Host–guest binding, despite the relatively simple structural and chemical features of individual components, still poses a challenge in computational modelling. The extreme underperformance of standard end-point methods in host–guest binding makes them practically useless. In the current work, we explore a potentially promising modification of the three-trajectory realization. The alteration couples the binding-induced structural reorganization into free energy estimation and suffers from dramatic fluctuations in internal energies in protein–ligand situations. Fortunately, the relatively small size of host–guest systems minimizes the magnitude of internal fluctuations and makes the three-trajectory realization practically suitable. Due to the incorporation of intra-molecular interactions in free energy estimation, a strong dependence on the force field parameters could be incurred. Thus, a term-specific investigation of transferable GAFF derivatives is presented, and noticeable differences in many aspects are identified between commonly applied GAFF and GAFF2. These force-field differences lead to different dynamic behaviors of the macrocyclic host, which ultimately would influence the end-point sampling and binding thermodynamics. Therefore, the three-trajectory end-point free energy calculations are performed with both GAFF versions. Additionally, due to the noticeable differences between host dynamics under GAFF and GAFF2, we add additional benchmarks of the single-trajectory end-point calculations. When only the ranks of binding affinities are pursued, the three-trajectory realization performs very well, comparable to and even better than the regressed PBSA_E scoring function and the dielectric constant-variable regime. With the GAFF parameter set, the TIP3P water in explicit solvent sampling and either PB or GB implicit solvent model in free energy estimation, the predictive power of the three-trajectory realization in ranking calculations surpasses all existing end-point methods on this dataset. We further combine the three-trajectory realization with another promising modified end-point regime of varying the interior dielectric constant. The combined regime does not incur sizable improvements for ranks and deviations from experiment exhibit non-monotonic variations.  End-point free energy techniques as pivotal tools in drug discovery are commonly recognized as regimes with intermediate accuracy and efficiency lying between molecular docking and  free energy calculation. In protein–ligand and protein–protein interactions, such a computational ladder is believed to be solid, with a huge library of case reports supporting its usage ,5,6,7,8.There are generally two sampling protocols in end-point free energy calculations. The first one, the ‘normal’ and popular single-trajectory realization, samples only the protein-ligand/host–guest bound state and extracts free energy of binding by computing inter-molecular potentials, solvation and entropic contributions. The main motivation of this single-trajectory realism is the exact cancellation of internal energies, and the end-point sampling only needs to converge the fluctuations in inter-molecular components. In complexes involving biomacromolecular species, the internal energies summed over a large amount of atoms are huge in magnitude, and the fluctuations in internal energies are overwhelmingly high. However, the smaller sizes of host–guest systems lead to smaller internal energies and fluctuations, which makes them potentially suitable for the second end-point sampling protocol named the three-trajectory realization. As suggested by its name, the three-trajectory realization samples three ensembles including both the bound and unbound states ,16,17,18The single-trajectory realization of end-point free energy calculations only samples the host–guest complex in the bound state, where the host cavity is occupied by the guest molecule due to the formation of favorable host–guest coordination. Thus, it is unlikely to observe a squashed host or a significant conformational change, and the intra-molecular interactions  could be of relatively little relevance. By contrast, the three-trajectory realization requires the sampling of both the bound complex and the free/unbound host-only and guest-only systems. In the unbound state (free host and free guest in solvent), the host–guest coordination is replaced by host/guest–water and also host/guest–ion interactions. If force field parameters do not produce a balanced description of the (de)solvation phenomenon and the backbone stiffness, the water- and ion-filled host cavity could possibly be squashed in the unbound state. Similar observations have been reported in our previous works on other host molecules including cucurbiturils and cyclodextrins ,21,22. T2-COO− tails, the stiffnesses of the host cavity produced by the two GAFF derivatives are expected to be similar, but the orientations of the polar -CH2-COO− tails are expected to exhibit different behaviors. We have compared the other bonded terms  of GAFF