Cross-Polarization of Insensitive Nuclei from Water Protons for Detection of Protein-Ligand Binding.

Hyperpolarization derived from water protons enhances the NMR signal of 15N nuclei in a small molecule, enabling the sensitive detection of a protein-ligand interaction. The water hyperpolarized by dissolution dynamic nuclear polarization (D-DNP) acts as a universal signal enhancement agent. The 15N signal of benzamidine was increased by 1480-fold through continuous polarization transfer by J-coupling-mediated cross-polarization (J-CP) via the exchangeable protons. The signal enhancement factor favorably compares to factors of 110- or 17-fold using non-CP-based polarization transfer mechanisms. The hyperpolarization enabled detection of the binding of benzamidine to the target protein trypsin with a single-scan measurement of 15N R2 relaxation. J-CP provides an efficient polarization mechanism for 15N or other low-frequency nuclei near an exchangeable proton. The hyperpolarization transfer sustained within the relaxation time limit of water protons additionally can be applied for the study of macromolecular structure and biological processes.

Preparation of micron-sized benzamidine-modified magnetic agarose beads for trypsin purification from fish viscera.

The effective method for trypsin purification should be established because trypsin has important economic value. In this work, a novel and simple strategy was proposed for fabricating micron-sized magnetic Fe3O4@agarose-benzamidine beads (MABB) with benzamidine as a ligand, which can efficiently and selectively capture trypsin. The micro-sized MABB, with clear spherical core-shell structure and average particle size of 6.6 µm, showed excellent suspension ability and magnetic responsiveness in aqueous solution. The adsorption capacity and selectivity of MABB towards target trypsin were significantly better than those of non-target lysozyme. According to the Langmuir equation, the maximum adsorption capacity of MABB for trypsin was 1946 mg g-1 at 25 °C, and the adsorption should be a physical sorption process. Furthermore, the initial adsorption rate and half equilibrium time of MABB toward trypsin were 787.4 mg g-1 min-1 and 0.71 min, respectively. To prove the practicability, MABB-based magnetic solid-phase extraction (MSPE) was proposed, and the related parameters were optimized in detail to improve the purification efficiency. With Tris-HCl buffer (50 mM, 10 mM CaCl2, pH 8.0) as extraction buffer, Tris-HCl buffer (50 mM, 100 mM CaCl2, pH 8.0) as rinsing buffer, acidic eluent (0.01 M HCl, 0.5 M NaCl, pH 2.0) as eluent buffer and alkaline buffer (1 M Tris-HCl buffer, pH 10.0) as neutralization solution, the MABB-based MSPE was successfully used for trypsin purification from the viscera of grass carp (Ctenopharyngodon idella). The molecular weight of purified trypsin was determined as approximate 23 kDa through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified trypsin was highly active from 30 °C to 60 °C, with an optimum temperature of 50 °C, and was tolerant to pH variation, exhibiting 85 % of maximum enzyme activity from pH 7.0 to 10.0. The results demonstrated that the proposed MABB-based MSPE could effectively purify trypsin and ensure the biological activity of purified trypsin. Therefore, we believe that the novel MABB could be applicable for efficient purification of trypsin from complex biological systems.

Structural basis for the inhibition of ßFXIIa by garadacimab.

Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of hereditary angioedema (HAE). Here, we describe a high-resolution cryoelectron microscopy (cryo-EM) structure of the beta-chain from FXIIa (ßFXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to ßFXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This structural mechanism is likely the primary contributor to the inhibition of activated FXIIa proteolytic activity in HAE. Garadacimab Fab-ßFXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXIIa. Structural analysis with other bona fide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a surprisingly similar mechanism of ßFXIIa inhibition by garadacimab. In summary, the garadacimab Fab-ßFXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes.

Accelerated Screening of Protein-Ligand Interactions via Parallel T2-Weighted 19F-MRI.

In drug discovery, ligands are sought that modulate the (mal-)function of medicinally relevant target proteins. In order to develop new drugs, typically a multitude of potential ligands are initially screened for binding and subsequently characterized for their affinity. Nuclear magnetic resonance (NMR) is a well-established and highly sensitive technology for characterizing such interactions. However, it has limited throughput, because only one sample can be measured at a time. In contrast, magnetic resonance imaging (MRI) is inherently parallel and MR parameters can conveniently be encoded in its images, potentially offering increased sample throughput. We explore this application using a custom-built 9-fold sample holder and a 19F-MRI coil. With this setup, we show that ligand binding can be detected by T2-weighted 19F-MRI using 4-(trifluoromethyl)benzamidine (TFBA) and trypsin as the reporter ligand and target protein, respectively. Furthermore, we demonstrate that the affinity of nonfluorinated ligands can be determined in a competition format by monitoring the dose-dependent displacement of TFBA. By comparing 19F-T2-weighted MR images of TFBA in the presence of different benzamidine (BA) concentrations-all recorded in parallel-the affinity of BA could be derived. Therefore, this approach promises parallel characterization of protein-ligand interactions and increased throughput of biochemical assays, with potential for increased sensitivity when combined with hyperpolarization techniques.

