Reactivities of the Front Pocket N-Cap Cysteines in Human Kinases

Discovery of targeted covalent inhibitors directed at nucleophilic cysteines is attracting enormous interest. The front pocket (FP) N-cap cysteine has been the most popular site of covalent modification in kinases. Curiously, a long-standing hypothesis associates the N-cap position with cysteine hyper-reactivity; however, traditional computational methods suggest that the FP N-cap cysteines in all human kinases are predominantly unreactive at physiological pH. Here we applied a newly developed GPU-accelerated continuous constant pH molecular dynamics (CpHMD) tool to test the N-cap hypothesis and elucidate the cysteine reactivities. Simulations showed that the N-cap cysteines in BTK/BMX/TEC/ITK/TXK, JAK3, and MKK7 sample the reactive thiolate form to varying degrees at physiological pH; however, those in BLK and EGFR/ERBB2/ERBB4 which contain an Asp at the N-cap+3 position adopt the unreactive thiol form. The latter argues in favor of the base-assisted thiol-Michael addition mechanisms as suggested by the quantum mechanical calculations and experimental structure-function studies of EGFR inhibitors. Analysis revealed that the reactive N-cap cysteines are stabilized by hydrogen bond as well as electrostatic interactions, and in their absence a N-cap cysteine is unreactive due to desolvation. To test a corollary of the N-cap hypothesis, we also examined the reactivities of the FP N-cap+2 cysteines in JNK1/JNK2/JNK3 and CASK. Additionally, our simulations predicted the reactive cysteine and lysine locations in all 15 kinases. Our findings offer a systematic understanding of cysteine reactivities in kinases and demonstrate the predictive power and physical insights CpHMD can provide to guide the rational design of targeted covalent inhibitors.

Synthesis and characterization of Fe(III) complex with thiosemicarbazide-based ligand

Complex iron(III) salicylaldehyde thiosemicarbazone with a formula Na[Fe(STSC)2] have been synthesized. Ligand, salicylaldehyde thiosemicarbazone (SCT) was prepared through the condensation reaction of salycilaldehide with thiosemicarbazide. Na[Fe(STSC)2] has been synthesized by reacting FeCl3 with thiosemicarbazide based ligand bearing ONS donor atoms. Complex has been formulated and characterized by mass spectrometry, infrared and UV/visible spectroscopy. The facts showed the formation of a complex in metal:ligand stoichiometric ratio 1:2. The ligand is coordinated as an ONS tridentate dianion via oxygen atom after the deprotonation of the phenolic OH-group, azomethine nitrogen and sulphur in thiol form of the deprotonated thiosemicarbazide residue. Antioxidant activity was determined, where ligand showed significant activity, while complex, at low concentration, exhibited almost no activity.

Spectral study of thione-thiol tautomerization of thiourea in aqueous alcohol solution

In this work we studied the equilibrium of thione–thiol tautomerization by Raman and UV spectroscopies. This type of tautomerization influences on the course and direction of the reaction between thiourea and other organic compounds. The studies were carried out in water and aqueous alcohol medium. Мethanol, ethanol, propanol-1 and propanol-2 were used as alcohols. Hydrochloric acid was used to protonate thiourea in water and aqueous alcoholic solutions. UV spectroscopy made it possible to establish the tautomer ratio in water and aqueous alcohol solutions as the ratio of the intensities of absorption bands at 236 and 200 nm. There is an increase in the content of the thiol form and a decrease of the thione form observed in the row water-methanol-ethanol-propanol-isopropyl alcohol. The addition of hydrochloric acid to the thiourea water or aqueous alcohol solutions leads to the increase of the thione form and to the decrease of the thiol form in the composition. The thione form of thiourea can be determine by Raman spectra of –C=S group. The thiol form of thiourea is difficult to detect by Raman spectroscopy due to the overlap of the –S–H bond absorption band with alcohols absorption bands.

