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Computer simulation of protein systems
Ligand binding to dihydrofolate reductase (DHFR) is discussed. This is an extremely important enzyme, as it is the target of several drugs (inhibitors) which are used clinically as antibacterials, antiprotozoals and in cancer chemotherapy. DHFR catalyzes the NADPH (reduced nicotinamide adenine dinucleotide phosphate) dependent reduction of dihydrofolate to tetrahydrofolate, which is used in several pathways of purine and pyrimidine iosynthesis, including that of thymidylate. Since DNA synthesis is dependent on a continuing supply of thymidylate, a blockade of DHFR resulting in a depletion of thymidylate can lead to the cessation of growth of a rapidly proliferating cell line. DHFR exhibits a significant species to species variability in its sensitivity to various inhibitors. For example, trimethoprim, an inhibitor of DHFR, binds to bacterial DHFR's 5 orders of magnitude greater than to vertebrate DHFR's. The structural mechanics, dynamics and energetics of a family of dihydrofolate reductases are studied to rationalize the basis for the inhibitor of these enyzmes and to understand the molecular basis of the difference in the binding constants between the species. This involves investigating the conformational changes induced in the protein on binding the ligand, the internal strain imposed by the enzyme on the ligand, the restriction of fluctuations in atom positions due to binding and the consequent change in entropy.
Expanding the Landscape of Dual Action Antifolate Antibacterials through 2,4-Diamino-1,6-dihydro-1,3,5-triazines
Antibiotics that operate via multiple mechanisms of action are a promising strategy to combat growing resistance. Previous studies have shown that dual action antifolates formed from a pyrroloquinazolinediamine core can inhibit the growth of bacterial pathogens without developing resistance. Here, in this work, we expand the scope of dual action antifolates by repurposing the 2,4-diamino-1,6-dihydro-1,3,5-triazine (DADHT) cycloguanil scaffold to a variety of derivatives designed to inhibit dihydrofolate reductase (DHFR) and disrupt bacterial membranes. Dual mechanism DADHTs have activity against a variety of target pathogens, including Mycobacterium tuberculosis, Mycobacterium abscessus, and Pseudomonas aeruginosa, among other ESKAPEE organisms. Through X-ray crystallography, we confirmed engagement of the Escherichia coli DHFR target and found that some DADHTs stabilize a previously unobserved conformation of the enzyme but, broadly, bind in the occluded conformation. Using in vitro inhibition of purified E. coli and Staphylococcus aureus DHFR and disruption of E. coli membranes, we determined that alkyl substitution of dihydrotriazine at the 6-position best optimizes the DADHT's two mechanisms of action. By employing both mechanisms, the DADHT spectrum of activity was extended beyond the scope of traditional antifolates. Finally, we are optimistic that the dual mechanism approach, particularly through the action of antifolates, offers a unique means of combating hard-to-treat bacterial infections.
Perturbative diffraction methods resolve a conformational switch that facilitates a two-step enzymatic mechanism
Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to poor experimental access. This shortcoming is evident with Escherichia coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we describe ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments to map the conformational dynamics of the Michaelis complex of DHFR. We resolve coupled global and local motions and find that these motions are engaged by the protonated substrate to promote efficient catalysis. This result suggests a fundamental design principle for multistep enzymes in which pre-existing dynamics enable intermediates to drive rapid electrostatic reorganization to facilitate subsequent chemical steps.
The Sun Radio Interferometer Space Experiment (SunRISE) Mission
The Sun Radio Interferometer Space Experiment (SunRISE) will provide an entirely new view on particle acceleration and transport in the inner heliosphere by creating the first low radio frequency interferometer in space to localize heliospheric radio emissions. By imaging and determining the location of decametric-hectometric (DH) radio bursts from 0.1 MHz–25 MHz, SunRISE will provide key information on particle acceleration mechanisms associated with coronal mass ejections (CMEs) and the magnetic field topology from active regions into interplanetary space. Six small spacecraft, of a 6U form factor, will fly in a supersynchronous geosynchronous Earth orbit (GEO) orbit within about 10 km of each other, in a passive formation, and image the Sun in a portion of the spectrum that is blocked by the ionosphere and cannot be observed from Earth. Key aspects that enable this mission are that only position knowledge of the spacecraft is required, not active control, and that the architecture involves a modest amount of on-board processing coupled with significant ground-based processing for navigation, position determination, and science operations. Mission-enabling advances in software-defined radios, GPS navigation and timing, and small spacecraft technologies, developed and flown on the DARPA High Frequency Research (DHFR), the Community Initiative for Continuing Earth Radio Occultation (CICERO), and the Mars Cube One (MarCO) missions, have made this mission affordable and low-risk. The SunRISE mission will involve utilizing commercial access to space, in which the SunRISE spacecraft will be carried to their target orbit as a secondary payload in conjunction with a larger host spacecraft intended for GEO.
The Sun Radio Interferometer Space Experiment (SunRISE) Mission
The Sun Radio Interferometer Space Experiment (SunRISE) will provide an entirely new view on particle acceleration and transport in the inner heliosphere by creating the first low radio frequency interferometer in space to localize heliospheric radio emissions. By imaging and determining the location of decametric-hectometric (DH, 0.1 MHz–23 MHz) solar radio bursts, SunRISE will provide key information on particle acceleration mechanisms associated with coronal mass ejections (CMEs) and the magnetic field topology from active regions into interplanetary space. The SunRISE Observatory will consist of six space vehicles in a passive formation, in orbits designed to keep them within approximately 10 km of each other, and flying in a supersynchronous geosynchronous Earth orbit (GEO). Each space vehicle consists of a Solar DH-GNSS payload and a 6U form factor spacecraft. The SunRISE Observatory together with significant ground-based processing, will enable imaging of the Sun in a portion of the spectrum that is blocked by the ionosphere and cannot be observed from Earth. Key aspects that enable this mission are that only position knowledge of the space vehicles is required, not active control, and that the architecture involves a modest amount of on-board processing coupled with significant ground-based processing for navigation, position determination, and science operations. Mission-enabling advances in software-defined radios, GPS navigation and timing, and small spacecraft technologies, developed and flown on the DARPA High Frequency Research (DHFR) and the Community Initiative for Continuing Earth Radio Occultation (CICERO) have made this mission affordable and low-risk. The SunRISE mission will exploit the multiple spacecraft per aperture (MSPA) capability of NASA’s Deep Space Network (DSN), for more efficient data transfers of larger data volumes, and utilize commercial access to space, in which the SunRISE space vehicles will be carried to their target orbit as secondary payloads in conjunction with a larger host spacecraft intended for GEO.