New Class of Benzodiazepinone Derivatives as Pro-Death Agents Targeting BIR Domains in Cancer Cells.

Inhibitor of Apoptosis Proteins (IAPs) are validated targets for cancer therapy, and the deregulation of their activities within the NF-κB pathway correlates with chemoresistance events, even after treatment with IAPs-antagonists in the clinic (Smac-mimetics). The molecule FC2 was identified as a NF-κB pathway modulator in MDA-MB-231 adenocarcinoma cancer cells after virtual screening of the Chembridge library against the Baculoviral IAP Repeat 1 (BIR1) domain of cIAP2 and XIAP. An improved cytotoxic effect is observed when FC2 is combined with Smac-mimetics or with the cytokine Tumor Necrosis Factor (TNF). Here, we propose a library of 22 derivatives of FC2, whose scaffold was rationally modified starting from the position identified as R1. The cytotoxic effect of FC2 derivatives was evaluated in MDA-MB-231 and binding to the cIAP2- and XIAP-BIR1 domains was assessed in fluorescence-based techniques and virtual docking. Among 22 derivatives, 4m and 4p display improved efficacy/potency in MDA-MB-231 cells and low micromolar binding affinity vs the target proteins. Two additional candidates (4b and 4u) display promising cytotoxic effects in combination with TNF, suggesting the connection between this class of molecules and the NF-κB pathway. These results provide the rationale for further FC2 modifications and the design of novel IAP-targeting candidates supporting known therapies.

Gelsolin pathogenic Gly167Arg mutation promotes domain-swap dimerization of the protein.

AGel amyloidosis is a genetic degenerative disease characterized by the deposition of insoluble gelsolin protein aggregates in different tissues. Until recently, this disease was associated with two mutations of a single residue (Asp187 to Asn/Tyr) in the second domain of the protein. The general opinion is that pathogenic variants are not per se amyloidogenic but rather that the mutations trigger an aberrant proteolytic cascade, which results in the production of aggregation prone fragments. Here, we report the crystal structure of the second domain of gelsolin carrying the recently identified Gly167Arg mutation. This mutant dimerizes through a three-dimensional domain swapping mechanism, forming a tight but flexible assembly, which retains the structural topology of the monomer. To date, such dramatic conformational changes of this type have not been observed. Structural and biophysical characterizations reveal that the Gly167Arg mutation alone is responsible for the monomer to dimer transition and that, even in the context of the full-length protein, the pathogenic variant is prone to form dimers. These data suggest that, in addition to the well-known proteolytic-dependent mechanism, an alternative oligomerization pathway may participate in gelsolin misfolding and aggregation. We propose to integrate this alternative pathway into the current model of the disease that may also be relevant for other types of AGel amyloidosis, and other related diseases with similar underlying pathological mechanisms.

The structure of N184K amyloidogenic variant of gelsolin highlights the role of the H-bond network for protein stability and aggregation properties.

Mutations in the gelsolin protein are responsible for a rare conformational disease known as AGel amyloidosis. Four of these mutations are hosted by the second domain of the protein (G2): D187N/Y, G167R and N184K. The impact of the latter has been so far evaluated only by studies on the isolated G2. Here we report the characterization of full-length gelsolin carrying the N184K mutation and compare the findings with those obtained on the wild type and the other variants. The crystallographic structure of the N184K variant in the Ca2+-free conformation shows remarkable similarities with the wild type protein. Only minimal local rearrangements can be observed and the mutant is as efficient as the wild type in severing filamentous actin. However, the thermal stability of the pathological variant is compromised in the Ca2+-free conditions. These data suggest that the N to K substitution causes a local disruption of the H-bond network in the core of the G2 domain. Such a subtle rearrangement of the connections does not lead to significant conformational changes but severely affects the stability of the protein.

Computational and Experimental Characterization of NF023, A Candidate Anticancer Compound Inhibiting cIAP2/TRAF2 Assembly.

