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While apolipoprotein E (APOE) is the strongest genetic modifier for late-onset Alzheimer's disease (LOAD), the molecular mechanisms underlying isoform-dependent risk and the relevance of ApoE-associated lipids remain elusive. Here, we report that impaired low-density lipoprotein (LDL) receptor (LDLR) binding of lipidated ApoE2 (lipApoE2) avoids LDLR recycling defects observed with lipApoE3/E4 and decreases the uptake of cholesteryl esters (CEs), which are lipids linked to neurodegeneration. In human neurons, the addition of ApoE carrying polyunsaturated fatty acids (PUFAs)-CE revealed an allelic series (ApoE4 > ApoE3 > ApoE2) associated with lipofuscinosis, an age-related lysosomal pathology resulting from lipid peroxidation. Lipofuscin increased lysosomal accumulation of tau fibrils and was elevated in the APOE4 mouse brain with exacerbation by tau pathology. Intrahippocampal injection of PUFA-CE-lipApoE4 was sufficient to induce lipofuscinosis in wild-type mice. Finally, the protective Christchurch mutation also reduced LDLR binding and phenocopied ApoE2. Collectively, our data strongly suggest decreased lipApoE-LDLR interactions minimize LOAD risk by reducing the deleterious effects of endolysosomal targeting of ApoE and associated pathogenic lipids., Competing Interests: Declaration of interests All authors, except N.N., A.L., and D.M.H., are full-time employees and/or shareholders of Denali Therapeutics. J.L.G., D.B., G.A.F., Y.-T.H., M. Steffek, and S.J.H. are currently full-time employees and shareholders of NICO Therapeutics. D.M.H. is on the scientific advisory board of Denali Therapeutics, Genentech, Cajal Neuroscience, C2N Diagnostics, and Cell. D.M.H. consults for Asteroid., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an ∼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis -isomer ( 2 ) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27 , which demonstrated measurable cell activity, albeit only at high concentrations., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance., (© 2023. The Author(s).)

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.

Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRAS G12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRAS G12C covalent modification in vivo and can be used to investigate response heterogeneity to KRAS G12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRAS G12C switch II region (SWII) by stabilizing a specific conformation of KRAS G12C , thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model., Competing Interests: Declaration of Interests All of the authors were employees of and shareholders in Roche at the time of this work. Patent WO2020051099 is affiliated with this work., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

LpxD, acyl-ACP-dependent N -acyltransferase, is the third enzyme of lipid A biosynthesis in Gram-negative bacteria. A recent probe-based screen identified several compounds, including 6359-0284 (compound 1 ), that inhibit the enzymatic activity of Escherichia coli ( E. coli ) LpxD. Here, we use these inhibitors to chemically validate LpxD as an attractive antibacterial target. We first found that compound 1 was oxidized in solution to the more stable aromatized tetrahydro-pyrazolo-quinolinone compound 1o . From the Escherichia coli strain deficient in efflux, we isolated a mutant that was less susceptible to compound 1o and had an lpxD missense mutation (Gly268Cys), supporting the cellular on-target activity. Using surface plasma resonance, we showed direct binding to E. coli LpxD for compound 1o and other reported LpxD inhibitors in vitro . Furthermore, we determined eight cocrystal structures of E. coli LpxD/inhibitor complexes. These costructures pinpointed the 4'-phosphopantetheine binding site as the common ligand binding hotspot, where hydrogen bonds to Gly269 and/or Gly287 were important for inhibitor binding. In addition, the LpxD/compound 1o costructure rationalized the reduced activity of compound 1o in the LpxD Gly268Cys mutant. Moreover, we obtained the LpxD structure in complex with a previously reported LpxA/LpxD dual targeting peptide inhibitor, RJPXD33, providing structural rationale for the unique dual targeting properties of this peptide. Given that the active site residues of LpxD are conserved in multidrug resistant Enterobacteriaceae, this work paves the way for future LpxD drug discovery efforts combating these Gram-negative pathogens.

Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2 ) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli . These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.

The RAS-RAF-MEK-ERK signaling axis is frequently activated in human cancers. Physiological concentrations of ATP prevent formation of RAF kinase-domain (RAF KD ) dimers that are critical for activity. Here we present a 2.9-Å-resolution crystal structure of human BRAF KD in complex with MEK and the ATP analog AMP-PCP, revealing interactions between BRAF and ATP that induce an inactive, monomeric conformation of BRAF KD . We also determine how 14-3-3 relieves the negative regulatory effect of ATP through a 2.5-Å-resolution crystal structure of the BRAF KD -14-3-3 complex, in which dimeric 14-3-3 enforces a dimeric BRAF KD assembly to increase BRAF activity. Our data suggest that most oncogenic BRAF mutations alter interactions with ATP and counteract the negative effects of ATP binding by lowering the threshold for RAF dimerization and pathway activation. Our study establishes a framework for rationalizing oncogenic BRAF mutations and provides new avenues for improved RAF-inhibitor discovery.

