Discovery of a Novel DCAF1 Ligand Using a Drug-Target Interaction Prediction Model: Generalizing Machine Learning to New Drug Targets.

DCAF1 functions as a substrate recruitment subunit for the RING-type CRL4DCAF1 and the HECT family EDVPDCAF1 E3 ubiquitin ligases. The WDR domain of DCAF1 serves as a binding platform for substrate proteins and is also targeted by HIV and SIV lentiviral adaptors to induce the ubiquitination and proteasomal degradation of antiviral host factors. It is therefore attractive both as a potential therapeutic target for the development of chemical inhibitors and as an E3 ligase that could be recruited by novel PROTACs for targeted protein degradation. In this study, we used a proteome-scale drug-target interaction prediction model, MatchMaker, combined with cheminformatics filtering and docking to identify ligands for the DCAF1 WDR domain. Biophysical screening and X-ray crystallographic studies of the predicted binders confirmed a selective ligand occupying the central cavity of the WDR domain. This study shows that artificial intelligence-enabled virtual screening methods can successfully be applied in the absence of previously known ligands.

Synthesis of Carvone Derivatives and In Silico and In Vitro Screening of Anti-Inflammatory Activity in Murine Macrophages.

The chemical modification of natural compounds is a promising strategy to improve their frequently poor bioavailability and low potency. This study aimed at synthesizing chemical derivatives of carvone, a natural monoterpene with anti-inflammatory properties, which we recently identified, and evaluating their potential anti-inflammatory activity. Fourteen chemical derivatives of carvone were synthesized, purified and their chemical structures confirmed. Noncytotoxic concentrations of the test compounds were selected based on the resazurin reduction assay. Among the tested compounds, four significantly reduced the lipopolysaccharides-induced protein levels of the inducible isoform of the nitric oxide synthase and nitric oxide production and showed a dual effect on pro-IL-1 protein levels in the Raw 264.7 cell line. The Ligand Express drug discovery platform was used to predict the targets of the test compounds, and an enrichment analysis was performed to group the different biological processes and molecular and cellular functions of the tested compounds. Moreover, Ligand Express also predicted that all chemicals evaluated have intestinal and blood-brain barrier permeability, do not inhibit P-gp and do not interact with major receptors. Although presenting anti-inflammatory and some advantageous ADME properties, the tested compounds still have low potency and specificity but may provide novel structures the further chemical modification of which may yield more promising drugs.

Immunotherapy with CD25/CD71-allodepleted T cells to improve T-cell reconstitution after matched unrelated donor hematopoietic stem cell transplant: a randomized trial.

BACKGROUND AIMS: Delayed immune reconstitution is a major challenge after matched unrelated donor (MUD) stem cell transplant (SCT). In this randomized phase 2 multi-center trial, Adoptive Immunotherapy with CD25/71 allodepleted donor T cells to improve immunity after unrelated donor stem cell transplant (NCT01827579), the authors tested whether allodepleted donor T cells (ADTs) can safely be used to improve immune reconstitution after alemtuzumab-based MUD SCT for hematological malignancies. METHODS: Patients received standard of care or up to three escalating doses of ADTs generated through CD25+/CD71+ immunomagnetic depletion. The primary endpoint of the study was circulating CD3+ T-cell count at 4 months post-SCT. Twenty-one patients were treated, 13 in the ADT arm and eight in the control arm. RESULTS: The authors observed a trend toward improved CD3+ T-cell count at 4 months in the ADT arm versus the control arm (230/µL versus 145/µL, P = 0.18), and three ADT patients achieved normal CD3+ T-cell count at 4 months (>700/µL). The rates of significant graft-versus-host disease (GVHD) were comparable in both cohorts, with grade ≥2 acute GVHD in seven of 13 and four of eight patients and chronic GVHD in three of 13 and three of eight patients in the ADT and control arms, respectively. CONCLUSIONS: These data suggest that adoptive transfer of ADTs is safe, but that in the MUD setting the benefit in terms of T-cell reconstitution is limited. This approach may be of more use in the context of more rigorous T-cell depletion.

Primary graft failure, but not relapse, may be identified by early chimerism following double cord blood unit transplantation.

