obese, and those living in the Northeast and South. Smoking status and family history did not consistently modify the association between PM and CVD, and risks were most elevated with exposures in the previous 12 months.
In this nationwide cohort, women with diabetes were identified as the subpopulation most sensitive to the adverse cardiovascular health effects of PM.
The clinical significance of pneumonia visualized on CT scan in the setting of a normal chest radiograph is uncertain.
In a multicenter prospective surveillance study of adults hospitalized with ...community-acquired pneumonia (CAP), we compared the presenting clinical features, pathogens present, and outcomes of patients with pneumonia visualized on a CT scan but not on a concurrent chest radiograph (CT-only pneumonia) and those with pneumonia visualized on a chest radiograph. All patients underwent chest radiography; the decision to obtain CT imaging was determined by the treating clinicians. Chest radiographs and CT images were interpreted by study-dedicated thoracic radiologists blinded to the clinical data.
The study population included 2,251 adults with CAP; 2,185 patients (97%) had pneumonia visualized on chest radiography, whereas 66 patients (3%) had pneumonia visualized on CT scan but not on concurrent chest radiography. Overall, these patients with CT-only pneumonia had a clinical profile similar to those with pneumonia visualized on chest radiography, including comorbidities, vital signs, hospital length of stay, prevalence of viral (30% vs 26%) and bacterial (12% vs 14%) pathogens, ICU admission (23% vs 21%), use of mechanical ventilation (6% vs 5%), septic shock (5% vs 4%), and inhospital mortality (0 vs 2%).
Adults hospitalized with CAP who had radiological evidence of pneumonia on CT scan but not on concurrent chest radiograph had pathogens, disease severity, and outcomes similar to patients who had signs of pneumonia on chest radiography. These findings support using the same management principles for patients with CT-only pneumonia and those with pneumonia seen on chest radiography.
Home Blood Pressure Monitoring Weinfeld, Jeffrey M; Hart, Kathryn M; Vargas, Jose D
American family physician,
09/2021, Letnik:
104, Številka:
2
Journal Article
Recenzirano
Home blood pressure monitoring can confirm the diagnosis of hypertension after an elevated in-office blood pressure measurement. Although ambulatory blood pressure monitoring is the diagnostic ...standard for measurement, home blood pressure monitoring is more practical and accessible to patients.
Proper folding of proteins is critical to producing the biological machinery essential for cellular function. The rates and energetics of a protein’s folding process, which is described by its energy ...landscape, are encoded in the amino acid sequence. Over the course of evolution, this landscape must be maintained such that the protein folds and remains folded over a biologically relevant time scale. How exactly a protein’s energy landscape is maintained or altered throughout evolution is unclear. To study how a protein’s energy landscape changed over time, we characterized the folding trajectories of ancestral proteins of the ribonuclease H (RNase H) family using ancestral sequence reconstruction to access the evolutionary history between RNases H from mesophilic and thermophilic bacteria. We found that despite large sequence divergence, the overall folding pathway is conserved over billions of years of evolution. There are robust trends in the rates of protein folding and unfolding; both modern RNases H evolved to be more kinetically stable than their most recent common ancestor. Finally, our study demonstrates how a partially folded intermediate provides a readily adaptable folding landscape by allowing the independent tuning of kinetics and thermodynamics.
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