derivatives and also identified noticeable differences. For instance, the averaged variation of the bond-stretching force constants is ~15%, but the equilibrium length is minimally perturbed (~0.02% on average). Aside from the bonded parameters, non-bonded terms are also entering intra-molecular interactions between atoms separated by more than two chemical bonds. The 1–4 scaling constants in the two GAFF versions are the same, but the other parameters do show some differences. As the atomic charges are obtained with molecule-specific fitting, in the constructed models only the vdW terms differ. Through a detailed comparison of vdW terms, we confirm that all vdW parameters  are altered in GAFF2 compared with GAFF, but the magnitudes of the variations are insignificant. For example, the difference between the GAFF and GAFF2 σ averaged over all host atoms is ~2%. As these differing terms of bond-stretching, angle-bending, and intra-molecular vdW interactions are not expected to have significant influences on conformational preferences or be involved in substantial reorganizations/rearrangements upon host–guest binding, the main difference between GAFF and GAFF2 would be triggered by the torsional terms in Two GAFF derivatives are commonly employed in modern computational investigations. The earlier GAFF v1.81 (or simply GAFF) is a parameter set and minorly updated from the original GAFF, while a newer GAFF version 2.11, or simply GAFF2, is the latest component of the second generation of GAFF, with majorly re-fitted transferable parameters. We first present a detailed comparison between the two GAFF versions in order to identify their differences and similarities. The main components of intra-molecular interactions, the bonded terms, include bond stretching, angle bending, and torsional potentials. Among them, the torsional terms contribute the largest portion of variations during conformational rearrangements in the presence of bond-length constraints. Through a detailed comparison between definitions of torsional terms in GAFF and GAFF2, we identify two terms that exhibit noticeable differences, as shown in The detailed analyses of the force field parameters and energetics presented above are informative, providing hints on the similarities and differences between different parameter sets. However, whether these factors are directly impacting the dynamics of the macrocyclic host remains to be revealed. Thus, we perform unbiased sampling in explicit solvent in order to probe the dynamic behaviors produced by both parameter sets. Note that the counter ions (sodium cations) are added for neutralization. Thus, the host cavity could be occupied by water molecules and/or cations, forming host–water or host–ion coordination. The solvated host experiences 5000 steps minimization, 0.1 ns NVT equilibration, and then 1 ns NPT equilibration. After that, the 500 ns NPT production run with a sampling interval of 0.5 ns is spawned. 2-COO− rims do exhibit significant differences. Under GAFF, the WP6 host does not exhibit any conformational preference and its -CH2-COO− tails rotate freely. By contrast, a directional rotational preference featuring a fraction of -CH2-COO− tails stabilizing at the cavity entrance could be identified from the GAFF2 overlay. This difference in the dynamic behaviors of -CH2-COO− tails agrees with the detailed analyses of torsional terms in the previous section, where the GAFF2 parameter set produces a more regular description of the -CH2-COO− rotation than GAFF. By including the solvent and ions in the visualization, we observe that this unusual rotational behavior is related to the formation of host–cation coordination. Thus, the regular/localized behavior under GAFF2 is caused by the interplay of host–solvent/ion interactions and intra-molecular torsional potentials. The host–solvent/ion coordination also exists under GAFF but does not incur significant conformational change like the GAFF2 situation. Overall, the dynamic behaviors of the WP6 host have been confirmed to be different under the two popular GAFF derivatives, which would obviously impact the sampling trajectories in end-point free energy calculations.The superpositions of unbiased trajectories are presented in Crucial influencing factors of model construction in end-point calculations investigated in our previous works include the charge scheme, water model, and docking procedure for the initial condition, all of which do not have significant impacts on the performance of end-point calculations given a selected implicit solvent model ,14. HoweThe experiment–calculation correlation under GAFF and GAFF2 is presented in We then turn to the cross-comparison of quality metrics in 2COO− tails stay at the entrance of the host entrance. As discussed in the comparison of host dynamics in 2COO− groups. This structural rearrangement of the host surface definitions serves as a critical factor causing the huge variations in the implicit solvent contributions . In The three-trajectory realization samples both the bound and unbound states. The