Tautomeric Equilibrium Modeling: Stability and Reactivity of Benzothiazole and Derivatives

Benzothiazoles are organic compounds with multiple biological activities. Due to their biological interests, these are synthesized on a large scale at the industrial level and used in various fields. Their release into waters causes environmental problems which leads to public health problems. Finding solution which can help for their degradation become necessary. That is the reason why a theoretical study of the reactivity of five benzothiazole derivatives has been initiated in order to understand some aspect of their biodegradation. The calculations were carried out in gaseous and aqueous phase with B3LYP functional associated with bases 6-311G(d) and 6-31+G(d). The results revealed that the thione tautomer of the MBT derivative is more stable than the thiol form. These results are in agreement with previous experimental work which showed that the thiones forms in MBT metal complexes are the most stable. Moreover, the study of the reactivity based on the computation of the global indices of reactivity reveals that the benzothiazoles BT, OBT and MBT are the most reactive. The most electrophilic is BT and the least electrophilic is MTBT. In addition, the thermodynamic parameters and the energy barriers predict a possibility of coexistence of tautomers ol and one of OBT derivative. Fukui dual descriptors have shown that the carbon C2 of BT is the most electrophilic. In substituted derivatives, it is the C6 carbon that is the most electrophilic. N3 nitrogen remains the most nucleophilic site in all the studied molecules.

S–H rotamerizationviatunneling in a thiol form of thioacetamide

Rotamerization of the S–H groupviahydrogen tunneling is reported for the first time.

Spinal disulfide HMGB1, but not all-thiol HMGB1, induces mechanical hypersensitivity in a TLR4-dependent manner

Aims Increasing evidence indicates that extracellular high mobility group box-1 protein (HMGB1) is involved in the pathogenesis of inflammatory and autoimmune disease. Data from our laboratory demonstrates that HMGB1 contributes to nociceptive behavior in a model of rheumatoid arthritis-induced pain. HMGB1 binds to multiple receptors, including toll like receptor (TLR) 2, TLR4 and receptor for advanced glycation end products (RAGE). When the cysteine in position C106 is in the reduced thiol form and C23 and C45 are engaged in a disulfide bridge (disulfide HMGB1), the molecule functions as a cytokine-inducing TLR4 ligand. In contrast, when these three cysteines are all reduced (all-thiol HMGB1), HMGB1 exclusively potentiates chemotactic activity via CXCR4. It is currently not well understood which receptor and which redox form of HMGB1 that mediates pain hypersensitivity and is therefore the aim of this study. Methods All animal work was carried out in accordance with protocol approved by the local ethics committee for animal experiments in Sweden. Balb/c, C57B/l6 (WT), Tlr2–/–, Tlr4–/– and Rage–/– male mice were used for this study. Disulfide (ds) and all thiol (at) form of HMGB1 were injected intrathecally (1 μg) and mechanical hypersensitivity assessed by von Frey filaments. Lumbar spinal cords were collected after i.t. injection of atHMGB1 and ds HMGB1 and mRNA levels for cytokine and glia markers assessed by quantitative PCR. Results In Balb/c and C57Bl/6 WT mice, i.t injection of dsHMGB1, but not atHMGB1, led to a significant reduction in mechanical thresholds. dsHMGB1 induced mechanical hypersensitivity 6 h after i.t. injection, which lasted for 5 days, compared to i.t. injection of saline. When dsHMGB1 was injected i.t. to Tlr4 deficient mice it did not induce mechanical hypersensitivity. In contrast Tlr2 and Rage deficient mice were still susceptible to dsHMGB1-induced mechanical hypersensitivity. Analysis of mRNA for cytokines and glial cell-associated factors in lumbar spinal cords revealed increased levels of Tnf, Ccl2, Cxcl1, Cxcl2, Gfap and Cd11b in mice injected with dsHMGB1, but not atHMGB1, with exception for Il1β and Cxcr3 that was induced also by atHMGB1. Intrathecal injection of dsHMGB1 to Tlr4–/– deficient mice, did not increase mRNA levels for Tnf, Il1β, Ccl2, Gfap and Cd11b. Conclusions We found the i.t. injection of the disulfide, but not the all-thiol, form of HMGB1 to induce pronouncedand long-lasting mechanical hypersensitivity, glial reactivity and cytokine induction in a TLR4-, but not TLR2- or RAGE-dependent manner. Thus our data indicates that, the redox state of HMGB1 is key for determining its nociceptive property and receptor usage and thus also the functional consequences of HMGB1 release. Agents interfering with extracellular HMGB1 may be considered in the development of new pain relieving therapeutics.