Protein-protein interactions are the basis of many important physiological processes and are currently promising, yet difficult, targets for drug discovery. In this context, inhibitor of apoptosis proteins (IAPs)-mediated interactions are pivotal for cancer cell survival; the interaction of the BIR1 domain of cIAP2 with TRAF2 was shown to lead the recruitment of cIAPs to the TNF receptor, promoting the activation of the NF-κB survival pathway. In this work, using a combined in silico-in vitro approach, we identified a drug-like molecule, NF023, able to disrupt cIAP2 interaction with TRAF2. We demonstrated in vitro its ability to interfere with the assembly of the cIAP2-BIR1/TRAF2 complex and performed a thorough characterization of the compound's mode of action through 248 parallel unbiased molecular dynamics simulations of 300 ns (totaling almost 75 µs of all-atom sampling), which identified multiple binding modes to the BIR1 domain of cIAP2 via clustering and ensemble docking. NF023 is, thus, a promising protein-protein interaction disruptor, representing a starting point to develop modulators of NF-κB-mediated cell survival in cancer. This study represents a model procedure that shows the use of large-scale molecular dynamics methods to typify promiscuous interactors.

Structural and functional characterization of TgpA, a critical protein for the viability of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen associated with severe diseases, such as cystic fibrosis. During an extensive search for novel essential genes, we identified tgpA (locus PA2873) in P. aeruginosa PAO1, as a gene playing a critical role in bacterial viability. TgpA, the translated protein, is an internal membrane protein with a periplasmic soluble domain, predicted to be endowed with a transglutaminase-like fold, hosting the Cys404, His448, and Asp464 triad. We report here that Cys404 mutation hampers the essential role of TgpA in granting P. aeruginosa viability. Moreover, we present the crystal structure of the TgpA periplasmic domain at 1.6 Šresolution as a first step towards structure-activity analysis of a new potential target for the discovery of antibacterial compounds.

High-resolution crystal structure of gelsolin domain 2 in complex with the physiological calcium ion.

The second domain of gelsolin (G2) hosts mutations responsible for a hereditary form of amyloidosis. The active form of gelsolin is Ca2+-bound; it is also a dynamic protein, hence structural biologists often rely on the study of the isolated G2. However, the wild type G2 structure that have been used so far in comparative studies is bound to a crystallographic Cd2+, in lieu of the physiological calcium. Here, we report the wild type structure of G2 in complex with Ca2+ highlighting subtle ion-dependent differences. Previous findings on different G2 mutations are also briefly revised in light of these results.

Identification of a Small Molecule That Compromises the Structural Integrity of Viroplasms and Rotavirus Double-Layered Particles.

Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries, causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a nonenveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as an RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and to hamper the formation of new ones without the need for de novo transcription of cellular RNAs. This phenotype was correlated with a reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5, and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed dose-dependent inhibitory activity, indicating the viral nature of its target. ML was found to interfere with the formation of higher-order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential, not only for DLP stability, but also for the structural integrity of viroplasms in infected cells.IMPORTANCE Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in the countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified that is able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point in the development of more potent and less cytotoxic compounds against rotavirus infection.

A Single Nucleoside Viral Polymerase Inhibitor Against Norovirus, Rotavirus, and Sapovirus-Induced Diarrhea.

A safe and highly efficient antiviral is needed for the prophylaxis and/or treatment of viral diarrhea. We here demonstrate the in vitro antiviral activity of four 2'-C-methyl nucleoside analogues against noro-, rota-, and sapoviruses. The most potent nucleoside analogue, 7-deaza-2'-C-methyladenosine, inhibits replication of these viruses with a 50% effective concentration < 5 µM. Mechanistically, we demonstrate that the 2'-C-methyl nucleoside analogues act by inhibiting transcription of the rotavirus genome. This provides the first evidence that a single viral-diarrhea-targeted treatment can be developed through a viral-polymerase-targeting small molecule.

Structural bases of the altered catalytic properties of a pathogenic variant of apoptosis inducing factor.

