Computational Design of Novel Cyclic Peptides Endowed with Autophagy-Inhibiting Activity on Cancer Cell Lines.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

PI3Kδ activation, IL6 over-expression, and CD37 loss cause resistance to the targeting of CD37-positive lymphomas with the antibody-drug conjugate naratuximab emtansine.

Purpose: The transmembrane protein CD37 is expressed almost exclusively in lymphoid tissues, with the highest abundance in mature B cells. CD37-directed antibody- and, more recently, cellular-based approaches have shown preclinical and promising early clinical activity. Naratuximab emtansine (Debio 1562, IMGN529) is an antibodydrug conjugate (ADC) that incorporates an anti-CD37 monoclonal antibody conjugated to the maytansinoid DM1 as payload. Naratuximab emtansine has shown activity as a single agent and in combination with the anti-CD20 monoclonal antibody rituximab in B cell lymphoma patients. Experimental Design: We assessed the activity of naratuximab emtansine using in vitro models of lymphomas, correlated its activity with CD37 expression levels, characterized two resistance mechanisms to the ADC, and identified combination partners providing synergy. Results: The anti-tumor activity of naratuximab emtansine was tested in 54 lymphoma cell lines alongside its free payload. The median IC 50 of naratuximab emtansine was 780 pM, and the activity, primarily cytotoxic, was more potent in B than in T cell lymphoma cell lines. In the subgroup of cell lines derived from B cell lymphoma, there was some correlation between sensitivity to DM1 and sensitivity to naratuximab emtansine (r=0.28, P = 0.06). After prolonged exposure to the ADC, one diffuse large B cell lymphoma (DLBCL) cell line developed resistance to the ADC due to the biallelic loss of the CD37 gene. After CD37 loss, we also observed upregulation of IL6 (IL-6) and other transcripts from MYD88/IL6-signaling. Recombinant IL6 led to resistance to naratuximab emtansine, while the anti-IL6 antibody tocilizumab improved the cytotoxic activity of the ADC in CD37-positive cells. In a second model, resistance was sustained by an activating mutation in the PIK3CD gene, associated with increased sensitivity to PI3K δ inhibition and a switch from functional dependence on the anti-apoptotic protein MCL1 to reliance on BCL2. The addition of idelalisib or venetoclax to naratuximab emtansine overcame resistance to the ADC in the resistant derivative while also improving the cytotoxic activity of the ADC in the parental cells. Conclusions: Targeting B cell lymphoma with the CD37 targeting ADC naratuximab emtansine showed vigorous anti-tumor activity as a single agent, which was also observed in models bearing genetic lesions associated with inferior outcomes, such as MYC translocations and TP53 inactivation or resistance to R-CHOP. Resistance DLBCL models identified active combinations of naratuximab emtansine with drugs targeting IL6, PI3K δ , and BCL2. Despite notable progress in recent decades, we still face challenges in achieving a cure for a substantial number of lymphoma patients (1,2). A pertinent example is diffuse large B cell lymphoma (DLBCL), the most prevalent type of lymphoma (3). More than half of DLBCL patients can achieve remission, but around 40% of them experience refractory disease or relapse following an initial positive response (3). Regrettably, the prognosis for many of these cases remains unsatisfactory despite introducing the most recent antibody-based or cellular therapies (3,4), underscoring the importance of innovating new therapeutic strategies and gaining insights into the mechanisms of therapy resistance. CD37 is a transmembrane glycoprotein belonging to the tetraspanin family, primarily expressed on the surface of immune cells, principally in mature B cells but also, at lower levels, in T cells, macrophages/monocytes, granulocytes and dendritic cells (5) (6-8). CD37 plays a crucial role in various immune functions, including B cell activation, proliferation, and signaling, although its precise role still needs to be fully elucidated. CD37 interacts with multiple molecules, including SYK, LYN, CD19, CD22, PI3K δ , PI3K γ , and different integrins, among others (6-8). In mice, the lack of CD37 is paired with reduced T cell-dependent antibody-secreting cells and memory B cells, apparently due to the loss of CD37-mediated clustering of α 4 ß 1 integrins (VLA-4) on germinal center B cells and decreased downstream activation of PI3K/AKT signaling and cell survival (5). Reflecting the expression pattern observed in normal lymphocytes, CD37 exhibits elevated expression in all mature B-cell lymphoid neoplasms, including most lymphoma subtypes, and absence in early progenitor cells or terminally differentiated plasma cells (6,8-14). In DLBCL, CD37 expression has been reported between 40% and 90% of cases across multiple studies performed using different antibodies (10,14-16). CD37-directed antibody- and, more recently, cellular-based approaches have shown preclinical (7,10-14,17-23) and early promising clinical activity (24-32). Among the CD37-targeting agents, naratuximab emtansine (Debio 1562, IMGN529) is an antibody-drug conjugate (ADC) that incorporates the anti-CD37 humanized IgG1 monoclonal antibody K7153A conjugated to the maytansinoid DM1, as payload, via the thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (10). Based on the initial in vitro and in vivo evidence of anti-tumor activity in lymphoma and chronic lymphocytic leukemia (CLL) (7,10), naratuximab emtansine entered the clinical evaluation as a single agent. The phase 1 study exploring naratuximab emtansine enrolled 39 patients with relapsed/refractory B cell lymphoma (27). The overall response rate (ORR) was 13% across all patients and 22% in DLBCL patients, including the only observed complete remission (CR) (27). In preliminary results of a phase 2 trial exploring the combination of naratuximab emtansine with the anti-CD20 monoclonal antibody rituximab (18), based on positive preclinical data (18), the ORR was 45% in 76 patients with DLBCL with 24 CRs (32%), 57% in 14 patients with follicular lymphoma (five CR), 50% in four MCL patients (2 CR) (31). Here, we studied the pattern of activity of naratuximab emtansine across a large panel of cell lines derived from DLBCL and other lymphoma subtypes and characterized two resistance mechanisms to the ADC.