detailed energetic contributions in individual ensembles are presented in We then turn to the comparison of the calculated binding affinities. The correlograms of the three-trajectory estimates under different parameter sets are given in Considering the successes in predicting the ranking information with both the three-trajectory realization and the dielectric constant-variable regime in the single-trajectory case, we combine the two non-conflicting regimes to form a more advanced modification, i.e., the three-trajectory dielectric constant scheme. We select three parameter combinations to test this method, including the best-performing set in the three-trajectory realization GAFF+TIP3P see d, the GAThe affinity variations of Individual host–guest pairs under two of the three parameter sets (GAFF+SPC/E and GAFF2+SPC/E) are presented as illustrative examples in The first term in Equation (3) denotes the three-trajectory bound-state inter-molecular electrostatic contribution, which is exactly the same as that in Equation (1). The second row includes the variation in intra-molecular interactions upon the binding event, and is not zero (cancelled out) due to the incorporation of the unbound state in the free energy calculation. This is also the term that the three-trajectory realization is believed to differ significantly from in the single-trajectory form. The third row, the implicit solvent contribution upon the binding event, corresponds to the second term in Equation (1) (substituting the subscript unbound with bound). Compared with the single-trajectory case, the incorporation of the unbound-state sampling alters this implicit solvent contribution to some extent. Comparing Equation (1) with Equation (3), we know that the differences between the single-trajectory and three-trajectory responses to variations in the interior dielectric constant are caused by the intra-molecular electrostatics and the implicit solvent contribution. Then, an interesting question to ask is which contribution  is playing a more critical role in the three-trajectory realization. To investigate this issue, we extract the detailed components in both the bound and unbound states and observe that the negative implicit solvent term is larger in magnitude than the electrostatic counterpart for all systems investigated . As for the other parameters , following our previous works [+ or Cl− are added to neutralize the simulation cell. Periodic boundary conditions are employed to replicate the unit cell in the whole space. In the modelling of host–guest complexes, the combination of the GAFF version and the solvent model gives four protocols for model construction. The model construction follows our WP6 end-point series works ,14. The us works ,14, we tus works  and a reus works , formingus works ,32 and Sus works , which dus works . Specifius works ,35. Non-us works ,37 of NaAside from the solvated host–guest complex, the unbound state containing the decoupled host–guest pair is also sampled in the three-trajectory realization of end-point free energy calculation. This leads to two independent simulations, each of which contains either the solvated host or the guest. The modelling protocol of these unbound systems is similar to the complex system. Namely, we use the HF/6-31G*-targeted RESP charges to describe electrostatics, employ GAFF or GAFF2 for host and GAFF2 for guest, and add solvents with either of the two water models and monovalent counter ions for neutralization.−1 for temperature regulation, and the Berendsen barostat with isotropic scaling for pressure regulation. The cutoff for non-covalent interactions is set to 8 Å (AMBER default), and the PME method [The constructed solvated host, guest, and host–guest complex first experience 5000 steps minimization, 300 ps constant-volume heating with weak harmonic restraints on heavy atoms of solutes, and 1-ns NPT relaxation to reach an equilibrated state at 300 K and 1 atm. The sampling time of host–guest complexes in our previous works is 100 ns, which is already long in end-point free energy calculations. However, the fluctuations in the free energy estimates from the three-trajectory realization are often much larger than the single-trajectory counterpart due to the inclusion of the reorganization free energy  ,16,38, tE method  is used E method  is used OBC with the second set of modified Bondi radii [The three-trajectory end-point method estimates the free energy of binding by taking the difference between the bound and unbound states. The former corresponds to the solvated host–guest complex, while the latter is sampled with the host-only and the guest-only simulations. The calculation of the energetics of the individual systems involves the gas-phase force-field energy estimation, the implicit solvent treatment, and normal mode analysis (NMA)  for the di radii ,47 and tdi radii . As for Host–guest binding remains rather challenging in the modern computational community. End-point free energy calculations with established predictive power in protein–ligand complexes are not