DON/DRP-104 as potent serine protease inhibitors implicated in SARS-CoV-2 infection: Comparative binding modes with human TMPRSS2 and novel therapeutic approach.

Human transmembrane serine protease 2 (TMPRSS2) is an important member of the type 2 transmembrane serine protease (TTSP) family with significant therapeutic markings. The search for potent TMPRSS2 inhibitors against severe acute respiratory syndrome coronavirus 2 infection with favorable tissue specificity and off-site toxicity profiles remains limited. Therefore, probing the anti-TMPRSS2 potential of enhanced drug delivery systems, such as nanotechnology and prodrug systems, has become compelling. We report the first in silico study of TMPRSS2 against a prodrug, [isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate] also known as DRP-104 synthesized from 6-Diazo-5-oxo-l-norleucine (DON). We performed comparative studies on DON and DRP-104 against a clinically potent TMPRSS2 inhibitor, nafamostat, and a standard serine protease inhibitor, 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) against TMPRSS2 and found improved TMPRSS2 inhibition through synergistic binding of the S1/S1' subdomains. Both DON and DRP-104 had better thermodynamic profiles than AEBSF and nafamostat. DON was found to confer structural stability with strong positive correlated inter-residue motions, whereas DRP-104 was found to confer kinetic stability with restricted residue displacements and reduced loop flexibility. Interestingly, the Scavenger Receptor Cysteine-Rich (SRCR) domain of TMPRSS2 may be involved in its inhibition mechanics. Two previously unidentified loops, designated X (270-275) and Y (293-296) underwent minimal and major structural transitions, respectively. In addition, residues 273-277 consistently transitioned to a turn conformation in all ligated systems, whereas unique transitions were identified for other transitioning residue groups in each TMPRSS2-inhibitor complex. Intriguingly, while both DON and DRP-104 showed similar loop transition patterns, DRP-104 preserved loop structural integrity. As evident from our systematic comparative study using experimentally/clinically validated inhibitors, DRP-104 may serve as a potent and novel TMPRSS2 inhibitor and warrants further clinical investigation.

Azafuramidines as potential anticancer Agents: Pro-apoptotic profile and cell cycle arrest.

The current study aimed to test the antiproliferative activity of three azafuramidines (X, Y, and Z) against three different human cell lines; liver HepG2, breast MCF-7, and bone U2OS. And to explore the molecular mechanism(s) of the antiproliferative activity of these derivatives. The three new azafuramidines demonstrated a potent cytotoxicity at < 2 µM against the three cell lines investigated. The azafuramidines were highly selective with selectivity index âˆ¼ 47 - 61 folds indicating safety to the normal cells. In the scratch assay, azafuramidines significantly reduced the percentage of wound healing indicating ability to prevent or reduce metastasis. Derivatives X and Z arrested the HepG2 cells at S and G2/M phases detected by the flow cytometry. Derivatives X, Y, and Z elevated the apoptosis of HepG2 cells by âˆ¼ 71 %, 66 %, and 59 %, respectively. Derivatives X and Z were superior to derivative Y. The potent antiproliferative, cell cycle arrest, and pro-apoptotic efficacy of these chlorophenyl derivatives could be attributed to their ability of inducing the overexpression of p53, p21, and p27. These derivatives had the potential to act as anticancer agents and merit further investigations.

Investigation of the effect of structure modification of furamidine on the DNA minor groove binding and antiprotozoal activity.

New analogs of the antiprotozoal agent Furamidine were prepared utilizing Stille coupling reactions and amidation of the bisnitrile intermediate using lithium bis-trimethylsilylamide. Both the phenyl groups and the furan moiety of furamidine were replaced by heterocycles including thiophene, selenophene, indole or benzimidazole. Based upon the ΔTm and the CD results, the new compounds showed strong binding to the DNA minor groove. The new analogues are also more active both in vitro and in vivo than furamidine. Compounds 7a, 7b, and 7f showed the highest activity in vivo by curing 75% of animals, and this merits further evaluation.

