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Proteases function within sophisticated networks. Altering the activity of one protease can have sweeping effects on other proteases, leading to changes in their activity, structure, specificity, localisation, stability, and expression. Using a suite of chemical tools, we investigated the impact of cathepsin X, a lysosomal cysteine protease, on the activity and expression of other cysteine proteases and their inhibitors in dendritic cells. Among all proteases examined, cathepsin X gene deletion specifically altered cathepsin L levels; pro-cathepsin L and its single chain accumulated while the two-chain form was unchanged. This effect was recapitulated by chemical inhibition of cathepsin X, suggesting a dependence on its catalytic activity. We demonstrated that accumulation of pro- and single chain cathepsin L was not due to a lack of direct cleavage by cathepsin X or altered glycosylation, secretion, or mRNA expression but may result from changes in lysosomal oxidative stress or pH. In the absence of active cathepsin X, nuclear cathepsin L and cleavage of the known nuclear cathepsin L substrate, Lamin B1, were diminished. Thus, cathepsin X activity selectively regulates cathepsin L, which has the potential to impact the degree of cathepsin L proteolysis, the nature of substrates that it cleaves, and the location of cleavage.

Peptide receptor radionuclide therapy (PRRT) is a promising form of systemic radiation therapy designed to eradicate cancer. Cholecystokinin-2 receptor (CCK2R) is an important molecular target that is highly expressed in a range of cancers. This study describes the synthesis and in vivo characterization of a novel series of 177Lu-labeled peptides ([177Lu]Lu-2b-4b) in comparison with the reference CCK2R-targeting peptide CP04 ([177Lu]Lu-1b). [177Lu]Lu-1b-4b showed high chemical purity (HPLC ≥ 94%), low Log D7.4 (-4.09 to -4.55) with strong binding affinity to CCK2R (KD 0.097-1.61 nM), and relatively high protein binding (55.6-80.2%) and internalization (40-67%). Biodistribution studies of the novel 177Lu-labeled peptides in tumors (AR42J and A431-CCK2R) showed uptake one- to eight-fold greater than the reference compound CP04 at 1, 24, and 48 h. Rapid clearance and high tumor uptake and retention were established for [177Lu]Lu-2b-4b, making these compounds excellent candidates for theranostic applications against CCK2R-expressing tumors.

The emergence of multidrug-resistant (MDR) Gram-negative pathogens is an urgent global medical challenge. The old polymyxin lipopeptide antibiotics (polymyxin B and colistin) are often the only therapeutic option due to resistance to all other classes of antibiotics and the lean antibiotic drug development pipeline. However, polymyxin B and colistin suffer from major issues in safety (dose-limiting nephrotoxicity, acute toxicity), pharmacokinetics (poor exposure in the lungs) and efficacy (negligible activity against pulmonary infections) that have severely limited their clinical utility. Here we employ chemical biology to systematically optimize multiple non-conserved positions in the polymyxin scaffold, and successfully disconnect the therapeutic efficacy from the toxicity to develop a new synthetic lipopeptide, structurally and pharmacologically distinct from polymyxin B and colistin. This resulted in the clinical candidate F365 (QPX9003) with superior safety and efficacy against lung infections caused by top-priority MDR pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.

Thalidomide analogues (or immunomodulatory imide drugs, IMiDs) are cornerstones in the treatment of multiple myeloma (MM). These drugs bind Cereblon (CRBN), a receptor for the Cullin-ring 4 ubiquitin-ligase (CRL4) complex, to modify its substrate specificity. IMiDs mediate CRBN-dependent engagement and proteasomal degradation of 'neosubstrates', Ikaros (IKZF1) and Aiolos (IKZF3), conveying concurrent antimyeloma activity and T-cell costimulation. There is now a greater understanding of physiological CRBN functions, including endogenous substrates and chaperone activity. CRISPR Cas9-based genome-wide screening has further elucidated the complex cellular machinery implicated in IMiD sensitivity, including IKZF1/3-independent mechanisms. New-generation IMiD derivatives with more potent anti-cancer properties-the CELMoDs (Cereblon E3 ligase modulators)-are now being evaluated. Rational drug design also allows 'hijacking' of CRL4CRBN utilising proteolysis targeting chimeras (PROTACs) to convey entirely distinct substrate repertoires. As all these chemotypes-thalidomide, IMiDs, CELMoDs and PROTACs-engage CRBN and modify its functions, we describe them here in aggregate as 'CRBN-interacting small molecules' (CISMs). In this review, we provide a contemporary summary of the biological consequences of CRBN modulation by CISMs. Detailed molecular insight into CRBN-CISM interactions now provides an opportunity to more effectively target previously elusive cancer dependencies, representing a new and powerful tool for the implementation of precision medicine.