ETS1 phosphorylation at threonine 38 is associated with the cell of origin of diffuse large B cell lymphoma and sustains the growth of tumour cells.

The transcriptional factor ETS1 is upregulated in 25% of diffuse large B cell lymphoma (DLBCL). Here, we studied the role of ETS1 phosphorylation at threonine 38, a marker for ETS1 activation, in DLBCL cellular models and clinical specimens. p-ETS1 was detected in activated B cell-like DLBCL (ABC), not in germinal centre B-cell-like DLBCL (GCB) cell lines and, accordingly, it was more common in ABC than GCB DLBCL diagnostic biopsies. MEK inhibition decreased both baseline and IgM stimulation-induced p-ETS1 levels. Genetic inhibition of phosphorylation of ETS1 at threonine 38 affected the growth and the BCR-mediated transcriptome program in DLBCL cell lines. Our data demonstrate that ETS1 phosphorylation at threonine 38 is important for the growth of DLBCL cells and its pharmacological inhibition could benefit lymphoma patients.

Mechanistic Insights into the Antibiofilm Mode of Action of Ellagic Acid.

Bacterial biofilm is a major contributor to the persistence of infection and the limited efficacy of antibiotics. Antibiofilm molecules that interfere with the biofilm lifestyle offer a valuable tool in fighting bacterial pathogens. Ellagic acid (EA) is a natural polyphenol that has shown attractive antibiofilm properties. However, its precise antibiofilm mode of action remains unknown. Experimental evidence links the NADH:quinone oxidoreductase enzyme WrbA to biofilm formation, stress response, and pathogen virulence. Moreover, WrbA has demonstrated interactions with antibiofilm molecules, suggesting its role in redox and biofilm modulation. This work aims to provide mechanistic insights into the antibiofilm mode of action of EA utilizing computational studies, biophysical measurements, enzyme inhibition studies on WrbA, and biofilm and reactive oxygen species assays exploiting a WrbA-deprived mutant strain of Escherichia coli. Our research efforts led us to propose that the antibiofilm mode of action of EA stems from its ability to perturb the bacterial redox homeostasis driven by WrbA. These findings shed new light on the antibiofilm properties of EA and could lead to the development of more effective treatments for biofilm-related infections.

Tau protein binds to the P53 E3 ubiquitin ligase MDM2.

Tau gene mutations cause a progressive dementia and neurotoxic Tau forms deposited in neurofibrillary tangles are hallmarks of neurodegenerative tauopathies. Loss of non-canonical Tau functions may contribute to disease. In fact, Tau depletion affects the cellular response to DNA damage and tauopathies exhibit the accumulation of DNA lesions. Moreover, Tau modulates P53 activity and cell fate. Considering that MDM2 is the main antagonist of P53, we investigated, using orthogonal assays, if Tau interacts with MDM2. We report the existence in cells and brain of a Tau-MDM2 complex that, in vitro, exhibits reduced P53 ubiquitination activity in a manner sensitive to a Tau mutation. The Tau-MDM2 interaction involves the microtubule-binding domain of Tau and the acidic domain of MDM2, reminiscent of the binding of Tau to negatively charged microtubules. Notably, MDM2 accumulates aberrantly in neurofibrillary tangles. Aging-associated insults may expose a novel loss-of-function of Tau in neurodegeneration and cancer.

