Enabling structure-based drug discovery utilizing predicted models.

High-quality predicted structures enable structure-based approaches to an expanding number of drug discovery programs. We propose that by utilizing free energy perturbation (FEP), predicted structures can be confidently employed to achieve drug design goals. We use structure-based modeling of hERG inhibition to illustrate this value of FEP.

Benchmarking Refined and Unrefined AlphaFold2 Structures for Hit Discovery.

The recently developed AlphaFold2 (AF2) algorithm predicts proteins' 3D structures from amino acid sequences. The open AlphaFold protein structure database covers the complete human proteome. Using an industry-leading molecular docking method (Glide), we investigated the virtual screening performance of 37 common drug targets, each with an AF2 structure and known holo and apo structures from the DUD-E data set. In a subset of 27 targets where the AF2 structures are suitable for refinement, the AF2 structures show comparable early enrichment of known active compounds (avg. EF 1%: 13.0) to apo structures (avg. EF 1%: 11.4) while falling behind early enrichment of the holo structures (avg. EF 1%: 24.2). With an induced-fit protocol (IFD-MD), we can refine the AF2 structures using an aligned known binding ligand as the template to improve the performance in structure-based virtual screening (avg. EF 1%: 18.9). Glide-generated docking poses of known binding ligands can also be used as templates for IFD-MD, achieving similar improvements (avg. EF 1% 18.0). Thus, with proper preparation and refinement, AF2 structures show considerable promise for in silico hit identification.

Capsid antibodies to different adeno-associated virus serotypes bind common regions.

Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies.

Using AlphaFold and Experimental Structures for the Prediction of the Structure and Binding Affinities of GPCR Complexes via Induced Fit Docking and Free Energy Perturbation.

Free energy perturbation (FEP) remains an indispensable method for computationally assaying prospective compounds in advance of synthesis. However, before FEP can be deployed prospectively, it must demonstrate retrospective recapitulation of known experimental data where the subtle details of the atomic ligand-receptor model are consequential. An open question is whether AlphaFold models can serve as useful initial models for FEP in the regime where there exists a congeneric series of known chemical matter but where no experimental structures are available either of the target or of close homologues. As AlphaFold structures are provided without a bound ligand, we employ induced fit docking to refine the AlphaFold models in the presence of one or more congeneric ligands. In this work, we first validate the performance of our latest induced fit docking technology, IFD-MD, on a retrospective set of public experimental GPCR structures with 95% of cross-docks producing a pose with a ligand RMSD ≤ 2.5 Å in the top two predictions. We then apply IFD-MD and FEP on AlphaFold models of the somatostatin receptor family of GPCRs. We use AlphaFold models produced prior to the availability of any experimental structure from this family. We arrive at FEP-validated models for SSTR2, SSTR4, and SSTR5, with RMSE around 1 kcal/mol, and explore the challenges of model validation under scenarios of limited ligand data, ample ligand data, and categorical data.

Structure-Activity Relationship of a Pyrrole Based Series of PfPKG Inhibitors as Anti-Malarials.

Controlling malaria requires new drugs against Plasmodium falciparum. The P. falciparum cGMP-dependent protein kinase (PfPKG) is a validated target whose inhibitors could block multiple steps of the parasite's life cycle. We defined the structure-activity relationship (SAR) of a pyrrole series for PfPKG inhibition. Key pharmacophores were modified to enable full exploration of chemical diversity and to gain knowledge about an ideal core scaffold. In vitro potency against recombinant PfPKG and human PKG were used to determine compound selectivity for the parasite enzyme. P. berghei sporozoites and P. falciparum asexual blood stages were used to assay multistage antiparasitic activity. Cellular specificity of compounds was evaluated using transgenic parasites expressing PfPKG carrying a substituted "gatekeeper" residue. The structure of PfPKG bound to an inhibitor was solved, and modeling using this structure together with computational tools was utilized to understand SAR and establish a rational strategy for subsequent lead optimization.