Multidrug-resistant Gram-negative bacteria represent a major medical challenge worldwide. New antibiotics are desperately required with 'old' polymyxins often being the only available therapeutic option. Here, we systematically investigated the structure-activity relationship (SAR) of polymyxins using a quantitative lipidomics-informed outer membrane (OM) model of Acinetobacter baumannii and a series of chemically synthesized polymyxin analogs. By integrating chemical biology and all-atom molecular dynamics simulations, we deciphered how each residue of the polymyxin molecule modulated its conformational folding and specific interactions with the bacterial OM. Importantly, a novel designed polymyxin analog FADDI-287 with predicted stronger OM penetration showed improved in vitro antibacterial activity. Collectively, our study provides a novel chemical biology and computational strategy to expedite the discovery of new-generation polymyxins against life-threatening Gram-negative 'superbugs'.

The p75 splice variant of lens epithelium-derived growth factor (LEDGF) is a 75 kDa protein, which is recruited by the human immunodeficiency virus (HIV) to tether the pre-integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We designed a series of small cyclic peptides that are structural mimics of the LEDGF binding domain, which interact with integrase as potential binding inhibitors. Herein we present the X-ray crystal structures, NMR studies, SPR analysis, and conformational studies of four cyclic peptides bound to the HIV-1 integrase core domain. Although the X-ray studies show that the peptides closely mimic the LEDGF binding loop, the measured affinities of the peptides are in the low millimolar range. Computational analysis using conformational searching and free energy calculations suggest that the low affinity of the peptides is due to mismatch between the low-energy solution and bound conformations.

Polymyxins (polymyxin B and colistin) are cyclic lipopeptide antibiotics that serve as a last-line defence against Gram-negative "superbugs". In the present study, two novel fluorescent polymyxin probes were designed through regio-selective modifications of the polymyxin B core structure at the N-terminus and the hydrophobic motif at positions 6 and 7. The resulting probes, FADDI-285 and FADDI-286 demonstrated comparable antibacterial activity (MICs 2-8 mg/L) to polymyxin B and colistin (MICs 0.5-8 mg/L) against a panel of gram-negative clinical isolates of Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. These probes should prove to be of considerable utility for imaging cellular uptake and mechanistic investigations of these important last-line antibiotics.

SPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2-iNOS interaction are expected to prolong iNOS lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox-stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind with low nanomolar affinity to the iNOS binding site on SPSB, as determined by SPR and (19)F NMR, and efficiently displace full-length iNOS from binding to SPSB2 in macrophage cell lysates. These peptides provide a foundation for future development of redox-stable, potent ligands for SPSB proteins as a potential novel class of anti-infectives.

Polymyxin B and colistin are exclusively active against Gram-negative pathogens and have been used in the clinic as a last-line therapy. In this study, we investigated the antimicrobial activity of a novel polymyxin, FADDI-019, against Staphylococcus aureus. MIC and time-kill assays were employed to measure the activity of FADDI-019 against S. aureus ATCC 700699. Cell morphology was examined with scanning electron microscopy (SEM), and cell membrane polarity was measured using flow cytometry. Transcriptome changes caused by FADDI-019 treatment were investigated using transcriptome sequencing (RNA-Seq). Pathway analysis was conducted to examine the mechanism of the antibacterial activity of FADDI-019 and to rationally design a synergistic combination. Polymyxin B and colistin were not active against S. aureus strains with MICs of >128 mg/liter; however, FADDI-019 had a MIC of 16 mg/liter. Time-kill assays revealed that no S. aureus regrowth was observed after 24 h at 2× to 4× MIC of FADDI-019. Scanning electron microscopy (SEM) and flow cytometry results indicated that FADDI-019 treatment had no effect on cell morphology but caused membrane depolarization. The vancomycin resistance genes vraRS, as well as the VraRS regulon, were activated by FADDI-019. Virulence determinants controlled by SaeRS and the expression of enterotoxin genes yent2, sei, sem, and seo were significantly downregulated by FADDI-019. Pathway analysis of transcriptomic data was predictive of a synergistic combination comprising FADDI-019 and sulfamethoxazole. Our study is the first to examine the mechanism of the killing of a novel polymyxin against S. aureus. We also show the potential of transcriptomic and pathway analysis as tools to design synergistic antibiotic combinations. IMPORTANCE S. aureus is currently one of the most pervasive multidrug-resistant pathogens and commonly causes nosocomial infections. Clinicians are faced with a dwindling armamentarium to treat infections caused by S. aur

