Effects of Rho-Associated Kinase (Rock) Inhibitors (Alternative to Y-27632) on Primary Human Corneal Endothelial Cells.

(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds-AR-13324 (Netarsudil) and its active metabolite, AR-13503-and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 µM; * p < 0.05); (ii) untreated vs. AR-13503 (10 µM; *** p < 0.001); (iii) Y-27632 vs. AR-13503 (10 µM; ** p < 0.005); (iv) AR-13324 (1 µM) vs. AR-13503 (10 µM; ** p < 0.005); and (v) AR-13324 (0.1 µM) vs. AR-13503 (10 µM; * p < 0.05). Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans.

Cannabinoid Biosynthesis Using Noncanonical Cannabinoid Synthases.

We report enzymes from the berberine bridge enzyme (BBE) superfamily that catalyze the oxidative cyclization of the monoterpene moiety in cannabigerolic acid (CBGA) to form cannabielsoin (CBE). The enzymes are from a variety of organisms and are previously uncharacterized. Out of 232 homologues chosen from the enzyme superfamily, four orthologues were shown to accept CBGA as a substrate and catalyze the biosynthesis of CBE. The four enzymes discovered in this study were recombinantly expressed and purified in Pichia pastoris. These enzymes are the first report of heterologous expression of BBEs that did not originate from the Cannabis plant that catalyze the production of cannabinoids using CBGA as substrate. This study details a new avenue for discovering and producing natural and unnatural cannabinoids.

Biosynthesis of Nature-Inspired Unnatural Cannabinoids.

Natural products make up a large proportion of medicine available today. Cannabinoids from the plant Cannabis sativa is one unique class of meroterpenoids that have shown a wide range of bioactivities and recently seen significant developments in their status as therapeutic agents for various indications. Their complex chemical structures make it difficult to chemically synthesize them in efficient yields. Synthetic biology has presented a solution to this through metabolic engineering in heterologous hosts. Through genetic manipulation, rare phytocannabinoids that are produced in low yields in the plant can now be synthesized in larger quantities for therapeutic and commercial use. Additionally, an exciting avenue of exploring new chemical spaces is made available as novel derivatized compounds can be produced and investigated for their bioactivities. In this review, we summarized the biosynthetic pathways of phytocannabinoids and synthetic biology efforts in producing them in heterologous hosts. Detailed mechanistic insights are discussed in each part of the pathway in order to explore strategies for creating novel cannabinoids. Lastly, we discussed studies conducted on biological targets such as CB1, CB2 and orphan receptors along with their affinities to these cannabinoid ligands with a view to inform upstream diversification efforts.

Recent Advances in Structure, Function, and Pharmacology of Class A Lipid GPCRs: Opportunities and Challenges for Drug Discovery.

Great progress has been made over the past decade in understanding the structural, functional, and pharmacological diversity of lipid GPCRs. From the first determination of the crystal structure of bovine rhodopsin in 2000, much progress has been made in the field of GPCR structural biology. The extraordinary progress in structural biology and pharmacology of GPCRs, coupled with rapid advances in computational approaches to study receptor dynamics and receptor-ligand interactions, has broadened our comprehension of the structural and functional facets of the receptor family members and has helped usher in a modern age of structure-based drug design and development. First, we provide a primer on lipid mediators and lipid GPCRs and their role in physiology and diseases as well as their value as drug targets. Second, we summarize the current advancements in the understanding of structural features of lipid GPCRs, such as the structural variation of their extracellular domains, diversity of their orthosteric and allosteric ligand binding sites, and molecular mechanisms of ligand binding. Third, we close by collating the emerging paradigms and opportunities in targeting lipid GPCRs, including a brief discussion on current strategies, challenges, and the future outlook.

Modeling p K Shift in DNA Triplexes Containing Locked Nucleic Acids.

The protonation states for nucleic acid bases are difficult to assess experimentally. In the context of DNA triplex, the protonation state of cytidine in the third strand is particularly important, because it needs to be protonated in order to form Hoogsteen hydrogen bonds. A sugar modification, locked nucleic acid (LNA), is widely used in triplex forming oligonucleotides to target sites in the human genome. In this study, the parameters for LNA are developed in line with the CHARMM nucleic acid force field and validated toward the available structural experimental data. In conjunction, two computational methods were used to calculate the protonation state of the third strand cytidine in various DNA triplex environments: λ-dynamics and multiple pH regime. Both approaches predict p K of this cytidine shifted above physiological pH when cytidine is in the third strand in a triplex environment. Both methods show an upshift due to cytidine methylation, and a small downshift when the sugar configuration is locked. The predicted p K values for cytidine in DNA triplex environment can inform the design of better-binding oligonucleotides.

Computational Study of Uracil Tautomeric Forms in the Ribosome: The Case of Uracil and 5-Oxyacetic Acid Uracil in the First Anticodon Position of tRNA.