Reliable and Accurate Prediction of Single-Residue pKa Values through Free Energy Perturbation Calculations.

Accurate prediction of the pKa's of protein residues is crucial to many applications in biological simulation and drug discovery. Here, we present the use of free energy perturbation (FEP) calculations for the prediction of single-protein residue pKa values. We begin with an initial set of 191 residues with experimentally determined pKa values. To isolate sampling limitations from force field inaccuracies, we develop an algorithm to classify residues whose environments are significantly affected by crystal packing effects. We then report an approach to identify buried histidines that require significant sampling beyond what is achieved in typical FEP calculations. We therefore define a clean data set not requiring algorithms capable of predicting major conformational changes on which other pKa prediction methods can be tested. On this data set, we report an RMSE of 0.76 pKa units for 35 ASP residues, 0.51 pKa units for 44 GLU residues, and 0.67 pKa units for 76 HIS residues.

Induced-Fit Docking Enables Accurate Free Energy Perturbation Calculations in Homology Models.

Homology models have been used for virtual screening and to understand the binding mode of a known active compound; however, rarely have the models been shown to be of sufficient accuracy, comparable to crystal structures, to support free-energy perturbation (FEP) calculations. We demonstrate here that the use of an advanced induced-fit docking methodology reliably enables predictive FEP calculations on congeneric series across homology models ≥30% sequence identity. Furthermore, we show that retrospective FEP calculations on a congeneric series of drug-like ligands are sufficient to discriminate between predicted binding modes. Results are presented for a total of 29 homology models for 14 protein targets, showing FEP results comparable to those obtained using experimentally determined crystal structures for 86% of homology models with template structure sequence identities ranging from 30 to 50%. Implications for the use and validation of homology models in drug discovery projects are discussed.

Prospective observational study of young adult ischemic stroke patients.

OBJECTIVE: Ischemic stroke (IS) in young patients may differ in etiology and prognosis from later-life IS, which is much more common. A number of single-center and population-based cohorts of affected individuals have been published, but information on the long-term prognosis of these patients is limited. METHODS: IS patients (≤55 years), discharged over a 10-year period, were evaluated and prospectively followed. Subgroups were evaluated for type of stroke, antecedent risk factors (RF), long-term outcomes, and occupational status over several years of follow-up. RESULTS: 178 IS individuals from 2001-2010 were divided into older (46-55, n = 118) and younger (18-45, n = 60) age groups. Traditional RF-hypertension, diabetes mellitus, hyperlipidemia-were significantly associated with IS, and increased with age. The distribution and type of IS were similar in both groups, except for an increase in small vessel IS among the older subgroup (p = .003). Of the evaluable patients at 5.1 ± 2.5 years of follow-up (n = 138), a similar proportion of patients in both subgroups had a recurrent IS, but no significant differences were found in most disability indices. Approximately one third of patients suffered from moderate to severe disability, and were unable to return to their prior work. CONCLUSIONS: Younger and older IS patients are generally predisposed from the same traditional RF which progress with age. Long-term disabilities tend to worsen over time due to recurrent vascular events. These data emphasize the need for a strategy for early identification of the already well-known stroke RF in younger stroke patients.

Reliable and Accurate Solution to the Induced Fit Docking Problem for Protein-Ligand Binding.

We present a reliable and accurate solution to the induced fit docking problem for protein-ligand binding by combining ligand-based pharmacophore docking, rigid receptor docking, and protein structure prediction with explicit solvent molecular dynamics simulations. This novel methodology in detailed retrospective and prospective testing succeeded to determine protein-ligand binding modes with a root-mean-square deviation within 2.5 Å in over 90% of cross-docking cases. We further demonstrate these predicted ligand-receptor structures were sufficiently accurate to prospectively enable predictive structure-based drug discovery for challenging targets, substantially expanding the domain of applicability for such methods.

Efficacy of ertapenem for secondary treatment of diabetic foot infections .