Polymyxin is the last-line therapy against Gram-negative 'superbugs'; however, dose-limiting nephrotoxicity can occur in up to 60% of patients after intravenous administration. Understanding the accumulation and concentration of polymyxin within renal tubular cells is essential for the development of novel strategies to ameliorate its nephrotoxicity and to develop safer, new polymyxins. We designed and synthesized a novel dual-modality iodine-labeled fluorescent probe for quantitative mapping of polymyxin in kidney proximal tubular cells. Measured by synchrotron X-ray fluorescence microscopy, polymyxin concentrations in single rat (NRK-52E) and human (HK-2) kidney tubular cells were approximately 1930- to 4760-fold higher than extracellular concentrations. Our study is the first to quantitatively measure the significant uptake of polymyxin in renal tubular cells and provides crucial information for the understanding of polymyxin-induced nephrotoxicity. Importantly, our approach represents a significant methodological advancement in determination of drug uptake for single-cell pharmacology.

The antimicrobial lipopeptides polymyxin B and E (colistin) are being used as a 'last-line' therapy for infections caused by multidrug-resistant Gram-negative pathogens. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections caused by the Gram-negative 'superbugs'. This report details the structure-activity relationships (SAR) based design, in toto synthesis, and preclinical evaluation of a series of novel polymyxin lipopeptides with better antibacterial activity against polymyxin-resistant Gram-negative bacteria.

The dry antibiotic development pipeline coupled with the emergence of multidrug resistant Gram-negative 'superbugs' has driven the revival of the polymyxin lipopeptide antibiotics. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections. The lack of molecular imaging probes that possess native polymyxin-like antibacterial activity is a barrier to understanding the resistance mechanisms and the development of a new generation of polymyxin lipopeptides. Here we report the regioselective modification of the polymyxin B core scaffold at the N-terminus with the dansyl fluorophore to generate an active probe that mimics polymyxin B pharmacologically. Time-lapse laser scanning confocal microscopy imaging of the penetration of probe (1) into Gram-negative bacterial cells revealed that the probe initially accumulates in the outer membrane and subsequently penetrates into the inner membrane and finally the cytoplasm. The implementation of this polymyxin-mimetic probe will advance the development of platforms for the discovery of novel polymyxin lipopeptides with efficacy against polymyxin-resistant strains.

Polymyxin B and colistin were examined for their ability to inhibit the type II NADH-quinone oxidoreductases (NDH-2) of three species of Gram-negative bacteria. Polymyxin B and colistin inhibited the NDH-2 activity in preparations from all of the isolates in a concentration-dependent manner. The mechanism of NDH-2 inhibition by polymyxin B was investigated in detail with Escherichia coli inner membrane preparations and conformed to a mixed inhibition model with respect to ubiquinone-1 and a non-competitive inhibition model with respect to NADH. These suggest that the inhibition of vital respiratory enzymes in the bacterial inner membrane represents one of the secondary modes of action for polymyxins.

The peptides angiotensin IV and LVV-hemorphin 7 were found to enhance memory in a number of memory tasks and reverse the performance deficits in animals with experimentally induced memory loss. These peptides bound specifically to the enzyme insulin-regulated aminopeptidase (IRAP), which is proposed to be the site in the brain that mediates the memory effects of these peptides. However, the mechanism of action is still unknown but may involve inhibition of the aminopeptidase activity of IRAP, since both angiotensin IV and LVV-hemorphin 7 are competitive inhibitors of the enzyme. IRAP also has another functional domain that is thought to regulate the trafficking of the insulin-responsive glucose transporter GLUT4, thereby influencing glucose uptake into cells. Although the exact mechanism by which the peptides enhance memory is yet to be elucidated, IRAP still represents a promising target for the development of a new class of cognitive enhancing agents.

