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.

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.

(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.

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.

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.