Esmethadone-HCl (REL-1017): a promising rapid antidepressant.

This review article presents select recent studies that form the basis for the development of esmethadone into a potential new drug. Esmethadone is a promising member of the pharmacological class of uncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonists that have shown efficacy for major depressive disorder (MDD) and other diseases and disorders, such as Alzheimer's dementia and pseudobulbar affect. The other drugs in the novel class of NMDAR antagonists with therapeutic uses that are discussed for comparative purposes in this review are esketamine, ketamine, dextromethorphan, and memantine. We present in silico, in vitro, in vivo, and clinical data for esmethadone and other uncompetitive NMDAR antagonists that may advance our understanding of the role of these receptors in neural plasticity in health and disease. The efficacy of NMDAR antagonists as rapid antidepressants may advance our understanding of the neurobiology of MDD and other neuropsychiatric diseases and disorders.

Autoantibodies against chemokines post-SARS-CoV-2 infection correlate with disease course.

Infection with severe acute respiratory syndrome coronavirus 2 associates with diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse coronavirus disease 2019 (COVID-19) outcomes. Here we discovered that antibodies against specific chemokines were omnipresent post-COVID-19, were associated with favorable disease outcome and negatively correlated with the development of long COVID at 1 yr post-infection. Chemokine antibodies were also present in HIV-1 infection and autoimmune disorders, but they targeted different chemokines compared with COVID-19. Monoclonal antibodies derived from COVID-19 convalescents that bound to the chemokine N-loop impaired cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising chemokine antibodies may modulate the inflammatory response and thus bear therapeutic potential.

Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein.

Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization, and, similar to fp.006 and hr2.016, protects mice expressing human angiotensin-converting enzyme 2 against infection when present as a bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.

CEMIP (HYBID, KIAA1199): structure, function and expression in health and disease.

CEMIP (cell migration-inducing protein), also known as KIAA1199 or HYBID, is a protein involved in the depolymerisation of hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix. CEMIP was originally described in patients affected by nonsyndromic hearing loss and has subsequently been shown to play a key role in tumour initiation and progression, as well as arthritis, atherosclerosis and idiopathic pulmonary fibrosis. Despite the vast literature associating CEMIP with these diseases, its biology remains elusive. The present review article summarises all the major scientific evidence regarding its structure, function, role and expression, and attempts to cast light on a protein that modulates EMT, fibrosis and tissue inflammation, an unmet key aspect in several inflammatory disease conditions.

A monoclonal antibody targeting nonjunctional claudin-1 inhibits fibrosis in patient-derived models by modulating cell plasticity.

Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2.

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera , including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae , including SARS-CoV-2 variants. One sentence summary: Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

Pharmacological Comparative Characterization of REL-1017 (Esmethadone-HCl) and Other NMDAR Channel Blockers in Human Heterodimeric N-Methyl-D-Aspartate Receptors.

Excessive Ca2+ currents via N-methyl-D-aspartate receptors (NMDARs) have been implicated in many disorders. Uncompetitive NMDAR channel blockers are an emerging class of drugs in clinical use for major depressive disorder (MDD) and other neuropsychiatric diseases. The pharmacological characterization of uncompetitive NMDAR blockers in clinical use may improve our understanding of NMDAR function in physiology and pathology. REL-1017 (esmethadone-HCl), a novel uncompetitive NMDAR channel blocker in Phase 3 trials for the treatment of MDD, was characterized together with dextromethorphan, memantine, (±)-ketamine, and MK-801 in cell lines over-expressing NMDAR subtypes using fluorometric imaging plate reader (FLIPR), automated patch-clamp, and manual patch-clamp electrophysiology. In the absence of Mg2+, NMDAR subtypes NR1-2D were most sensitive to low, sub-µM glutamate concentrations in FLIPR experiments. FLIPR Ca2+ determination demonstrated low µM affinity of REL-1017 at NMDARs with minimal subtype preference. In automated and manual patch-clamp electrophysiological experiments, REL-1017 exhibited preference for the NR1-2D NMDAR subtype in the presence of 1 mM Mg2+ and 1 µM L-glutamate. Tau off and trapping characteristics were similar for (±)-ketamine and REL-1017. Results of radioligand binding assays in rat cortical neurons correlated with the estimated affinities obtained in FLIPR assays and in automated and manual patch-clamp assays. In silico studies of NMDARs in closed and open conformation indicate that REL-1017 has a higher preference for docking and undocking the open-channel conformation compared to ketamine. In conclusion, the pharmacological characteristics of REL-1017 at NMDARs, including relatively low affinity at the NMDAR, NR1-2D subtype preference in the presence of 1 mM Mg2+, tau off and degree of trapping similar to (±)-ketamine, and preferential docking and undocking of the open NMDAR, could all be important variables for understanding the rapid-onset antidepressant effects of REL-1017 without psychotomimetic side effects.