Umbilical cord blood transplantation (UCBT) has increased access to potentially curative therapy for patients with life-threatening disorders of the bone marrow and immune system. The introduction of reduced intensity conditioning (RIC) regimens and double umbilical cord unit infusions (DUCBT) has broadened the applicability of UCBT to more frail or larger recipients. The kinetics of chimerism after RIC DUCBT and their clinical utility are poorly understood. The RIC CBT trial reported here sought to prospectively evaluate the role of lineage-specific chimerism after DUCBT in adult patients with hematologic malignancies in the United Kingdom. Fifty-eight patients with a median age of 52 years were recruited, with overall and progression-free survivals of 59% (95% confidence interval [CI], 45%-71%) and 52% (95% CI, 39%-64%), respectively, at 2 years. Nonrelapse mortality was 4% (95% CI, 1%-13%) at day 100, and the relapse rate was 31% (95% CI, 21%-45%) at 1 year. Peripheral blood lineage-specific chimerism was feasible from day 7 after transplant onward. Five patterns of chimerism were observed including (1) complete single unit dominance (39 patients), (2) sustained donor-donor mixed chimerism (3 patients), (3) sustained donor-recipient mixed chimerism (5 patients), (4) dominance reversion (1 patient), and (5) primary graft failure (4 patients). The RIC CBT trial enabled adult patients with high-risk hematologic malignancies to safely access UCBT in the United Kingdom and provided novel insights into the kinetics of donor and recipient chimerism after RIC DUCBT that are clinically relevant. This trial was registered at https://www.clinicaltrialsregister.eu/ctr-search/trial/2004-003845-41/GB as #NCT00959231 and EudraCT 2004-003845-41.

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease.

The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cell-based experimental assessment reveals several clinically-relevant drug repurposing candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

Proteome-Scale Drug-Target Interaction Predictions: Approaches and Applications.

Drug-Target interaction predictions are an important cornerstone of computer-aided drug discovery. While predictive methods around individual targets have a long history, the application of proteome-scale models is relatively recent. In this overview, we will provide the context required to understand advances in this emerging field within computational drug discovery, evaluate emerging technologies for suitability to given tasks, and provide guidelines for the design and implementation of new drug-target interaction prediction models. We will discuss the validation approaches used, and propose a set of key criteria that should be applied to evaluate their validity. We note that we find widespread deficiencies in the existing literature, making it difficult to judge the practical effectiveness of some of the techniques proposed from their publications alone. We hope that this review may help remedy this situation and increase awareness of several sources of bias that may enter into commonly used cross-validation methods. © 2021 Cyclica Inc. Current Protocols published by Wiley Periodicals LLC.

Predictors of recovery following allogeneic CD34+-selected cell infusion without conditioning to correct poor graft function.

Poor graft function is a serious complication following allogeneic hematopoietic stem cell transplantation. Infusion of CD34+-selected stem cells without pre-conditioning has been used to correct poor graft function, but predictors of recovery are unclear. We report the outcome of 62 consecutive patients who had primary or secondary poor graft function who underwent a CD34+-selected stem cell infusion from the same donor without further conditioning. Forty-seven of 62 patients showed hematological improvement and became permanently transfusion and growth factor-independent. In multivariate analysis, parameters significantly associated with recovery were shared CMV seronegative status for recipient/donor, the absence of active infection and matched recipient/donor sex. Recovery was similar in patients with mixed and full donor chimerism. Five -year overall survival was 74.4% (95% CI 59-89) in patients demonstrating complete recovery, 16.7% (95% CI 3-46) in patients with partial recovery and 22.2% (CI 95% 5-47) in patients with no response. In patients with count recovery, those with poor graft function in 1-2 lineages had superior 5-year overall survival (93.8%, 95% CI 82-99) than those with tri-lineage failure (53%, 95% CI 34-88). New strategies including cytokine or agonist support, or second transplant need to be investigated in patients who do not recover.

Presence of donor-encoded centromeric KIR B content increases the risk of infectious mortality in recipients of myeloablative, T-cell deplete, HLA-matched HCT to treat AML.