UNLABELLED:  Background. The diabetic foot is a common site of infections that frequently result in significant patient morbidity and mortality. Antibiotic treatment is an important part of therapy with selection of the specific agent depending on the stage of ulceration. Recently, ertapenem has entered use as first line therapy for moderate to severe diabetic foot infections. The following prospective study recounts the experience of using ertapenem as tertiary salvage therapy following failure of first and second-line antibiotics. METHODS: Forty consecutive patients with diabetic foot ulcers (IDSA classification moderate-severe), 28 with soft tissue infections alone, 12 with concomitant osteomyelitis, were included in the study. Patients were referred from the diabetic foot clinic at the authors' institution after failure of treatment with cephalexin and ciprofloxacin/clindamycin combination, and were treated with ertapenem 1-g daily intravenously (IV). Endpoints were significant clinical improvement (resolution of fever, leukocytosis, surrounding erythema, and purulent discharge). RESULTS: Clinical improvement was noted in all 28 individuals with soft tissue infections-all of these individuals had complete ulcer closure after a mean of 30 ± 8 treatment days. Of the 12 patients with concomitant osteomyelitis, only 25% required surgical intervention. Nine individuals were cured following a mean of 60 ± 15 treatment days of home IV antibiotic therapy. CONCLUSION: Ertapenem is an efficacious, easy to use, and cost saving antibiotic for the treatment of diabetic foot infections that have failed therapy with traditional antibiotics.

[Cefepime versus piperacillin/tazobactam with or without amikacin in the treatment of febrile hematological and oncological neutropenic patients in an internal medicine ward].

BACKGROUND: The efficacy of conventional doses of piperacillin/ tazobactam (PTZ) plus amikacin (AMK) were compared retrospectively to low doses of cefepime (CEF) plus amikacin in high risk febrile neutropenic patients with an underlying hematologic malignancy, and CEF versus PTZ (without AMK) in low risk individuals with febrile neutropenia and underlying solid tumor malignancies. METHODS: Fifty-six high risk hematologic malignancy patients received a combination of PTZ 4.5 grams administered every 8 hours plus AMK 15 mg/kg/day, while 46 received CEF 1 gram administered every 12 hours plus AMK 15 mg/kg/day. In addition, 19 febrile neutropenic individuals with underlying solid malignancies received PTZ 4.5 grams every 8 hours and 25 received CEF 1 gram every 12 hours. All patients were treated in an isolation unit section of a general internal medicine ward. RESULTS: There was no significant difference between the groups in terms of age, depth of neutropenia, microbiologic result, morbidity, length of hospital stay or mortality. CONCLUSION: PTZ and CEF in combination with AMK were equally efficacious in neutropenic patients with hematologic malignancies. In addition, monotherapy with CEF or PTZ proved to be equally efficacious in neutropenic patients with solid tumors. Low dose CEF is safe and allows a reduction of cost and less antibiotic exposure.

On the validity of the Born-Oppenheimer separation and the accuracy of diagonal corrections in anharmonic molecular vibrations.

The energies and wave functions of several lowest-lying vibrational states of FHF(-), ClHCl(-), and BrHBr(-) have been computed by a finite-difference method with and without the Born-Oppenheimer (BO) separation between the heavy (halogen) and light (hydrogen) particle motion. The so-called diagonal BO correction (DBOC), which includes the effect of the heavy particles' kinetic energy operator acting on the light particles' wave functions, has also been made to the energies. The errors caused by the BO approximation are found to be remarkably small (ca. 10(-5) au) and can be systematically and effectively reduced by the DBOC except for states excited in the heavy particle motion. When the bare mass of the light particle is used instead of the reduced mass in the BO approximation and, therefore, the translational degrees of freedom are not correctly decoupled, the errors in the BO treatment become greater by a factor of 2-7. However, these additional errors are almost completely erased by the DBOC. Analytical and numerical results suggest that the remaining errors in the BO and DBOC treatments be proportional to epsilon(1) and epsilon(2), where epsilon is the mass ratio of the light to heavy particles, when the corrections are made to the potential energy surfaces and the wave functions for these surfaces are determined variationally. When the DBOC is applied in the first-order perturbation approximation, the remaining errors scale as epsilon(3/2).

