Theoretical Studies on Selectivity of HPK1/JAK1 Inhibitors by Molecular Dynamics Simulations and Free Energy Calculations.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T cell receptor, which has been regarded as a potential target for immunotherapy. Yu et al. observed the off-target effect of the high-throughput screening HPK1 kinase inhibitor hits on JAK1 kinase. The off-target effect is usually due to the lack of specificity of the drug, resulting in toxic side effects. Therefore, exploring the mechanisms to selectively inhibit HPK1 is critical for developing effective and safe inhibitors. In this study, two indazole compounds as HPK1 inhibitors with different selectivity towards JAK1 were used to investigate the selectivity mechanism using multiple computational methods, including conventional molecular dynamics simulations, binding free energy calculations and umbrella sampling simulations. The results indicate that the salt bridge between the inhibitor and residue Asp101 of HPK1 favors their selectivity towards HPK1 over JAK1. Information obtained from this study can be used to discover and design more potent and selective HPK1 inhibitors for immunotherapy.

Multiple rapid-responsive probes for hypochlorite detection based on dioxetane luminophore derivatives.

In recent years, various methods for detecting exogenous and endogenous hypochlorite have been studied, considering its essential role as a biomolecule. However, the existing technologies still pose obstacles such as their invasiveness, high costs, and complicated operation. In the current study, we developed a glow-type chemiluminescent probe, hypochlorite chemiluminescence probe (HCCL)-1, based on the scaffold of Schaap's 1,2-dioxetane luminophores. To better explore the physiological and pathological functions of hypochlorite, we modified the luminophore scaffold of HCCL-1 to develop several probes, including HCCL-2, HCCL-3, and HCCL-4, which amplify the response signal of hypochlorite. By comparing the luminescent intensities of the four probes using the IVIS® system, we determined that HCCL-2 with a limit of detection of 0.166 µM has enhanced sensitivity and selectivity for tracking hypochlorite both in vitro and in vivo.

Aminoluciferin 4-hydroxyphenyl amide enables bioluminescence detection of endogenous tyrosinase.

Tyrosinase, a copper-containing enzyme existing widely in plants, animals and microorganisms, usually serves as an important biomarker in melanoma, and is also related to hyperpigmentation of the skin, melasma, age spots and albinism. At present, only one bioluminescent probe has been applied to image tyrosinase in cells. Thus, it's of great significance to develop a new bioluminescent probe that can detect tyrosinase in living cells and in live animals. In the current work, we report a new BL probe, TyrBP-3, which not detect tyrosinase in vitro and in living cells, but can also visualize the level of tyrosinase activity in tumors of living animals. In summary, TyrBP-3 is the first bioluminescent probe that can image tyrosinase on a cellular level. Hence, we anticipate that TyrBP-3 can be a good tool to monitor tyrosinase in complex biosystems in the future.

Bioluminescent Probe for Detection of Starvation-Induced Pantetheinase Upregulation.

Pantetheinase, a glycosylphosphatidylinositol (GPI) anchored enzyme, overexpresses in intestine, liver, and kidney with various biological functions such as its linkage to the inflammation and some metabolic diseases. It can hydrolyze pantetheine to cysteamine, an antioxidant, and pantothenic acid (Vitamin B5) that is an essential component of coenzyme A (CoA). Until now, very few analytic methods were developed for this enzyme, hampering the further investigation of its biological functions. In this work, we report the design, synthesis, and biological examination of a highly sensitive bioluminogenic probe for pantetheinase with a limit of detection of 1.14 ng/mL. Furthermore, animal experiments validated that our probe can be applied to detect the endogenous pantetheinase activity. To the best of our knowledge, this is the first bioluminogenic probe achieving the detection of pantetheinase level in vivo.

Novel photoactivatable substrates for Renilla luciferase imaging in vitro and in vivo.

To develop a photoactivatable bioluminescence imaging technique, a set of high and efficient photoactivatable substrates for Renilla luciferase has been well designed and synthesized. Surprisingly, all of them could release the free luciferin that presented robust bioluminescent signals ex vivo and in living animals after UV irradiation at 365 nm.

Bioluminescent probe for detecting endogenous hypochlorite in living mice.

As a kind of biologically important reactive oxygen species (ROS), hypochlorite (ClO-) plays a crucial role in many physiological processes. As such, endogenous ClO- is a powerful antibacterial agent during pathogen invasion. Nonetheless, excessive endogenous ClO- could pose a health threat to mammalian animals including humans. However, the detection of endogenous ClO- by bioluminescence probes in vivo remains a considerable challenge. Herein, based on a caged strategy, we developed a turn-on bioluminescent probe 1 for the highly selective detection of ClO-in vitro and imaging endogenous ClO- in a mouse inflammation model. We anticipate that such a probe could help us understand the role of endogenous ClO- in a variety of physiological and pathological processes.

A highly sensitive and rapidly responding fluorescent probe based on a rhodol fluorophore for imaging endogenous hypochlorite in living mice.

Hypochlorous (HOCl) acid is generated as a defense tool in the immune system and plays a vital role in killing a wide range of pathogens. There is therefore great interest in developing fluorescent probes that can endogenously respond to the change in concentration of HOCl in vivo. To address this challenge, we here present a rapidly responding fluorescent probe RO610 to image endogenous HOCl in living mice. The development of RO610 was based on a novel water-soluble and pH-independent fluorescent xanthene dye, 2'-formylrhodol ROA, which exhibits highly selective and sensitive responses to HOCl/ClO- over other reactive species. Moreover, adding a little more than 5 equiv. of ClO- to the solution of RO610 resulted in a clearly observable fluorescence enhancement (48-fold) within 30 s. Based on these properties, RO610 was used to detect ClO- in A549 cells without interference by other oxidants. It was applied for the imaging of endogenous HOCl in living nude mice with satisfactory results.

Novel caged luciferin derivatives can prolong bioluminescence imaging in vitro and in vivo.

Based on N-cyclobutylaminoluciferin (cybLuc), a set of high and efficient caged bioluminescent derivatives (Clucs) as firefly luciferase pro-substrates has been developed herein. After careful examination, these molecules exhibited low cytotoxicity and prolonged bioluminescence imaging up to 6 h in vitro and in vivo. Importantly, these caged luciferin derivatives have the potential to serve as long-term tracking tools to explore some biological process by using bioluminescent imaging.

cybLuc: An Effective Aminoluciferin Derivative for Deep Bioluminescence Imaging.

To enhance the efficiency of firefly luciferase/luciferin bioluminescence imaging, a series of N-cycloalkylaminoluciferins (cyaLucs) were developed by introducing lipophilic N-cycloalkylated substitutions. The experimental results demonstrate that these cyaLucs are effective substrates for native firefly luciferase (Fluc) and can produce elevated bioluminescent signals in vitro, in cellulo, and in vivo. It should be noted that, in animal studies, N-cyclobutylaminoluciferin (cybLuc) at 10 µM (0.1 mL), which is 0.01% of the standard dose of d-luciferin (dLuc) used in mouse imaging, can radiate 20-fold more bioluminescent light than d-luciferin (dLuc) or aminoluciferin (aLuc) at the same concentration. Longer in vivo emission imaging using cybLuc suggests that it can be used for long-time observation. Regarding the mechanism of cybLuc, our cocrystal structure data from firefly luciferase with oxidized cybLuc suggested that oxidized cybLuc fits into the same pocket as oxyluciferin. Most interestingly, our results demonstrate that the sensitivity of cybLuc in brain tumor imaging contributes to its extended application in deep tissues.