The reported influence of donor Killer-cell Immunoglobulin-like Receptor (KIR) genes on the outcomes of haematopoietic cell transplantation (HCT) are contradictory, in part due to diversity of disease, donor sources, era and conditioning regimens within and between different studies. Here, we describe the results of a retrospective clinical analysis establishing the effect of donor KIR motifs on the outcomes of 119 HLA-matched, unrelated donor HCT for adult acute myeloid leukaemia (AML) using myeloablative conditioning (MAC) in a predominantly T-cell deplete (TCD) cohort. We observed that HCT involving donors with at least one KIR B haplotype were more likely to result in non-relapse mortality (NRM) than HCT involving donors with two KIR A haplotypes (p = 0.019). Upon separation of KIR haplotypes into their centromeric (Cen) and telomeric (Tel) motif structures, we demonstrated that the Cen-B motif was largely responsible for this effect (p = 0.001). When the cause of NRM was investigated further, infection was the dominant cause of death (p = 0.006). No evidence correlating donor KIR B haplotype with relapse risk was observed. The results from this analysis confirm previous findings in the unrelated, TCD, MAC transplant setting and imply a protective role for donor-encoded Cen-A motifs against infection in allogeneic HCT recipients.

Allo-HSCT in transplant-naïve patients with Hodgkin lymphoma: a single-arm, multicenter study.

We evaluated the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in transplant-naïve patients with relapsed/refractory Hodgkin lymphoma (HL) who failed to attain metabolic complete response (mCR) to 1 to 2 lines of salvage chemotherapyThose with residual but nonprogressive disease assessed by positron emission tomography/computed tomography scanning were eligible. An additional 1 to 2 cycles of salvage therapy were permissible in those with progressive disease or when required to bridge to allo-HSCT, with additional imaging at baseline before transplantation. Conditioning consisted of carmustine, etoposide, cytarabine, melphalan, and alemtuzumab. Donor lymphocyte infusions (DLI) were administered for mixed chimerism or residual or relapsed disease. Eleven patients had sibling donors, 13 had HLA-matched unrelated donors, and 7 had HLA-mismatched unrelated donors. There were no graft failures, and no episodes of grade 4 acute graft-versus-host disease (GVHD); only 19.4% of patients had grade 2 to 3 GVHD, and 22.2% had extensive chronic GVHD. The non-relapse mortality rate was 16.1% (95% confidence interval [CI], 7.1%-34.5%). Relapse incidence was 18.7% (95% CI, 8.2%-39.2%). The study met its primary objective, with a 3-year progression-free survival of 67.7% (95% CI, 48.4%-81.2%). Survival outcomes were equivalent in those with residual metabolically active disease immediately before transplantation (n = 24 [70.8%; 95% CI, 17.2%-83.7%]). Two of the 5 patients who relapsed received DLI and remained in mCR at latest follow-up, with a 3-year overall survival of 80.7% (95% CI, 61.9%-90.8%). We demonstrate encouraging results that establish a potential role for allo-HSCT in selected high-risk patients with HL. This trial was registered at www.clinicaltrials.gov as #NCT00908180.

Recipients Receiving Better HLA-Matched Hematopoietic Cell Transplantation Grafts, Uncovered by a Novel HLA Typing Method, Have Superior Survival: A Retrospective Study.

HLA matching at an allelic-level resolution for volunteer unrelated donor (VUD) hematopoietic cell transplantation (HCT) results in improved survival and fewer post-transplant complications. Limitations in typing technologies used for the hyperpolymorphic HLA genes have meant that variations outside of the antigen recognition domain (ARD) have not been previously characterized in HCT. Our aim was to explore the extent of diversity outside of the ARD and determine the impact of this diversity on transplant outcome. Eight hundred ninety-one VUD-HCT donors and their recipients transplanted for a hematologic malignancy in the United Kingdom were retrospectively HLA typed at an ultra-high resolution (UHR) for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 using next-generation sequencing technology. Matching was determined at full gene level for HLA class I and at a coding DNA sequence level for HLA class II genes. The HLA matching status changed in 29.1% of pairs after UHR HLA typing. The 12/12 UHR HLA matched patients had significantly improved 5-year overall survival when compared with those believed to be 12/12 HLA matches based on their original HLA typing but were found to be mismatched after UHR HLA typing (54.8% versus 30.1%, P = .022). Survival was also significantly better in 12/12 UHR HLA-matched patients when compared with those with any degree of mismatch at this level of resolution (55.1% versus 40.1%, P = .005). This study shows that better HLA matching, found when typing is done at UHR that includes exons outside of the ARD, introns, and untranslated regions, can significantly improve outcomes for recipients of a VUD-HCT for a hematologic malignancy and should be prospectively performed at donor selection.