Transient affinity binding interactions are central to life, composing the fundamental elements of biological networks including cell signaling, cell metabolism and gene regulation. Assigning a defined reaction mechanism to affinity binding interactions is critical to our understanding of the associated structure-function relationship, a cornerstone of biophysical characterization. Transient kinetics are currently measured using low throughput methods such as nuclear magnetic resonance, or stop-flow spectrometry-based techniques, which are not practical in many settings. In contrast, label-free biosensors measure reaction kinetics through direct binding, and with higher throughout, impacting life sciences with thousands of publications each year. Here we have developed a methodology enabling label-free biosensors to measure transient kinetic interactions towards providing a higher throughput approach suitable for mechanistic understanding of these processes. The methodology relies on hydrodynamic dispersion modeling of a smooth analyte gradient under conditions that maintain the quasi-steady-state boundary layer assumption. A transient peptide-protein interaction of relevance to drug discovery was analyzed thermodynamically using transition state theory and numerical simulations validated the approach over a wide range of operating conditions. The data establishes the technical feasibility of this approach to transient kinetic analyses supporting further development towards higher throughput applications in life science.

Prolonged drug-target occupancy has become increasingly important in lead optimization, and biophysical assays that measure residence time are in high demand. Here we report a practical label-free assay methodology that provides kinetic and affinity measurements suitable for most target classes without long preincubations and over comparatively short sample contact times. The method, referred to as a "chaser" assay, has been applied to three sets of unrelated kinase/inhibitor panels in order to measure the residence times, where correlation with observed efficacy was suspected. A lower throughput chaser assay measured a residence time of 3.6 days ±3.4% (95% CI) and provided single digit pM sensitivity. A higher throughput chaser methodology enabled a maximum capacity of 108 compounds in duplicate/day with an upper residence time limit of 9 h given an assay dissociation time of 34 min.

In the preceding manuscript [ Moreau et al. 2018 , 10.1021/acs.jmedchem.7b01691 ] we described a successful fragment-based lead discovery (FBLD) strategy for discovery of bacterial phosphopantetheine adenylyltransferase inhibitors (PPAT, CoaD). Following several rounds of optimization two promising lead compounds were identified: triazolopyrimidinone 3 and 4-azabenzimidazole 4. Here we disclose our efforts to further optimize these two leads for on-target potency and Gram-negative cellular activity. Enabled by a robust X-ray crystallography system, our structure-based inhibitor design approach delivered compounds with biochemical potencies 4-5 orders of magnitude greater than their respective fragment starting points. Additional optimization was guided by observations on bacterial permeability and physicochemical properties, which ultimately led to the identification of PPAT inhibitors with cellular activity against wild-type E. coli.

Drug-resistant Gram-negative bacteria are of increasing concern worldwide. Novel antibiotics are needed, but their development is complicated by the requirement to simultaneously optimize molecules for target affinity and cellular potency, which can result in divergent structure-activity relationships (SARs). These challenges were exemplified during our attempts to optimize inhibitors of the bacterial enzyme CoaD originally identified through a biochemical screen. To facilitate lead optimization, we developed mass spectroscopy assays based on the hypothesis that levels of CoA metabolites would reflect the cellular enzymatic activity of CoaD. Using these methods, we were able to monitor the effects of cellular enzyme inhibition at compound concentrations up to 100-fold below the minimum inhibitory concentration (MIC), a common metric of growth inhibition. Furthermore, we generated a panel of efflux pump mutants to dissect the susceptibility of a representative CoaD inhibitor to efflux. These approaches allowed for a nuanced understanding of the permeability and efflux liabilities of the series and helped guide optimization efforts to achieve measurable MICs against wild-type E. coli.

Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Stimulation of hepatocyte growth factor (HGF) signaling through the Met receptor is an attractive approach for promoting tissue repair and preventing fibrosis. Using structure-guided peptide phage display combined with an activity-based sorting strategy, we engineered allosteric activators of zymogen-like pro-HGF to bypass proteolytic activation and reversibly stimulate pro-HGF signaling through Met. Biochemical, structural and biological data showed that zymogen activator peptides (ZAPtides) potently and selectively bind the activation pocket within the serine protease-like β-chain of pro-HGF and display titratable activation of pro-HGF-dependent Met signaling, leading to cell survival and migration. To further demonstrate the versatility of our ZAPtide platform, we identified allosteric activators for pro-macrophage stimulating protein and a zymogen serine protease, Protein C, which also provides evidence for target selectivity. These studies reveal that ZAPtides use molecular mimicry of the trypsin-like N-terminal insertion mechanism and establish a new paradigm for selective pharmacological activation of plasminogen-related growth factors and zymogen serine proteases.

Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A highly ligand efficient, novel 8-oxo-pyridopyrimidine containing inhibitor of Jak1 and Jak2 isoforms with a pyridone moiety as the hinge-binding motif was discovered. Structure-based design strategies were applied to significantly improve enzyme potency and the polarity of the molecule was adjusted to gain cellular activity. The crystal structures of two representative inhibitors bound to Jak1 were obtained to enable SAR exploration., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: The complexity of ventricular septal defects in early infancy led to development of new mini-invasive techniques based on collaboration of cardiac surgeons with interventional cardiologists, called hybrid procedures. Hybrid therapies aim to combine the advantages of surgical and interventional techniques in an effort to reduce the invasiveness. The aim of this study was to present our approach with mVSD patients and initial results in the development of a mini-invasive hybrid procedure in the Gdansk Hybrid Heartlink Programme (GHHP) at the Department of Pediatric Cardiac Surgery, Pomeranian Centre of Traumatology in Gdansk, Poland., Material and Methods: The group of 11 children with mVSDs was enrolled in GHHP and 6 were finally qualified to hybrid trans-ventricular mVSD device closure. Mean age at time of hybrid procedure was 8.22 months (range: from 2.7 to 17.8 months, SD=5.1) and mean body weight was 6.3 kg (range: from 3.4 to 7.5 kg, SD=1.5)., Results: The implants of choice were Amplatzer VSD Occluder and Amplatzer Duct Occluder II (AGA Med. Corp, USA). The position of the implants was checked carefully before releasing the device with both transesophageal echocardiography and epicardial echocardiography. All patients survived and their general condition improved. No complications occurred. The closure of mVSD was complete in all children., Conclusions: Hybrid procedures of periventricular muscular VSD closure appear feasible and effective for patients with septal defects with morphology unsuitable for classic surgical or interventional procedures. The modern strategy of joint cardiac surgical and interventional techniques provides the benefits of close cooperation between cardiac surgeon and interventional cardiologist for selected patients in difficult clinical settings.

Ubiquitin is a highly conserved eukaryotic protein that interacts with a diverse set of partners to act as a cellular signaling hub. Ubiquitin's conformational flexibility has been postulated to underlie its multifaceted recognition. Here we use computational and library-based means to interrogate core mutations that modulate the conformational dynamics of human ubiquitin. These ubiquitin variants exhibit increased affinity for the USP14 deubiquitinase, with concomitantly reduced affinity for other deubiquitinases. Strikingly, the kinetics of conformational motion are dramatically slowed in these variants without a detectable change in either the ground state fold or excited state population. These variants can be ligated into substrate-linked chains in vitro and in vivo but cannot solely support growth in eukaryotic cells. Proteomic analyses reveal nearly identical interaction profiles between WT ubiquitin and the variants but identify a small subset of altered interactions. Taken together, these results show that conformational dynamics are critical for ubiquitin-deubiquitinase interactions and imply that the fine tuning of motion has played a key role in the evolution of ubiquitin as a signaling hub.

Herein we report on the structure-based discovery of a C-2 hydroxyethyl moiety which provided consistently high levels of selectivity for JAK1 over JAK2 to the imidazopyrrolopyridine series of JAK1 inhibitors. X-ray structures of a C-2 hydroxyethyl analogue in complex with both JAK1 and JAK2 revealed differential ligand/protein interactions between the two isoforms and offered an explanation for the observed selectivity. Analysis of historical data from related molecules was used to develop a set of physicochemical compound design parameters to impart desirable properties such as acceptable membrane permeability, potent whole blood activity, and a high degree of metabolic stability. This work culminated in the identification of a highly JAK1 selective compound (31) exhibiting favorable oral bioavailability across a range of preclinical species and robust efficacy in a rat CIA model.