Survey reaction of 2-amino-N-(aryl) benzimidamides with ninhydrin in different conditions and investigation of reaction mechanism using density functional theory (DFT).

In this investigation, firstly, 1-(2-amino-phenyl)-N-(aryl) methane diamine derivatives were synthesized by reaction of 2-aminobenzo nitrile with aromatic amines in the presence of aluminum chloride as the catalyst. Then, the reaction of these intermediates with ninhydrin in different conditions was investigated. The reaction between ninhydrin and 2-amino-N'-(aryl) benzimidamide derivatives in water as solvent under reflux conditions resulted in the synthesis of diazepine derivatives. The same results were obtained when the reaction was done in EtOH and in the presence of a few drops of sulfuric acid at room temperature. Also, this reaction was carried out in ethanol as solvent without the presence of sulfuric acid at room temperature which resulted in the synthesis of spiro [indene-2,2'-quinazoline] derivatives. And finally, the reaction was carried out in ethanol as solvent without the presence of sulfuric acid at the reflux conditions which resulted in the synthesis of isoquinolino-quinazoline derivatives. These N-heterocycles compounds are important biologically. Mild reaction conditions, simple procedure and purification and also product diversity with changing conditions are important advantages of this method. Also, to better understanding reaction mechanism on the condensation reactions of 2-amino-N-(aryl) benzimidamides with ninhydrin in different conditions, density functional theory (DFT)-based quantum chemical methods have been applied. Calculated atomic charges suggest that the C-1 (+ 0.54 a.u.) center of ninhydrin is a better electrophile than C-2 (+ 0.42 a.u.) center.

Water regulates the residence time of Benzamidine in Trypsin.

The process of ligand-protein unbinding is crucial in biophysics. Water is an essential part of any biological system and yet, many aspects of its role remain elusive. Here, we simulate with state-of-the-art enhanced sampling techniques the binding of Benzamidine to Trypsin which is a much studied and paradigmatic ligand-protein system. We use machine learning methods to determine efficient collective coordinates for the complex non-local network of water. These coordinates are used to perform On-the-fly Probability Enhanced Sampling simulations, which we adapt to calculate also the ligand residence time. Our results, both static and dynamic, are in good agreement with experiments. We find that the presence of a water molecule located at the bottom of the binding pocket allows via a network of hydrogen bonds the ligand to be released into the solution. On a finer scale, even when unbinding is allowed, another water molecule further modulates the exit time.

Assessing the Role of a Malonamide Linker in the Design of Potent Dual Inhibitors of Factor Xa and Cholinesterases.

The rational discovery of new peptidomimetic inhibitors of the coagulation factor Xa (fXa) could help set more effective therapeutic options (to prevent atrial fibrillation). In this respect, we explored the conformational impact on the enzyme inhibition potency of the malonamide bridge, compared to the glycinamide one, as a linker connecting the P1 benzamidine anchoring moiety to the P4 aryl group of novel selective fXa inhibitors. We carried out structure-activity relationship (SAR) studies aimed at investigating para- or meta-benzamidine as the P1 basic group as well as diversely decorated aryl moieties as P4 fragments. To this end, twenty-three malonamide derivatives were synthesized and tested as inhibitors of fXa and thrombin (thr); the molecular determinants behind potency and selectivity were also studied by employing molecular docking. The malonamide linker, compared to the glycinamide one, does significantly increase anti-fXa potency and selectivity. The meta-benzamidine (P1) derivatives bearing 2',4'-difluoro-biphenyl as the P4 moiety proved to be highly potent reversible fXa-selective inhibitors, achieving inhibition constants (Ki) in the low nanomolar range. The most active compounds were also tested against cholinesterase (ChE) isoforms (acetyl- or butyrylcholinesterase, AChE, and BChE), and some of them returned single-digit micromolar inhibition potency against AChE and/or BChE, both being drug targets for symptomatic treatment of mild-to-moderate Alzheimer's disease. Compounds 19h and 22b were selected as selective fXa inhibitors with potential as multimodal neuroprotective agents.

Nafamostat mesylate as a broad-spectrum candidate for the treatment of flavivirus infections by targeting envelope proteins.