Hepatitis E infection in stem cell and solid organ transplantpatients: A cross-sectional study: The importance of HEV RNA screening in peri-transplant period.

BACKGROUND: Hepatitis E Virus (HEV) is a common cause of acute viral hepatitis worldwide. Typically associated with a self-limiting illness, infection may persist in immunosuppressed populations with significant morbidity and mortality. Based on clinical data published world-wide, UK blood safety guidance recommends the universal screening for HEV RNA of blood donors and donors of tissue, organs and stem cells. OBJECTIVES: This cross-sectional study aimed to determine the point prevalence of HEV viraemia and clinical course of viraemic patients in the peri-transplant period in solid organ transplant (SOT) and haematopoietic stem cell transplant (HSCT) recipients transplanted over a 3-year period (2013-2015). STUDY DESIGN: Nucleic acid extracts of whole blood from patients undergoing SOT or HSCT were tested by an in-house real-time reverse-transcriptase polymerase chain reaction assay for HEV RNA. Samples were tested at baseline (time of transplant), 30, 60 and 90 days post-transplant. RESULTS: 870 patients (259 HSCT, 262 liver and 349 kidney transplant) were included with 2554 samples meeting the inclusion criteria. No kidney transplant patients had HEV viraemia at time of testing. One HSCT and three liver transplant patients were found to be HEV RNA positive. Overall this represented 0.46% of the patients testing positive for HEV viraemia. CONCLUSIONS: Prevalence of HEV viraemia in SOT and HSCT patients in U.K. although higher than in the general population is low at baseline and remains low throughout the early post-transplant phase. Clearance of viraemia can be maintained despite ongoing immunosuppression. Prospective U.K. studies are necessary to inform screening policies in this population.

Interplay of self-association and conformational flexibility in regulating protein function.

Many functional roles have been attributed to homodimers, the most common mode of protein self-association, notably in the regulation of enzymes, ion channels, transporters and transcription factors. Here we review findings that offer new insights into the different roles conformational flexibility plays in regulating homodimer function. Intertwined homodimers of two-domain proteins and their related family members display significant conformational flexibility, which translates into concerted motion between structural domains. This flexibility enables the corresponding proteins to regulate function across family members by modulating the spatial positions of key recognition surfaces of individual domains, to either maintain subunit interfaces, alter them or break them altogether, leading to a variety of functional consequences. Many proteins may exist as monomers but carry out their biological function as homodimers or higher-order oligomers. We present early evidence that in such systems homodimer formation primes the protein for its functional role. It does so by inducing elevated mobility in protein regions corresponding to the binding epitopes of functionally important ligands. In some systems this process acts as an allosteric response elicited by the self-association reaction itself. Our analysis furthermore suggests that the induced extra mobility likely facilitates ligand binding through the mechanism of conformational selection.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

Comprehensive mapping of cystic fibrosis mutations to CFTR protein identifies mutation clusters and molecular docking predicts corrector binding site.

Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, of which over 2000 have been reported to date. Mutations have yet to be analyzed in aggregate to assess their distribution across the tertiary structure of the CFTR protein, an approach that could provide valuable insights into the structure-function relationship of CFTR. In addition, the binding site of Class I correctors (VX-809, VX-661, and C18) is not well understood. In this study, exonic CFTR mutations and mutant allele frequencies described in 3 curated databases (ABCMdb, CFTR1, and CFTR2, comprising >130 000 data points) were mapped to 2 different structural models: a homology model of full-length CFTR protein in the open-channel state, and a cryo-electron microscopy core-structure of CFTR in the closed-channel state. Accordingly, residue positions of 6 high-frequency mutant CFTR alleles were found to spatially co-localize in CFTR protein, and a significant cluster was identified at the NBD1:ICL4 interdomain interface. In addition, immunoblotting confirmed the approximate binding site of Class I correctors, demonstrating that these small molecules act via a similar mechanism in vitro, and in silico molecular docking generated binding poses for their complex with the cryo-electron microscopy structure to suggest the putative corrector binding site is a multi-domain pocket near residues F374-L375. These results confirm the significance of interdomain interfaces as susceptible to disruptive mutation, and identify a putative corrector binding site. The structural pharmacogenomics approach of mapping mutation databases to protein models shows promise for facilitating drug discovery and personalized medicine for monogenetic diseases.