Anti-chemokine antibodies after SARS-CoV-2 infection correlate with favorable disease course.

Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 infection and autoimmune disorders, but they target different chemokines than those in COVID-19. Monoclonal antibodies derived from COVID- 19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID-19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential. One-Sentence Summary: Naturally arising anti-chemokine antibodies associate with favorable COVID-19 and predict lack of long COVID.

Focused Design of Novel Cyclic Peptides Endowed with GABARAP-Inhibiting Activity.

(1) Background: Disfunctions in autophagy machinery have been identified in various conditions, including neurodegenerative diseases, cancer, and inflammation. Among mammalian autophagy proteins, the Atg8 family member GABARAP has been shown to be greatly involved in the autophagy process of prostate cancer cells, supporting the idea that GABARAP inhibitors could be valuable tools to fight the progression of tumors. (2) Methods: In this paper, starting from the X-ray crystal structure of GABARAP in a complex with an AnkirinB-LIR domain, we identify two new peptides by applying in silico drug design techniques. The two ligands are synthesized, biophysically assayed, and biologically evaluated to ascertain their potential anticancer profile. (3) Results: Two cyclic peptides (WC8 and WC10) displayed promising biological activity, high conformational stability (due to the presence of disulfide bridges), and Kd values in the low micromolar range. The anticancer assays, performed on PC-3 cells, proved that both peptides exhibit antiproliferative effects comparable to those of peptide K1, a known GABARAP inhibitor. (4) Conclusions: WC8 and WC10 can be considered new GABARAP inhibitors to be employed as pharmacological tools or even templates for the rational design of new small molecules.

Systematic Development of Peptide Inhibitors Targeting the CXCL12/HMGB1 Interaction.

During inflammatory reactions, the production and release of chemotactic factors guide the recruitment of selective leukocyte subpopulations. The alarmin HMGB1 and the chemokine CXCL12, both released in the microenvironment, can form a heterocomplex, which exclusively acts on the chemokine receptor CXCR4, enhancing cell migration, and in some pathological conditions such as rheumatoid arthritis exacerbates the immune response. An excessive cell influx at the inflammatory site can be diminished by disrupting the heterocomplex. Here, we report the computationally driven identification of the first peptide (HBP08) binding HMGB1 and selectively inhibiting the activity of the CXCL12/HMGB1 heterocomplex. Furthermore, HBP08 binds HMGB1 with the highest affinity reported so far (Kd of 0.8 ± 0.4 µM). The identification of this peptide represents an important step toward the development of innovative pharmacological tools for the treatment of severe chronic inflammatory conditions characterized by an uncontrolled immune response.

Compounds targeting OSBPL7 increase ABCA1-dependent cholesterol efflux preserving kidney function in two models of kidney disease.

Impaired cellular cholesterol efflux is a key factor in the progression of renal, cardiovascular, and autoimmune diseases. Here we describe a class of 5-arylnicotinamide compounds, identified through phenotypic drug discovery, that upregulate ABCA1-dependent cholesterol efflux by targeting Oxysterol Binding Protein Like 7 (OSBPL7). OSBPL7 was identified as the molecular target of these compounds through a chemical biology approach, employing a photoactivatable 5-arylnicotinamide derivative in a cellular cross-linking/immunoprecipitation assay. Further evaluation of two compounds (Cpd A and Cpd G) showed that they induced ABCA1 and cholesterol efflux from podocytes in vitro and normalized proteinuria and prevented renal function decline in mouse models of proteinuric kidney disease: Adriamycin-induced nephropathy and Alport Syndrome. In conclusion, we show that small molecule drugs targeting OSBPL7 reveal an alternative mechanism to upregulate ABCA1, and may represent a promising new therapeutic strategy for the treatment of renal diseases and other disorders of cellular cholesterol homeostasis.