well understood in host–guest modelling. In our series works towards a thorough evaluation of host–guest modelling focusing on the latest SAMPL9 host–guest dataset involving a pillararene derivative, contrary to the over-optimistic view reported in numerous reports published in mainstream forums, we prove with solid numerical evidence that standard end-point free energy calculations are practically useless and achieve prediction qualities far from acceptable. Modifications are necessary to bring the computational results close to the experimental reference. The alterations considering linear regression and interior dielectric constant have been extensively validated in our previous works. In this paper, we focus on the specific modification of the three-trajectory realization. 2COO− tails. Directly applying the transferable parameter sets in dynamics simulation, we observe similar backbone stiffnesses for WP6 under GAFF and GAFF2, but the polar tails at both rims exhibit different conformational preferences. Structural analysis reveals that the directional orientation in the solvated host is related to ion coordination . Overall, the observed different dynamic behaviors produced by the two GAFF derivatives are expected to produce different conformational preferences in both the unbound host and the bound host–guest complex, and thus produce different fluctuating behaviors of energetics. The three-trajectory realization is rather uncommon due to the overwhelming fluctuations in internal energies in protein–ligand complexes but could be well-suitable for host–guest binding due to the relatively small sizes of the components involved in inter-molecular packing and thus the small energy fluctuations. Force field parameters, especially bonded terms, play a critical role here, and a thorough investigation of the similarities and differences between the popular GAFF derivatives is presented. The GAFF derivatives are found to share similar parameters for the pillararene backbone, and the differences mainly lie in torsional terms involving -CHDue to the observed significant differences between GAFF derivatives, this force-field selection could be a crucial influencing factor determining the prediction quality of host–guest binding affinity. As this conclusion could be applicable to both single-trajectory and three-trajectory realizations, we thus first benchmark single-trajectory end-point calculations under GAFF and GAFF2 as a critical addition to the extensive benchmark calculations presented in our previous work. The numerical results support the usage of the SPC/E water model, the GB implicit solvent, and the GAFF2 parameter set. Thus, in the popular single-trajectory realization, GAFF2 outperforms GAFF for this host family. 2COO− rims. Third, the predicted binding affinities under the three-trajectory realization exhibit systematic overestimation of the binding strength also due to the inclusion of the unbound-state sampling . This offset leads to significantly different RMSE values for different parameter sets but does not poison the ranking calculation. The best-performing parameter combination GAFF+TIP3P achieves a Kendall τ ~0.5, which surpasses all end-point predictions on this dataset and is even comparable to the costlier alchemical method. The three-trajectory end-point free energy calculations exhibit behaviors somehow different from the single-trajectory procedure. First, the shift to the three-trajectory realization further triggers an increase in energetic fluctuations and noisier statistics, which calls for a lengthened sampling time in order to minimize the statistical uncertainty to values similar to the single-trajectory estimates. A detailed look into the numerical data suggests that the uncertainty/fluctuation increase is mainly caused by the inclusion of the unbound states, especially the host-only system. Based on this fact, a general rule or recommendation for the three-trajectory end-point free energy calculation is to perform longer sampling in the unbound host-only state, while the unbound guest and the bound host–guest states could be less focused. Second, the comparison between the two GAFF versions suggests that the energetic fluctuations under GAFF2 are much more significant than GAFF. This difference in fluctuating behaviors could be specifically located on the implicit solvent energetics of the unbound host, which is highly related to the directional rotation of the -CHTo pursue a further improved performance and accumulate experiences of the three-trajectory realization, we combine two non-conflicting modified end-point procedures into a three-trajectory dielectric constant protocol. This newly constructed modification does not systematically better or worsen the reproduction of the absolute binding affinities, which differs from the single-trajectory situation. As for the ranking calculation, the combined regime leads to minor but not systematic improvements compared with individual modifications . Further, the prediction quality of the ranking information achieves the best performance when the interior dielectric constant reaches 2, which also differs from the single-trajectory case at ~6 dielectric constants. Overall, combining different non-conflicting protocols into more advanced end-point schemes could be helpful, but the improvements over individual baseline alterations could be marginal. To further explore the space of advanced end-point techniques and search for really usable protocols, we would attempt a series of other modifications (e.g., enhanced sampling ,21,22,49