Granulomatous hepatitis and Sjogren's syndrome: an association.

BACKGROUND: Sjogren's syndrome (SS) is a common autoimmune disorder in which liver involvement is frequent, but generally mild and subclinical. Multiple hepatic histologies have been reported, but to our knowledge an association with granulomatous hepatitis (GH) has never been described. We recently evaluated an individual in whom biopsy-proven GH was associated with concomitant SS. OBJECTIVE: To clarify the possible association between GH and SS. METHODS: We retrospectively reviewed all cases of biopsy-proven GH seen in our institution from 1991 to 2004. Overall, there were 16 individuals with GH identified of which 4 were considered idiopathic in origin. These individuals underwent an extensive evaluation for the presence of SS as well as other disorders known to be associated with GH. RESULTS: Of the 4 identified cases with a previously suspected idiopathic GH, 3 met criteria for primary SS. All 3 individuals underwent minor salivary gland biopsy, which was diagnostic for this condition. CONCLUSIONS: These results suggest a likely association between these 2 conditions. Further epidemiological studies will be necessary to confirm this finding.

Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 1.

Crystals of baculovirus-expressed adeno-associated virus serotype 1 (AAV1) capsids have been grown in the rhombohedral space group R32 (unit-cell parameters a = 254.7 A, alpha = 62.3 degrees) and shown to diffract X-rays to at least 2.5 A resolution. The diffraction data were subsequently processed and reduced with an overall R(sym) of 12.3% and a completeness of 89.0%. Based on the unit-cell volume, rotation-function and translation-function results and packing considerations, there is one virus capsid (60 viral proteins) per unit cell and there are ten viral proteins per crystallographic asymmetric unit. The AAV1 capsid shares both the twofold and threefold crystallographic symmetry operators. The AAV1 data have been initially phased using a polyalanine model (based on the crystal structure of AAV4) to 4.0 A resolution and the structure determination and refinement is in progress using tenfold noncrystallographic symmetry electron-density averaging.

Topical hyperbaric oxygen and low-energy laser for the treatment of chronic ulcers.

BACKGROUND: Diabetic foot ulcers (DFU) and chronic venous ulcers (CVU) are persistent cutaneous lesions that are difficult to treat and heal. Topical hyperbaric oxygen (THO) and low-energy laser (LEL) are therapies that have been employed separately for ulcer treatment, but their concomitant use has not been investigated. METHODS: In this unblinded, open-label non-randomized trial, we treated 374 consecutive patients with treatment-refractory chronic ulcers (218 patients with DFU and 156 individuals with CVU) with a combination of THO and LEL. THO was administered by pumping 100% oxygen into a disposable, sealed polyethylene chamber for 2 h, two to three times weekly. LEL was administered concurrently using a helium-neon laser at 4 J/cm(2) for 20 min. RESULTS: Complete ulcer closure was obtained in 78% of patients in each group (170 patients with DFU and 127 patients with CVU). Treatment failure resulting in amputation in DFU was seen in 48 patients (22%); non-closure of ulcers within 18 months in individuals with CVU was seen in 29 (22%). The length of therapy was also similar in the two groups (3.7+/-3 versus 4.1+/-3 months in DFU and CVU cohorts, respectively). However, the number of treatments required to affect healing was greater in the CVU group than among the DFU patients (40+/-25 versus 31.4+/-20 treatments). CONCLUSION: THO and LEL therapies are safe, effective, simple and inexpensive therapies for DFU and CVU. Confirmation must await the performance of double-blind, randomized, controlled trials currently under way.

Isolated abdominal aortitis following administration of granulocyte colony stimulating factor (G-CSF).