Epidemics caused by flaviviruses occur globally; however, no antiviral drugs treating flaviviruses infections have yet been developed. Nafamostat (NM) is a protease inhibitor approved for pancreatitis and anti-coagulation. The anti-flavivirus potential of NM has yet to be determined. Here, utilizing in vitro and in vivo infection assays, we present that NM effectively inhibits Zika virus (ZIKV) and other flaviviruses in vitro. NM inhibited the production of ZIKV viral RNA and proteins originating from Asia and African lineage in human-, mouse- and monkey-derived cell lines and the in vivo anti-ZIKV efficacy of NM was verified. Mode-of-action analysis using time-of-drug-addition assay, infectivity inhibition assay, surface plasmon resonance assay, and molecular docking revealed that NM interacted with viral particles and blocked the early stage of infection by targeting the domain III of ZIKV envelope protein. Analysing the anti-flavivirus effects of NM-related compounds suggested that the antiviral effect depended on the unique structure of NM. These findings suggest the potential use of NM as an anti-flavivirus candidate, and a novel drug design approach targeting the flavivirus envelope protein.

In Silico Analysis and Synthesis of Nafamostat Derivatives and Evaluation of Their Anti-SARS-CoV-2 Activity.

Inhibition of transmembrane serine protease 2 (TMPRSS2) is expected to block the spike protein-mediated fusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nafamostat, a potent TMPRSS2 inhibitor as well as a candidate for anti-SARS-CoV-2 drug, possesses the same acyl substructure as camostat, but is known to have a greater antiviral effect. A unique aspect of the molecular binding of nafamostat has been recently reported to be the formation of a covalent bond between its acyl substructure and Ser441 in TMPRSS2. In this study, we investigated crucial elements that cause the difference in anti-SARS-CoV-2 activity of nafamostat and camostat. In silico analysis showed that Asp435 significantly contributes to the binding of nafamostat and camostat to TMPRSS2, while Glu299 interacts strongly only with nafamostat. The estimated binding affinity for each compound with TMPRSS2 was actually consistent with the higher activity of nafamostat; however, the evaluation of the newly synthesized nafamostat derivatives revealed that the predicted binding affinity did not correlate with their anti-SARS-CoV-2 activity measured by the cytopathic effect (CPE) inhibition assay. It was further shown that the substitution of the ester bond with amide bond in nafamostat resulted in significantly weakened anti-SARS-CoV-2 activity. These results strongly indicate that the ease of covalent bond formation with Ser441 in TMPRSS2 possibly plays a major role in the anti-SARS-CoV-2 effect of nafamostat and its derivatives.

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

A Facile Method for the Quantification of Urinary Uracil Concentration by a Uracil-Specific Fluorescence Derivatization Reaction.

A facile and reliable fluorescence method for the quantification of urinary uracil concentration is proposed herein. The assay utilizes a specific fluorescence (FL) derivatization reaction for uracil using 3-methylbenzamidoxime as a fluorogenic reagent. Although the presence of urine inhibited the FL reaction, 10 µL of urine was sufficient for the detection of urinary uracil. The uracil derivative was successfully separated from other fluorescent impurities using simple reversed-phase LC with FL detection. Urinary uracil concentrations from 16 people were compared with the concentrations obtained by the traditional column-switching liquid chromatographic analysis with UV detection. The FL derivative of uracil appeared as a single peak in the chromatograms of all samples. However, several samples showed an additional peak overlapping the uracil peak when using the column-switching method because of UV-active impurities. These results indicated that that the present method is not affected by interfering substances in urine and affords a precise determination of urinary uracil. We expect the proposed method to be applicable for diagnosing dihydropyrimidine dehydrogenase deficiency in 5-fluorouracil chemotherapy.

Interrogating the Inhibition Mechanisms of Human Aldehyde Oxidase by X-ray Crystallography and NMR Spectroscopy: The Raloxifene Case.

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

Identification of 13 Guanidinobenzoyl- or Aminidinobenzoyl-Containing Drugs to Potentially Inhibit TMPRSS2 for COVID-19 Treatment.

Positively charged groups that mimic arginine or lysine in a natural substrate of trypsin are necessary for drugs to inhibit the trypsin-like serine protease TMPRSS2 that is involved in the viral entry and spread of coronaviruses, including SARS-CoV-2. Based on this assumption, we identified a set of 13 approved or clinically investigational drugs with positively charged guanidinobenzoyl and/or aminidinobenzoyl groups, including the experimentally verified TMPRSS2 inhibitors Camostat and Nafamostat. Molecular docking using the C-I-TASSER-predicted TMPRSS2 catalytic domain model suggested that the guanidinobenzoyl or aminidinobenzoyl group in all the drugs could form putative salt bridge interactions with the side-chain carboxyl group of Asp435 located in the S1 pocket of TMPRSS2. Molecular dynamics simulations further revealed the high stability of the putative salt bridge interactions over long-time (100 ns) simulations. The molecular mechanics/generalized Born surface area-binding free energy assessment and per-residue energy decomposition analysis also supported the strong binding interactions between TMPRSS2 and the proposed drugs. These results suggest that the proposed compounds, in addition to Camostat and Nafamostat, could be effective TMPRSS2 inhibitors for COVID-19 treatment by occupying the S1 pocket with the hallmark positively charged groups.