Mild gastrointestinal symptoms and mild abdominal pain often occur in association with COVID-19. However, acute abdomen and severe abdominal pain warranting urgent surgical treatment are rare. Here we present the case of a 40-year-old man who presented with the clinical picture of a perforated duodenal ulcer. He was eventually found to have COVID-19 and was treated conservatively. In this report, we discuss his course of treatment and review the relevant literature. Novel coronavirus disease COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first described in the Hubei region of China in 2019 and declared a pandemic by the World Health Organization (WHO) in March 2020 [9 caused A 40-year-old Saudi man was admitted to our emergency department with a sudden onset of severe epigastric pain associated with frequent vomiting. The pain had occurred after three days of flu-like symptoms for which he had not seen a doctor. He was otherwise healthy and had no chronic illnesses apart from recurrent dyspepsia, which usually resolved with antacids, and he had never had surgery. On examination, the patient looked ill and had tachycardia (pulse of 110 beats per minute), blood pressure of 128/82 mm Hg, fever (38.6°C), respiratory rate, and oxygen saturation were normal. Chest examination revealed no basal crepitations or wheezing on either side of the chest. Abdomen examination revealed severe tenderness and board-like muscle rigidity over the epigastrium. The rest of the abdomen was lax and not painful. Bowel sounds were audible. Laboratory investigations revealed a leukocyte count of 11.2 (109/liter), and a normal hemoglobin of 13.6 g/L. Serum creatinine was slightly elevated (136 mmol/L), blood urea and electrolyte levels, and liver function profile were within normal range. Pancreatic enzymes were within the normal range. The screening test for COVID-19 was initially negative. The C-reactive protein was 108 mg/L. Given the history of chronic dyspepsia, the sudden onset of severe epigastric pain, and the abdominal examination findings, a diagnosis of perforated peptic ulcer was made, but the X-ray of the abdomen and thorax in the upright position showed no air under the diaphragm Figures -1b.For further examination, a double contrast scan computed tomography (CT) was requested, which showed no free air in the abdomen and no intraperitoneal leakage of the oral contrast medium Figure . A chestConsidering the result of the radiological examination, we planned to treat the patient conservatively. He was admitted to the surgical ward, kept fasting, and given an intravenous antibiotic (cefuroxime) and intravenous fluids. The next day, the polymerase chain reaction (PCR) of nasopharyngeal swabs was positive for COVID-19. The patient was isolated and given permission for oral intake and continued antibiotics, resulting in a dramatic clinical response. He was discharged five days after admission.COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first described in the Hubei region of China in 2019 and declared a pandemic by the World Health Organization (WHO) in March 2020 [COVID-19 is primarily a respiratory disease affecting the lung parenchyma, but it may also affect several organs and cause various extrapulmonary symptoms. As the disease progressed, more and more reports of extrapulmonary disease emerged, reporting cardiovascular, GI, renal, neurological, and skeletal-muscular involvement, either in isolation or in association with a pulmonary manifestation of the disease.COVID-19 is primarily a respiratory infection causing fever, cough, shortness of breath, and loss of smell or taste. However, mild GI symptoms such as nausea, vomiting, and diarrhea have been reported in association with pulmonary manifestations of the disease, but acute abdomen requiring immediate life-saving surgical treatment has rarely been reported in association with the infection.The exact mechanism of how COVID-19 causes GI symptoms and acute abdomen is not yet fully understood. Still, some possible explanations include infection of the cells of the GI tract by binding of the virus to the angiotensin-converting enzyme 2 (ACE2) receptor expressed in the esophagus, stomach, small and large intestine, inflammation and damage to the mucous lining, and the triggering of an excessive immune response (cytokine storm) that can cause systemic inflammation and multi-organ failure and lead to ischemia and necrosis of the intestine or other abdominal organs, and the causation of coagulation abnormalities that can increase the risk of thrombosis