Thioester derivatives of the natural product psammaplin A as potent histone deacetylase inhibitors

There has been significant interest in the bioactivity of the natural product psammaplin A, most recently as a potent and isoform selective HDAC inhibitor. Here we report our preliminary studies on thioester HDAC inhibitors derived from the active monomeric (thiol) form of psammaplin A, as a means to improve compound delivery into cells. We have discovered that such compounds exhibit both potent cytotoxicity and enzymatic inhibitory activity against recombinant HDAC1. The latter effect is surprising since previous SAR suggested that modification of the thiol functionality should detrimentally affect HDAC potency. We therefore also report our preliminary studies on the mechanism of action of this observed effect.

A Crystallographic Study of Bis[S-benzyl-5-bromo-2-oxoindolin-3-ylidenemethanehydrazonthioate] Disulfide Solvated with Dimethylsulfoxide

The crystal structure of the title compound has been determined. The compound crystallized in the triclinic space group P -1, Z = 2, V = 1839 .42( 18) A3 and unit cell parameters a= 11. 0460( 6) A, b = 13 .3180(7) A, c=13. 7321 (8) A, a = 80.659(3 )0, b = 69 .800(3 )0 and g = 77 .007 (2)0 with one disordered dimethylsulfoxide solvent molecule with the sulfur and oxygen atoms are distributed over two sites; S101/S102 [site occupancy factors: 0.6035/0.3965] and 0130/0131 [site occupancy factor 0.3965/0.6035]. The C22-S2 l and C 19-S20 bond distances of 1. 779(7) A and 1. 788(8) A indicate that both of the molecules are connected by the disulfide bond [S20-S21 2.055(2) A] in its thiol form. The crystal structure reveals that both of the 5-bromoisatin moieties are trans with respect to the [S21-S20 and CI 9-Nl 8] and [S20-S21 and C22-N23] bonds whereas the benzyl group from the dithiocarbazate are in the cis configuration with respect to [S21-S20 and C19-S44] and [S20-S21 and C22-S36] bonds. The crystal structure is further stabilized by intermolecular hydrogen bonds of N9-H35···O16 formed between the two molecules and N28-H281 ···O130, N28-H281 ···O131 and C4 l-H4 l l ···O 131 with the solvent molecule.

A Crystallographic Study of Bis[S-benzyl-5-bromo-2-oxoindolin-3-ylidenemethanehydrazonthioate] DisuIfide Solvated with Dimethylsulfoxide

The crystal structure of the title compound has been determined. The compound crystallized in the triclinic space group P -1 , Z = 2, V = 1839.42(18) A3 and unit cell parameters a= 11.0460(6) A, b = 13.3180(7) A, c = 13.7321(8) A, a= 80.659(3)° , b= 69.800(3)° and g= 77.007(2)° with one disordered dimethylsulfoxide solvent molecule with the sulfur and oxygen atoms are distributed over two sites; S101/S102 [site occupancy factors: 0.6035/0.3965] and O130/O131 [site occupancy factor 0.3965/0.6035]. The C22-S21 and C19-S20 bond distances of1.779(7) A and 1.788(8) A indicate that both of the molecules are connected by the disulfide bond [S20-S21 2.055(2) A] in its thiol form. The crystal structure reveals that both of the 5-bromoisatin moieties are trans with respect to the [S21-S20 and C19-N18] and [S20-S21 and C22-N23] bonds whereas the benzyl group from the dithiocarbazate are in the cis configuration with respect to [S21-S20 and C19-S44] and [S20-S21 and C22-S36] bonds. The crystal structure is further stabilized by intermolecular hydrogen bonds of N9-H35...O16 formed between the two molecules and N28-H281...O130, N28-H281...O131 and C41-H411...O131 with the solvent molecule.