The apoptosis-inducing factor (AIF) is a FAD-containing protein playing critical roles in caspase-independent apoptosis and mitochondrial respiratory chain biogenesis and maintenance. While its lethal role is well known, the details of its mitochondrial function remain elusive. So far, nineteen allelic variants of AIF have been associated to human diseases, mainly affecting the nervous system. A strict correlation is emerging between the degree of impairment of its ability to stabilize the charge-transfer (CT) complex between FAD and NAD+ and the severity of the resulting pathology. Recently, we demonstrated that the G307E replacement in murine AIF (equivalent to the pathogenic G308E in the human protein) dramatically decreases the rate of CT complex formation through the destabilization of the flavoprotein interaction with NAD(H). To provide further insights into the structural bases of its altered functional properties, here we report the first crystal structure of an AIF pathogenic mutant variant in complex with NAD+ (murine AIF-G307ECT) in comparison with its oxidized form. With respect to wild type AIF, the mutation leads to an altered positioning of NAD+ adenylate moiety, which slows down CT complex formation. Moreover, the altered balance between the binding of the adenine/nicotinamide portions of the coenzyme determines a large drop in AIF-G307E ability to discriminate between NADH and NADPH.

The activator of apoptosis Smac-DIABLO acts as a tetramer in solution.

Smac-DIABLO in its mature form (20.8 kDa) binds to baculoviral IAP repeat (BIR) domains of inhibitor of apoptosis proteins (IAPs) releasing their inhibitory effects on caspases, thus promoting cell death. Despite its apparent molecular mass (∼100 kDa), Smac-DIABLO was held to be a dimer in solution, simultaneously targeting two distinct BIR domains. We report an extensive biophysical characterization of the protein alone and in complex with the X-linked IAP (XIAP)-BIR2-BIR3 domains. Our data show that Smac-DIABLO adopts a tetrameric assembly in solution and that the tetramer is able to bind two BIR2-BIR3 pairs of domains. Our small-angle x-ray scattering-based tetrameric model of Smac-DIABLO/BIR2-BIR3 highlights some conformational freedom of the complex that may be related to optimization of IAPs binding.

NF023 binding to XIAP-BIR1: searching drugs for regulation of the NF-κB pathway.

Inhibitor of Apoptosis Proteins (IAPs) are the target of extensive research in the field of cancer therapy since they regulate apoptosis and cell survival. Smac-mimetics, the most promising IAP-targeting compounds specifically recognize the IAP-BIR3 domain and promote apoptosis, competing with caspases for IAP binding. Furthermore, Smac-mimetics interfere with the NF-κB survival pathway, inducing cIAP1 and cIAP2 degradation through an auto-ubiquitination process. It has been shown that the XIAP-BIR1 (X-BIR1) domain is involved in the interaction with TAB1, an upstream adaptor for TAK1 kinase activation, which in turn couples with the NF-κB survival pathway. Preventing X-BIR1 dimerization abolishes XIAP-mediated NF-κB activation, thus implicating a proximity-induced mechanism for TAK1 activation. In this context, in a systematic search for a molecule capable of impairing X-BIR1/TAB1 assembly, we identified the compound NF023. Here we report the crystal structure of the human X-BIR1 domain in the absence and in the presence of NF023, as a starting concept for the design of novel BIR1-specific compounds acting synergistically with existing pro-apoptotic drugs in cancer therapy.

Stabilization of the retromer complex: Analysis of novel binding sites of bis-1,3-phenyl guanylhydrazone 2a to the VPS29/VPS35 interface.

The stabilization of the retromer protein complex can be effective in the treatment of different neurological disorders. Following the identification of bis-1,3-phenyl guanylhydrazone 2a as an effective new compound for the treatment of amyotrophic lateral sclerosis, in this work we analyze the possible binding sites of this molecule to the VPS35/VPS29 dimer of the retromer complex. Our results show that the affinity for different sites of the protein assembly depends on compound charge and therefore slight changes in the cell microenvironment could promote different binding states. Finally, we describe a novel binding site located in a deep cleft between VPS29 and VPS35 that should be further explored to select novel molecular chaperones for the stabilization of the retromer complex.

CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation.

Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.

Head or tail? A molecular dynamics approach to the complex structure of TNF-associated factor TRAF2.