G-CSF is a myeloid growth factor produced by monocytes, macrophages, fibroblasts, and endothelial cells. Clinical uses of G-CSF includes mobilization of peripheral blood progenitor cells from healthy donors before hematopoietic stem cell transplantation, acceleration of neutrophil recovery following chemotherapy, and in the management of neutropenia due to other causes including AIDS and genetic disorders of granulocyte production. G-CSF is well tolerated and reports to be safe in healthy donors, although follow-up studies are limited in duration (D'Souza et al. in Transfus Med Rev 22(4):280-290, 2008).Isolated abdominal aortitis (IAA) is a rare disorder most commonly caused by the large-vessel vasculitides giant cell arteritis (GCA) and Takayasu arteritis, although it may also be associated with several other rheumatologic diseases and infections (Gornik and Creager in Circulation 117:3039-3051, 2008). To our knowledge, there only two cases have been published of IAA occurring in patients who had received G-CSF therapy (Dariea et al. in Rev Med Interne 25(3):225-229, 2004; Adiga et al. in Clin Drug Investig 29:821-825, 2009).We describe a case of a 55-year-old male, with peripheral vascular disease who after receiving Neupogen (G-CSF) developed a latent case of IAA. After further investigation and exclusion of other possible causative factors, we conclude that the most probable etiology is induction by G-CSF.

Incidence of Profound Hypogammaglobulinemia and Infection Rate in Lymphoma Patients Following the Combination of Chemotherapy and Rituximab.

BACKGROUND: The Anti-CD20 monoclonal antibody Rituximab suppresses B-lymphocytes and may induce hypogammaglobulinemia in treated patients. The incidence and clinical significance of rituximab induced hypogammaglobulinemia in lymphoma patients is underestimated. METHODS: We retrospectively analyzed the rates of hypogammaglobulinemia, infection and infection-related mortality in 136 lymphoma patients who were treated with a combination of chemotherapy and rituximab. RESULTS: Rituximab given in more than 8 doses (OR 6.05, 95% CI: 1.24-29.5), relative hypogammaglobulinemia at time of lymphoma diagnosis (OR 4.2, 95% CI: 1.26-14.1) and the combination of fludarabine with rituximab (OR 3.4, 95% CI: 1.24-9.47) were factors significantly associated with prolonged (more than 6 months) hypogammaglobulinemia. The combination of fludarabine and rituximab (OR 6.4, 95% CI: 1.49-27.0) and secondarily prolonged hypogammaglobulinemia (OR 4.5, 95% CI: 1.19-18.5) were found to be predictive factors for severe infections and infection-related mortality. CONCLUSION: These data suggest the importance of following serum immunoglobulin levels before and after combination immuno-chemotherapy, particularly in patients with recurrent infections or relapsed/refractory disease.

Prediction of Protein-Ligand Binding Poses via a Combination of Induced Fit Docking and Metadynamics Simulations.

Ligand docking is a widely used tool for lead discovery and binding mode prediction based drug discovery. The greatest challenges in docking occur when the receptor significantly reorganizes upon small molecule binding, thereby requiring an induced fit docking (IFD) approach in which the receptor is allowed to move in order to bind to the ligand optimally. IFD methods have had some success but suffer from a lack of reliability. Complementing IFD with all-atom molecular dynamics (MD) is a straightforward solution in principle but not in practice due to the severe time scale limitations of MD. Here we introduce a metadynamics plus IFD strategy for accurate and reliable prediction of the structures of protein-ligand complexes at a practically useful computational cost. Our strategy allows treating this problem in full atomistic detail and in a computationally efficient manner and enhances the predictive power of IFD methods. We significantly increase the accuracy of the underlying IFD protocol across a large data set comprising 42 different ligand-receptor systems. We expect this approach to be of significant value in computationally driven drug design.

Recurrent herpes simplex encephalitis.

Recurrent herpes simplex virus (HSV) encephalitis is a rare disorder with only a few cases reported. We report a case of HSV encephalitis with documented recurrence in the same anatomic location, lending support to the theory of reactivation as the mechanism of disease.