Tautomerism is important in many biomolecular interactions, not least in RNA biology. Crystallographic studies show the possible presence of minor tautomer forms of transfer-RNA (tRNA) anticodon bases in the ribosome. The hydrogen positions are not resolved in the X-ray studies, and we have used ab initio calculations and molecular dynamics simulations to understand if and how the minor enol form of uracil (U), or the modified uracil 5-oxyacetic acid (cmo5U), can be accommodated in the tRNA-messenger-RNA interactions in the ribosome decoding center. Ab initio calculations on isolated bases show that the modification affects the keto-enol equilibrium of the uracil base only slightly; the keto form is dominant (>99.99%) in both U and cmo5U. Other factors such as interactions with the surrounding nucleotides or ions would be required to shift the equilibrium toward the enol tautomer. Classical molecular simulations show a better agreement with the X-ray structures for the enol form, but free energy calculations indicate that the most stable form is the keto. In the ribosome, the enol tautomers of U and cmo5U pair with a guanine forming two hydrogen bonds, which do not involve the enol group. The oxyacetic acid modification has a minor effect on the keto-enol equilibrium.

Role of Pseudoisocytidine Tautomerization in Triplex-Forming Oligonucleotides: In Silico and in Vitro Studies.

Pseudoisocytidine (ΨC) is a synthetic cytidine analogue that can target DNA duplex to form parallel triplex at neutral pH. Pseudoisocytidine has mainly two tautomers, of which only one is favorable for triplex formation. In this study, we investigated the effect of sequence on ΨC tautomerization using λ-dynamics simulation, which takes into account transitions between states. We also performed in vitro binding experiments with sequences containing ΨC and furthermore characterized the structure of the formed triplex using molecular dynamics simulation. We found that the neighboring methylated or protonated cytidine promotes the formation of the favorable tautomer, whereas the neighboring thymine or locked nucleic acid has a poor effect, and consecutive ΨC has a negative influence. The deleterious effect of consecutive ΨC in a triplex formation was confirmed using in vitro binding experiments. Our findings contribute to improving the design of ΨC-containing triplex-forming oligonucleotides directed to target G-rich DNA sequences.

Adsorption and folding dynamics of MPER of HIV-1 gp41 in the presence of DPC micelle.

Membrane-proximal ectodomain region (MPER) of HIV-1 gp41 is known to have several epitopes of monoclonal antibodies. It also plays an important role in the membrane fusion process that is well-evidenced, though not well-elucidated. There are also disputes over the true structure of MPER. In this study, MPER NMR structure in the presence of dodecylphosphatidylcholine micelle is used in the molecular dynamic simulation to elucidate structural dynamics and adsorption to model MPER interaction in a membrane environment. Polarized protein-specific charge derived from its NMR structure is found to better preserve the helical structure found in the NMR structure compared to AMBER03 calculation. The preserved helical structure also adsorb to the micelle using the hydrophobic side-chains, consistent to the NMR structure. Ab initio folding of MPER predicts a structure quite in well agreement with the NMR structure (RMSd 3.9 Å) and shows that the micelle plays a role in the folding process.

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41.

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: -15.28 kcal/mol for HK20 Fab (expt. -11.60 kcal/mol) and -17.90 kcal/mol for D5 Fab (expt. -11.70 kcal/mol). Alanine scanning at protein-protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of V(H) region, as well as R30' of V(L) region; whereas for D5 Fab, F54 of V(H) region, as well as W32' and Y94' of V(L) region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing.

Computational study of bindings of HL9, a nonapeptide fragment of human lysozyme, to HIV-1 fusion protein gp41.

HL9 is a nonapeptide fragment of human lysozyme which has been shown to have anti-HIV-1 activity in nanomolar concentration. This study aims to explain this inhibitory activity by using molecular dynamics (MD) simulation, focusing on the ectodomain of gp41, the envelope glycoprotein of HIV-1 crucial to membrane fusion. It was found that in HL9, two Trp residues separated by two others occupy the conserved hydrophobic pocket on gp41 and thus inhibit fusion in dominant-negative manner. Detailed HL9-gp41 binding interactions and free energies of binding were obtained through MD simulation and solvated interaction energies (SIE) calculation, giving a binding free energy of -8.25 kcal/mol which is in close agreement with the experimental value of -9.96 kcal/mol. Since C-helical region (C34) of gp41 also has two Trp residues separated by two others, this arrangement may be generalised and used to scan peptide library and to find those having similar manner of inhibition.

Molecular dynamics studies of human receptor molecule in hemagglutinin of 1918 and 2009 H1N1 influenza viruses.

Molecular dynamics (MD) simulations were carried out to study the behavior of human receptor molecule in the hemagglutinin (HA) of 1918 and 2009 H1N1 influenza viruses respectively. The 2009 HA model was obtained by virtually mutating the 1918 HA crystal structure based on A/Mexico City/MCIG01/2009(H1N1) segment 4 sequence. We found that human receptor molecule has no binding preference between the 2009 HA and the 1918 HA. In addition, among the four sugar moieties in the human receptor molecule, sialic acid contributes the most to the electrostatic and non-polar interaction energy during binding. Furthermore, the hydrogen bonds between sialic acid and the surrounding residues in 1918 HA are preserved in 2009 HA. We also found that the mutated residues contribute to a more favorable binding of hemagglutinin to the human receptor molecule.