Pim kinases are promising targets for the development of cancer therapeutics. Among the three Pim isoforms, Pim-2 is particularly important in multiple myeloma, yet is the most difficult to inhibit due to its high affinity for ATP. We identified compound 1 via high throughput screening. Using property-based drug design and co-crystal structures with Pim-1 kinase to guide analog design, we were able to improve potency against all three Pim isoforms including a significant 10,000-fold gain against Pim-2. Compound 17 is a novel lead with low picomolar potency on all three Pim kinase isoforms., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Herein we describe our successful efforts in obtaining C-2 substituted imidazo-pyrrolopyridines with improved JAK1 selectivity relative to JAK2 by targeting an amino acid residue that differs between the two isoforms (JAK1: E966; JAK2: D939). Efforts to improve cellular potency by reducing the polarity of the inhibitors are also detailed. The X-ray crystal structure of a representative inhibitor in complex with the JAK1 enzyme is also disclosed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Herein we report the discovery of the C-2 methyl substituted imidazopyrrolopyridine series and its optimization to provide potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2. The C-2 methyl substituted inhibitor 4 exhibited not only improved JAK1 potency relative to unsubstituted compound 3 but also notable JAK1 vs JAK2 selectivity (20-fold and >33-fold in biochemical and cell-based assays, respectively). Features of the X-ray structures of 4 in complex with both JAK1 and JAK2 are delineated. Efforts to improve the in vitro and in vivo ADME properties of 4 while maintaining JAK1 selectivity are described, culminating in the discovery of a highly optimized and balanced inhibitor (20). Details of the biological characterization of 20 are disclosed including JAK1 vs JAK2 selectivity levels, preclinical in vivo PK profiles, performance in an in vivo JAK1-mediated PK/PD model, and attributes of an X-ray structure in complex with JAK1.

The caspases are a family of cytosolic proteases with essential roles in inflammation and apoptosis. Drug discovery efforts have focused on developing molecules directed against the active sites of caspases, but this approach has proved challenging and has not yielded any approved therapeutics. Here we describe a new strategy for generating inhibitors of caspase-6, a potential therapeutic target in neurodegenerative disorders, by screening against its zymogen form. Using phage display to discover molecules that bind the zymogen, we report the identification of a peptide that specifically impairs the function of caspase-6 in vitro and in neuronal cells. Remarkably, the peptide binds at a tetramerization interface that is uniquely present in zymogen caspase-6, rather than binding into the active site, and acts via a new allosteric mechanism that promotes caspase tetramerization. Our data illustrate that screening against the zymogen holds promise as an approach for targeting caspases in drug discovery.

A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as rheumatoid arthritis (RA), by specific targeting of the JAK1 pathway. Examination of the preferred binding conformation of clinically effective, pan-JAK inhibitor 1 led to identification of a novel, tricyclic hinge binding scaffold 3. Exploration of SAR through a series of cycloamino and cycloalkylamino analogues demonstrated this template to be highly tolerant of substitution, with a predisposition to moderate selectivity for the JAK1 isoform over JAK2. This study culminated in the identification of subnanomolar JAK1 inhibitors such as 22 and 49, having excellent cell potency, good rat pharmacokinetic characteristics, and excellent kinase selectivity. Determination of the binding modes of the series in JAK1 and JAK2 by X-ray crystallography supported the design of analogues to enhance affinity and selectivity.

Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.

Background: Invasive fungal infections due to Candida species constitute an increasing clinical problem. There are no guidelines for the management of candidemia in children undergoing surgical procedures for congenital heart defects (CHD). The aim of the study was to draw attention to the problem of candidemia in children who are operated on due to congenital heart defects., Material/methods: We retrospectively analyzed medical documentation of 307 children with congenital heart defects treated in 1 clinical centre in Poland, from whom we selected those diagnosed with candidemia during the postoperative period. Next, we analyzed in detail the clinical course of invasive candidiasis in each individual and we performed an analysis of risk factors that lead to candidemia., Results: In the analyzed period, among 307 children who underwent surgical procedures, we observed 2 cases of candidemia (0.65%) which were effectively cured with caspofungin. No adverse effects were observed after treatment with the drug., Conclusions: Candidemia in children who undergo surgical treatment of congenital heart defects is an important factor that can influence final clinical results. Caspofungin may be an effective therapeutic option when treating candidemia in children after extensive cardiosurgical procedures.

There are several strategies of surgical approach for the repair of multiple muscular ventricular septal defects (mVSDs), but none leads to a fully predictable, satisfactory therapeutic outcome in infants. We followed a concept of treating multiple mVSDs consisting of a hybrid approach based on intraoperative perventricular implantation of occluding devices. In this report, we describe a 2-step procedure consisting of a final hybrid approach for multiple mVSDs in the infant following initial coarctation repair with pulmonary artery banding in the newborn. At 7 months, sternotomy and debanding were performed, the right ventricle was punctured under transesophageal echocardiographic guidance, and the 8-mm device was implanted into the septal defect. Color Doppler echocardiography results showed complete closure of all VSDs by 11 months after surgery, probably via a mechanism of a localized inflammatory response reaction, ventricular septum growth, and implant endothelization.