Heat-induced structural and chemical changes to a computationally designed miniprotein.

The de novo design of miniprotein inhibitors has recently emerged as a new technology to create proteins that bind with high affinity to specific therapeutic targets. Their size, ease of expression, and apparent high stability makes them excellent candidates for a new class of protein drugs. However, beyond circular dichroism melts and hydrogen/deuterium exchange experiments, little is known about their dynamics, especially at the elevated temperatures they seemingly tolerate quite well. To address that and gain insight for future designs, we have focused on identifying unintended and previously overlooked heat-induced structural and chemical changes in a particularly stable model miniprotein, EHEE_rd2_0005. Nuclear magnetic resonance (NMR) studies suggest the presence of dynamics on multiple time and temperature scales. Transiently elevating the temperature results in spontaneous chemical deamidation visible in the NMR spectra, which we validate using both capillary electrophoresis and mass spectrometry (MS) experiments. High temperatures also result in greatly accelerated intrinsic rates of hydrogen exchange and signal loss in NMR heteronuclear single quantum coherence spectra from local unfolding. These losses are in excellent agreement with both room temperature hydrogen exchange experiments and hydrogen bond disruption in replica exchange molecular dynamics simulations. Our analysis reveals important principles for future miniprotein designs and the potential for high stability to result in long-lived alternate conformational states.

Effects of Xylanase A double mutation on substrate specificity and structural dynamics.

While protein activity is traditionally studied with a major focus on the active site, the activity of enzymes has been hypothesized to be linked to the flexibility of adjacent regions, warranting more exploration into how the dynamics in these regions affects catalytic turnover. One such enzyme is Xylanase A (XylA), which cleaves hemicellulose xylan polymers by hydrolysis at internal ß-1,4-xylosidic linkages. It contains a "thumb" region whose flexibility has been suggested to affect the activity. The double mutation D11F/R122D was previously found to affect activity and potentially bias the thumb region to a more open conformation. We find that the D11F/R122D double mutation shows substrate-dependent effects, increasing activity on the non-native substrate ONPX2 but decreasing activity on its native xylan substrate. To characterize how the double mutant causes these kinetics changes, nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations were used to probe structural and flexibility changes. NMR chemical shift perturbations revealed structural changes in the double mutant relative to the wild-type, specifically in the thumb and fingers regions. Increased slow-timescale dynamics in the fingers region was observed as intermediate-exchange line broadening. Lipari-Szabo order parameters show negligible changes in flexibility in the thumb region in the presence of the double mutation. To help understand if there is increased energetic accessibility to the open state upon mutation, alchemical free energy simulations were employed that indicated thumb opening is more favorable in the double mutant. These studies aid in further characterizing how flexibility in adjacent regions affects the function of XylA.

Predicting binding affinity changes from long-distance mutations using molecular dynamics simulations and Rosetta.

Computationally modeling how mutations affect protein-protein binding not only helps uncover the biophysics of protein interfaces, but also enables the redesign and optimization of protein interactions. Traditional high-throughput methods for estimating binding free energy changes are currently limited to mutations directly at the interface due to difficulties in accurately modeling how long-distance mutations propagate their effects through the protein structure. However, the modeling and design of such mutations is of substantial interest as it allows for greater control and flexibility in protein design applications. We have developed a method that combines high-throughput Rosetta-based side-chain optimization with conformational sampling using classical molecular dynamics simulations, finding significant improvements in our ability to accurately predict long-distance mutational perturbations to protein binding. Our approach uses an analytical framework grounded in alchemical free energy calculations while enabling exploration of a vastly larger sequence space. When comparing to experimental data, we find that our method can predict internal long-distance mutational perturbations with a level of accuracy similar to that of traditional methods in predicting the effects of mutations at the protein-protein interface. This work represents a new and generalizable approach to optimize protein free energy landscapes for desired biological functions.

Free energy calculations of ALS-causing SOD1 mutants reveal common perturbations to stability and dynamics along the maturation pathway.

With over 150 heritable mutations identified as disease-causative, superoxide dismutase 1 (SOD1) has been a main target of amyotrophic lateral sclerosis (ALS) research and therapeutic efforts. However, recent evidence has suggested that neither loss of function nor protein aggregation is responsible for promoting neurotoxicity. Furthermore, there is no clear pattern to the nature or the location of these mutations that could suggest a molecular mechanism behind SOD1-linked ALS. Here, we utilize reliable and accurate computational techniques to predict the perturbations of 10 such mutations to the free energy changes of SOD1 as it matures from apo monomer to metallated dimer. We find that the free energy perturbations caused by these mutations strongly depend on maturational progress, indicating the need for state-specific therapeutic targeting. We also find that many mutations exhibit similar patterns of perturbation to native and non-native maturation, indicating strong thermodynamic coupling between the dynamics at various sites of maturation within SOD1. These results suggest the presence of an allosteric network in SOD1 which is vulnerable to disruption by these mutations. Analysis of these perturbations may contribute to uncovering a unifying molecular mechanism which explains SOD1-linked ALS and help to guide future therapeutic efforts.