Diastereoselective, Multicomponent Synthesis of Pyrrolopyrazinoquinazolinones via a Tandem Quinazolinone Rearrangement/Intramolecular Ring Closure of Tautomeric (Z)-Benzamidines.

An expedient route to enantiopure, diastereomeric pyrrolopyrazinoquinazolinones was developed following the discovery of a domino quinazolinone rearrangement-intramolecular cyclization of N-H benzamidines. A Ugi-Mumm-Staudinger sequence employing an optically pure proline derivative gave quinazolinones that, upon N-Boc deprotection, rearranged to tautomeric Z-benzamidines. Subsequent spontaneous cyclization afforded 15 diastereomeric pyrazinoquinazolinone pairs in up to 83% overall yield and 89:11 d.r which were separated easily via routine chromatographic purification-the only one required in the entire process.

A Novel Computational Approach for the Discovery of Drug Delivery System Candidates for COVID-19.

In order to treat Coronavirus Disease 2019 (COVID-19), we predicted and implemented a drug delivery system (DDS) that can provide stable drug delivery through a computational approach including a clustering algorithm and the Schrödinger software. Six carrier candidates were derived by the proposed method that could find molecules meeting the predefined conditions using the molecular structure and its functional group positional information. Then, just one compound named glycyrrhizin was selected as a candidate for drug delivery through the Schrödinger software. Using glycyrrhizin, nafamostat mesilate (NM), which is known for its efficacy, was converted into micelle nanoparticles (NPs) to improve drug stability and to effectively treat COVID-19. The spherical particle morphology was confirmed by transmission electron microscopy (TEM), and the particle size and stability of 300-400 nm were evaluated by measuring DLSand the zeta potential. The loading of NM was confirmed to be more than 90% efficient using the UV spectrum.

Possible Role of Electrolytes on the Formation of Precipitates during the Infusion of Nafamostat Mesilate in Hemodialysis.

Nafamostat mesilate (NFM) is used as an anticoagulant during hemodialysis in patients who have had complications due to hemorrhages. The formation of precipitates, which could lead to the interruption of hemodialysis has been reported when NFM is infused into blood during hemodialysis. We report herein on an examination of possible factors that could cause this. The effects of electrolytes such as phosphates, citrates or succinates on the formation of precipitates were examined by mixing NFM with aqueous solutions or plasma that contained these electrolytes. The formation of precipitates was observed in all electrolyte solutions when higher concentrations of NFM were mixed at around physiological pH. In the case of plasma, precipitates were observed when solutions containing higher concentrations of NFM were mixed with plasma that contained phosphate and citrate. In addition, the formation of precipitates under dynamic conditions where NFM was infused into flowing electrolyte solutions was also evaluated. The data suggested that such precipitates might be formed and disrupt the blood flow and/or an NFM infusion when NFM is infused into blood flowing in the hemodialysis circuit. The findings presented herein suggest the serum levels of anionic electrolytes (e.g., phosphate), the type of excipients present in pharmaceutical products (e.g., succinic acid or citric acid), the concentration of NFM used for the infusion or the rates of NFM infusion and blood flow are all factors that could affect precipitate formation during NFM infusions for hemodialysis.

Multisecond ligand dissociation dynamics from atomistic simulations.

Coarse-graining of fully atomistic molecular dynamics simulations is a long-standing goal in order to allow the description of processes occurring on biologically relevant timescales. For example, the prediction of pathways, rates and rate-limiting steps in protein-ligand unbinding is crucial for modern drug discovery. To achieve the enhanced sampling, we perform dissipation-corrected targeted molecular dynamics simulations, which yield free energy and friction profiles of molecular processes under consideration. Subsequently, we use these fields to perform temperature-boosted Langevin simulations which account for the desired kinetics occurring on multisecond timescales and beyond. Adopting the dissociation of solvated sodium chloride, trypsin-benzamidine and Hsp90-inhibitor protein-ligand complexes as test problems, we reproduce rates from molecular dynamics simulation and experiments within a factor of 2-20, and dissociation constants within a factor of 1-4. Analysis of friction profiles reveals that binding and unbinding dynamics are mediated by changes of the surrounding hydration shells in all investigated systems.