The Antiretroviral Agent Nelfinavir Mesylate: A Potential Therapy for Systemic Sclerosis.

OBJECTIVE: Transforming growth factor ß1 (TGFß1) is considered a key factor in fibrogenesis, and blocking TGFß1 signaling pathways diminishes fibrogenesis in animal models. The objective of this study was to determine whether nelfinavir mesylate (NFV), a drug approved by the Food and Drug Administration (FDA) for treating HIV infection, could be repurposed to treat pulmonary fibrosis in patients with systemic sclerosis (SSc). METHODS: Normal human lung, ventricular, and skin fibroblasts as well as lung fibroblasts from SSc patients were used to determine the effects of NFV on fibroblast-to-myofibroblast differentiation mediated by TGFß1. The efficacy of NFV was also evaluated in an animal model of SSc (bleomycin-induced pulmonary fibrosis). In addition, in silico analysis was performed to determine novel off-target effects of NFV. RESULTS: NFV inhibited TGFß1-mediated fibroblast-to-myofibroblast differentiation in lung fibroblasts through inhibition of the TGFß1 canonical pathway. NFV also inhibited differentiation of skin and ventricular fibroblasts and adipocyte precursors into myofibroblasts. Activation of the TGFß1/mechanistic target of rapamycin pathway inhibited autophagy in lung fibroblasts, favoring collagen deposition, and NFV counteracted this effect in a dose-dependent manner. Moreover, NFV significantly reduced lung injury and collagen deposition in an animal model of SSc. In silico analysis of NFV binding proteins revealed new putative beneficial mechanisms of action, consistent with known common pathways in fibrogenesis. CONCLUSION: NFV abrogates TGFß1-mediated fibroblast-to-myofibroblast differentiation and pulmonary fibrosis through off-target protein binding, a finding that supports consideration of this FDA-approved medication as an antifibrotic agent.

Successful outcome following allogeneic hematopoietic stem cell transplantation in adults with primary immunodeficiency.

The primary immunodeficiencies (PIDs), rare inherited diseases characterized by severe dysfunction of immunity, have been successfully treated by allogeneic hematopoietic stem cell transplantation (Allo-HSCT) in childhood. Controversy exists regarding optimal timing and use of Allo-HSCT in adults, due to lack of experience and previous poor outcomes. Twenty-nine consecutive adult patients, with a mean age at transplant of 24 years (range, 17-50 years), underwent Allo-HSCT. Reduced-intensity conditioning (RIC) included fludarabine (Flu)/melphalan/alemtuzumab (n = 20), Flu/busulfan (Bu)/alemtuzumab (n = 8), and Flu/Bu/antithymocyte globulin (n = 1). Stem cell donors were matched unrelated donors or mismatched unrelated donors (n = 18) and matched related donors (n = 11). Overall survival (OS), event-free survival, transplant-related mortality (TRM), acute and chronic graft-versus-host disease incidence and severity, time to engraftment, lineage-specific chimerism, immune reconstitution, and discontinuation of immunoglobulin replacement therapy were recorded. OS at 3 years for the whole cohort was 85.2%. The rarer PID patients without chronic granulomatous disease (CGD) achieved an OS at 3 years of 88.9% (n = 18), compared with 81.8% for CGD patients (n = 11). TRM was low with only 4 deaths observed at a median follow-up of 3.5 years. There were no cases of early or late rejection. In all surviving patients, either stable mixed chimerism or full donor chimerism were observed. At last follow-up, 87% of the surviving patients had no evidence of persistent or recurrent infections. Allo-HSCT is safe and effective in young adult patients with severe PID and should be considered the treatment of choice where an appropriate donor is available.

Correction: Randomized Controlled Trials to Define Viral Load Thresholds for Cytomegalovirus Pre-Emptive Therapy.

[This corrects the article DOI: 10.1371/journal.pone.0163722.].