Application of Off-Rate Screening in the Identification of Novel Pan-Isoform Inhibitors of Pyruvate Dehydrogenase Kinase.

Libraries of nonpurified resorcinol amide derivatives were screened by surface plasmon resonance (SPR) to determine the binding dissociation constant (off-rate, kd) for compounds binding to the pyruvate dehydrogenase kinase (PDHK) enzyme. Parallel off-rate measurements against HSP90 and application of structure-based drug design enabled rapid hit to lead progression in a program to identify pan-isoform ATP-competitive inhibitors of PDHK. Lead optimization identified selective sub-100-nM inhibitors of the enzyme which significantly reduced phosphorylation of the E1α subunit in the PC3 cancer cell line in vitro.

Targeting conserved water molecules: design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization.

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.

Combining hit identification strategies: fragment-based and in silico approaches to orally active 2-aminothieno[2,3-d]pyrimidine inhibitors of the Hsp90 molecular chaperone.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe novel 2-aminothieno[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, which were designed by combining structural elements of distinct low affinity hits generated from fragment-based and in silico screening exercises in concert with structural information from X-ray protein crystallography. Examples from this series have high affinity (IC50 = 50-100 nM) for Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Several examples (34a, 34d and 34i) caused tumor growth regression at well tolerated doses when administered orally in a human BT474 human breast cancer xenograft model.

4,5-diarylisoxazole Hsp90 chaperone inhibitors: potential therapeutic agents for the treatment of cancer.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic agents for cancer. Here, we describe the structure-based design, synthesis, structure-activity relationships and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold. Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP) competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound 40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC50 = 21 nM) and inhibits proliferation of various human cancer cell lines in vitro, with GI50 averaging 9 nM. Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by approximately 50%.

A vacinacao na pratica homeopatica: uma revisao bibliografica / Vaccination in homeopathic practice: a bibliographic review

Este trabalho teve como objetivo a abordagem do tema "vacianao" atraves de uma revisao bibliografica sob a visao da Alopatia e da Homeopatia, apresentando o posicionamento de diversos autores, com a finalidade de fornecer instrumentos... (AU)

A vacinação na pratica homeopatica: uma revisão bibliografica / Vaccination in homeopathic practice: a bibliographic review

Este trabalho teve como objetivo a abordagem do tema "vacianao" atraves de uma revisao bibliografica sob a visao da Alopatia e da Homeopatia, apresentando o posicionamento de diversos autores, com a finalidade de fornecer instrumentos...

Novel bromotyrosine alkaloids: inhibitors of mycothiol S-conjugate amidase.

[structure: see text] The novel alkaloids 1 and 4 were isolated from an Australian non-verongid sponge, Oceanapia sp. Compound 1 contains an unprecedented imidazolyl-quinolinone substructure attached to a bromotyrosine-derived spiro-isoxazoline. Three other known alkaloids were isolated in addition to 1 and 4 and together represent the first examples of inhibitors of a new mycobacterial enzyme mycothiol S-conjugate amidase (MCA).

Cortamidine oxide, a novel disulfide metabolite from the New Zealand basidiomycete (mushroom) Cortinarius species.

Three disulfide metabolites were isolated from the fruiting bodies of the basidiomycete (mushroom) Cortinarius sp., collected in the Catlins, New Zealand. The structures of these compounds were determined as the unsymmetrical disulfide cortamidine oxide (1), 2,2'-dithiobis(pyridine N-oxide) (2), and the symmetrical disulfide 3. Both 1 and 2 showed significant antimicrobial activity and cytotoxicity. 2,2'-Dithiobis(pyridine N-oxide) (2) and the symmetrical disulfide 3 are assumed to be artifacts of the isolation procedure.

Oceanapiside, an antifungal bis-alpha,omega-amino alcohol glycoside from the marine sponge Oceanapia phillipensis.

The structure of oceanapiside, an antifungal alpha, omega-bis-aminohydroxylipid glycoside from the temperate marine sponge Oceanapia sp., was elucidated by a combination of 2D NMR, chemical degradation/correlation, and MALDI MS-MS spectrometry. Oceanapiside exhibits antifungal activity against Candida glabrata at 10 micrograms/mL (MIC).