EZH2-induced lysine K362 methylation enhances TMPRSS2-ERG oncogenic activity in prostate cancer.

The TMPRSS2-ERG gene fusion is the most frequent alteration observed in human prostate cancer. However, its role in disease progression is still unclear. In this study, we uncover an important mechanism promoting ERG oncogenic activity. We show that ERG is methylated by Enhancer of zest homolog 2 (EZH2) at a specific lysine residue (K362) located within the internal auto-inhibitory domain. Mechanistically, K362 methylation modifies intra-domain interactions, favors DNA binding and enhances ERG transcriptional activity. In a genetically engineered mouse model of ERG fusion-positive prostate cancer (Pb-Cre4 Pten flox/flox Rosa26-ERG, ERG/PTEN), ERG K362 methylation is associated with PTEN loss and progression to invasive adenocarcinomas. In both ERG positive VCaP cells and ERG/PTEN mice, PTEN loss results in AKT activation and EZH2 phosphorylation at serine 21 that favors ERG methylation. We find that ERG and EZH2 interact and co-occupy several sites in the genome forming trans-activating complexes. Consistently, ERG/EZH2 co-regulated target genes are deregulated preferentially in tumors with concomitant ERG gain and PTEN loss and in castration-resistant prostate cancers. Collectively, these findings identify ERG methylation as a post-translational modification sustaining disease progression in ERG-positive prostate cancers.

Machine learning analyses of antibody somatic mutations predict immunoglobulin light chain toxicity.

In systemic light chain amyloidosis (AL), pathogenic monoclonal immunoglobulin light chains (LC) form toxic aggregates and amyloid fibrils in target organs. Prompt diagnosis is crucial to avoid permanent organ damage, but delayed diagnosis is common because symptoms usually appear only after strong organ involvement. Here we present LICTOR, a machine learning approach predicting LC toxicity in AL, based on the distribution of somatic mutations acquired during clonal selection. LICTOR achieves a specificity and a sensitivity of 0.82 and 0.76, respectively, with an area under the receiver operating characteristic curve (AUC) of 0.87. Tested on an independent set of 12 LCs sequences with known clinical phenotypes, LICTOR achieves a prediction accuracy of 83%. Furthermore, we are able to abolish the toxic phenotype of an LC by in silico reverting two germline-specific somatic mutations identified by LICTOR, and by experimentally assessing the loss of in vivo toxicity in a Caenorhabditis elegans model. Therefore, LICTOR represents a promising strategy for AL diagnosis and reducing high mortality rates in AL.

Computational Identification of a Putative Allosteric Binding Pocket in TMPRSS2.

Camostat, nafamostat, and bromhexine are inhibitors of the transmembrane serine protease TMPRSS2. The inhibition of TMPRSS2 has been shown to prevent the viral infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses. However, while camostat and nafamostat inhibit TMPRSS2 by forming a covalent adduct, the mode of action of bromhexine remains unclear. TMPRSS2 is autocatalytically activated from its inactive form, zymogen, through a proteolytic cleavage that promotes the binding of Ile256 to a putative allosteric pocket (A-pocket). Computer simulations, reported here, indicate that Ile256 binding induces a conformational change in the catalytic site, thus providing the atomistic rationale to the activation process of the enzyme. Furthermore, computational docking and molecular dynamics simulations indicate that bromhexine competes with the N-terminal Ile256 for the same binding site, making it a potential allosteric inhibitor. Taken together, these findings provide the atomistic basis for the development of more selective and potent TMPRSS2 inhibitors.

Exploring Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Autoproteolysis Process by Molecular Simulations: Hints for Drug Design.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a notable target for the treatment of hypercholesterolemia because it regulates the population of the low-density lipoprotein receptor (LDLR) on liver cells. The PCSK9 zymogen is a serine protease that spontaneously undergoes a double self-cleavage step. Available X-ray structures depict the PCSK9 mature state, but the atomic details of the zymogen state of the enzyme are still unknown. Additionally, why the protease activity of PCSK9 is blocked after the second autoprocessing step remains unclear, as this deviates from other members of the PCSK family. By performing constant-pH molecular dynamics (MD) simulations, we investigated the protonation state of the catalytic triad of PCSK9 and found that it strongly influences the catalytic properties of the enzyme. Moreover, we determined the final step of the maturation process by classical and steered MD simulations. This study could facilitate the identification of ligands capable of interfering with the PCSK9 maturation process.