in the mesenteric vessels and lead to intestinal ischemia and infarction.Safari et al. suggest that COVID-19 uses ACE2 as an entry receptor to interact with host cells. Therefore, tissues with high ACE2 expression, such as lungs and visceral adipose tissue, could be a potential target for this new virus .The positive correlation between COVID-19 and the incidence of peptic ulcers was suggested by the study of Dao et al., which showed that the incidence of peptic ulcers was significantly higher during the COVID-19 pandemic in 2020 compared to 2019 in the same health facility in Vietnam , and by A meta-analysis of 35 Chinese studies involving 6686 patients with COVID-19 found a pooled prevalence of digestive symptoms of 15%, with nausea or vomiting, diarrhea, and loss of appetite being the three most common symptoms. There was also a correlation between the presence of GI manifestations and clinical deterioration , while aMelazzini et al. reported five patients with GI symptoms without respiratory symptoms. All had positive PCR for COVID-19 and were diagnosed with peptic ulcer on admission with no history of peptic ulcer or Helicobacter pylori infection. They suggested that the pathogenesis of peptic ulcer disease could be explained by stress due to fear of a pandemic or acute illness, by direct damage to the gastric epithelium by the virus, or by active inflammation of the mucosa maintained by the cytokine storm [Acute abdomen is a rapid onset of severe symptoms that may indicate life-threatening intra-abdominal pathology requiring urgent attention and treatment . Common Diagnosis of COVID-19 with acute abdomen can be difficult as symptoms are non-specific and may mimic other causes of abdominal pain. The rare occurrence of COVID-19 as acute abdomen, especially in the absence of respiratory manifestations of the disease, leads to unnecessary harmful surgical interventions. In addition, surgical complications of GI disease such as perforations can be difficult to diagnose in critically ill, intubated patients, leading to misdiagnosis of severe cases requiring surgical intervention.The assessment of COVID-19-positive patients with acute abdomen and the decision to operate should be made with great caution, as unnecessary surgery in such patients may lead to a cytokine storm and worsen the patient’s condition. An active observation protocol with repeated clinical examination and repeated determination of inflammatory markers in the blood is more useful than a hasty negative exploration.There are several reports on the association of COVID-19 with the non-complicated peptic disease, but unfortunately, there are not a large number of published studies with large samples on this topic, probably because of the rarity of the association between COVID-19 and acute abdomen. Most of the available literature consists of sporadic case reports. To our knowledge, our case is the first case reported in which severe symptoms and signs of peptic ulcer perforation were the main features of the disease, without respiratory symptoms, and actual perforation.Merdad GA et al. reported a case of COVID-19 with classical complaints suggestive of complicated peptic ulcer disease without respiratory symptoms. After laboratory and radiographic investigations, the patient was diagnosed with a perforated duodenal ulcer and underwent laparotomy. The patient had a complicated postoperative course .Similarly, He L et al. reported the case of a COVID-19-positive patient with lung involvement on CT. Five days after admission, he developed clinical and radiological signs of a perforated duodenal ulcer. The patient underwent surgery and was successfully treated .Agnes A et al. reported on a COVID-19-positive patient with severe respiratory syndrome who was treated with multiple doses of IL-6 inhibitors and underwent successful emergency surgery for a perforated duodenal ulcer. They concluded that the association between repeated administration of IL-6 inhibitors and upper GI bleeding and perforation needs to be investigated and that the threshold for administration of prophylactic therapy with proton pump inhibitors in patients with severe COVID-19 should be carefully considered .COVID-19 is primarily a respiratory infection. However, mild GI symptoms such as nausea, vomiting, and diarrhea have been reported in association with the disease. Acute abdomen requiring immediate life-saving surgical decisions and treatment has rarely been reported in association with the infection. Diagnosis of acute abdomen in COVID-19 patients can be difficult as the symptoms may represent the main symptoms of the disease without intra-abdominal pathology being present. The situation is even more deceptive in the absence of respiratory symptoms of the disease and may lead to unfavorable surgical intervention. The assessment of COVID-19-positive patients with symptoms and signs of acute abdomen and the decision to proceed with surgery should be made with great caution, as surgery in these patients may lead to a cytokine storm and worsen the patient’s condition.