Tumor necrosis factor receptor-associated factor proteins (TRAFs) are trimeric proteins that play a fundamental role in signaling, acting as intermediaries between the tumor necrosis factor (TNF) receptors and the proteins that transmit the downstream signal. The monomeric subunits of all the TRAF family members share a common tridimensional structure: a C-terminal globular domain and a long coiled-coil tail characterizing the N-terminal section. In this study, the dependence of the TRAF2 dynamics on the length of its tail was analyzed in silico. In particular, we used the available crystallographic structure of a C-terminal fragment of TRAF2 (168 out of 501 a.a.), TRAF2-C, and that of a longer construct, addressed as TRAF2-plus, that we have re-constructed using the AlphaFold2 code. The results indicate that the longer N-terminal tail of TRAF2-plus has a strong influence on the dynamics of the globular regions in the protein C-terminal head. In fact, the quaternary interactions among the TRAF2-C subunits change asymmetrically in time, while the movements of TRAF2-plus monomers are rather limited and more ordered than those of the shorter construct. Such findings shed a new light on the dynamics of TRAF subunits and on the protein mechanism in vivo, since TRAF monomer-trimer equilibrium is crucial for several reasons (receptor recognition, membrane binding, hetero-oligomerization).

Flaviviral helicase: insights into the mechanism of action of a motor protein.

Motor proteins are involved in crucial cell activities, such as cargo transport or nucleic acid remodeling, by converting the free energy of ATP hydrolysis into motion or mechanical work. Flavivirus helicase is a motor protein involved in dsRNA separation during viral replication, thus essential for virus infection. Since a clear vision of the protein activity, in particular of the relationship between ATP cycling and dynamics, is missing, we carried over a molecular dynamics study on Dengue virus helicase in its ATP bound and unbound states. Our simulations show different opening levels of the ssRNA access site to the helicase core. Specifically, we show that ATP induces a closed state into the ssRNA access site, likely involved in the helicase unwinding activity.

Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug.

OBJECTIVES: Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed. METHODS: Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small molecules allowed us to identify a few selected compounds with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymatic assays. The effect on the in vitro replication of flaviviruses was then evaluated. RESULTS: Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC50 values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase. CONCLUSIONS: The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogues exerting potent activity against flaviviruses other than YFV.

Rational design, synthesis and characterization of potent, drug-like monomeric Smac mimetics as pro-apoptotic anticancer agents.

A set of phenyl-substituted Smac mimetics/IAP inhibitor analogues of lead compound 2a was synthesized, aiming to retain its strong cell-free potency while increasing its bioavailability. Seventeen compounds 2b-r were prepared and characterized in vitro, using cell-free and cellular assays. Among them, the p-CF(3) substituted analogue 2m showed the best permeability through cell membranes, and was selected for further in vitro and in vivo studies due to its strong, sub-micromolar cellular potency.

Homo- and heterodimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part I: Synthesis.

Novel pro-apoptotic, homo- and heterodimeric Smac mimetics/IAPs inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold were prepared from monomeric structures connected through a head-head (8), tail-tail (9) or head-tail (10) linker. The selection of appropriate decorating functions for the scaffolds, and of rigid and flexible linkers connecting them, is described. The synthesis, purification and analytical characterization of each prepared dimer 8-10 is thoroughly described.

Dimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part II: Structural and biological characterization.

Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head-head (8), tail-tail (9) or head-tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2-BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail-tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.

Virucidal Activity of the Pyridobenzothiazolone Derivative HeE1-17Y against Enveloped RNA Viruses.

Pyridobenzothiazolone derivatives are a promising class of broad-spectrum antivirals. However, the mode of action of these compounds remains poorly understood. The HeE1-17Y derivative has already been shown to be a potent compound against a variety of flaviviruses of global relevance. In this work, the mode of action of HeE1-17Y has been studied for West Nile virus taking advantage of reporter replication particles (RRPs). Viral infectivity was drastically reduced by incubating the compound with the virus before infection, thus suggesting a direct interaction with the viral particles. Indeed, RRPs incubated with the inhibitor appeared to be severely compromised in electron microscopy analysis. HeE1-17Y is active against other enveloped viruses, including SARS-CoV-2, but not against two non-enveloped viruses, suggesting a virucidal mechanism that involves the alteration of the viral membrane.