We have studied the mechanisms underlying stress-induced platelet formation during plasma hydrogenation. The stress is purposely introduced by a buried SiGe stained layer in a Si substrate. During plasma hydrogenation, diffusing H is trapped in the region of the SiGe layer and H platelets are formed. The platelet orientation is controlled by the in-plane compressive stress, which favors nucleation and growth of platelets in the plane of stress and parallel to the substrate surface, and ultimately leads to controlled fracture along the SiGe layer. Also, the SiSiGeSi structure is found to be more efficient in utilizing H for platelet formation and growth compared to H ion implanted Si because there are fewer defects to trap H (e.g., Vn Hm and In Hm); therefore, the total H dose needed for layer exfoliation is greatly reduced. © 2007 American Institute of Physics.

We have shown that the Si/Si interface produced by molecular-beam epitaxy (MBE) growth of Si on a Si substrate can significantly enhance the efficiency of ion cutting. MBE growth is performed at 650 °C. Samples are then implanted at room temperature by 62 keV H- to a dose of 7 × 10 16 ions/cm2. The implantation energy locates H-peak in the vicinity of the Si/Si interface, which is 600 nm below the Si surface. Scanning electron microscopy shows that, after post-implantation annealing at 300 °C for 50 min, the H implanted MBE Si has bubbles formed with an average diameters of 33 μm, which is around one order of magnitude larger than that observed in the control bulk silicon sample. It is also observed that the area covered with blisters is a factor of 2 larger for the MBE samples, a trend that is systematically observed for anneals carried out in the range of 300-550 °C. © 2005 Elsevier B.V. All rights reserved.

We have investigated hydrogen diffusion in hydrogenated 〈100〉 Si/Si homoepitaxial structures, which were grown by molecular beam epitaxy at various temperatures. The substrate growth temperature can significantly affect the H diffusion behavior, with higher growth temperatures resulting in deeper H diffusion. For the Si/Si structure grown at the highest temperature of 800°C, H trapping occurs at the epitaxial Si/Si substrate interface, which results in the formation of (100) oriented microcracks at the interface. The mechanism of H trapping and the potential application of these findings for the development of a method of transferring ultrathin Si layers are discussed. © 2006 American Institute of Physics.

Approximately 2% of mammalian genes encode proteases. Comparative genomics reveals that those involved in immunity and reproduction show the most interspecies diversity and evidence of positive selection during evolution. This is particularly true of granzymes, the cytotoxic proteases of natural killer cells and CD8+ T cells. There are 5 granzyme genes in humans and 10 in mice, and it is suggested that granzymes evolve to meet species-specific immune challenge through gene duplication and more subtle alterations to substrate specificity. We show that mouse and human granzyme B have distinct structural and functional characteristics. Specifically, mouse granzyme B is 30 times less cytotoxic than human granzyme B and does not require Bid for killing but regains cytotoxicity on engineering of its active site cleft. We also show that mouse granzyme A is considerably more cytotoxic than human granzyme A. These results demonstrate that even "orthologous" granzymes have species-specific functions, having evolved in distinct environments that pose different challenges.

An approach to transfer a high-quality Si layer for the fabrication of silicon-on-insulator wafers has been proposed based on the investigation of platelet and crack formation in hydrogenated epitaxial Si Si0.98 B0.02 Si structures grown by molecular-beam epitaxy. H-related defect formation during hydrogenation was found to be very sensitive to the thickness of the buried Si0.98 B0.02 layer. For hydrogenated Si containing a 130 nm thick Si0.98 B0.02 layer, no platelets or cracking were observed in the B-doped region. Upon reducing the thickness of the buried Si0.98 B0.02 layer to 3 nm, localized continuous cracking was observed along the interface between the Si and the B-doped layers. In the latter case, the strains at the interface are believed to facilitate the (100)-oriented platelet formation and (100)-oriented crack propagation. © 2006 American Institute of Physics.

We report a process for the lift-off of an ultrathin Si layer. By plasma hydrogenation of a molecular-beam-epitaxy-grown heterostructure of SiSb-doped-SiSi, ultrashallow cracking is controlled to occur at the depth of the Sb-doped layer. Prior to hydrogenation, an oxygen plasma treatment is used to induce the formation of a thin oxide layer on the surface of the heterostructure. Chemical etching of the surface oxide layer after hydrogenation further thins the thickness of the separated Si layer to be only 15 nm. Mechanisms of hydrogen trapping and strain-facilitated cracking are discussed. © 2005 American Institute of Physics.

We have developed an innovative approach without the use of ion implantation to transfer a high-quality thin Si layer for the fabrication of silicon-on-insulator wafers. The technique uses a buried strained SiGe layer, a few nanometers in thickness, to provide H trapping centers. In conjunction with H plasma hydrogenation, lift-off of the top Si layer can be realized with cleavage occurring at the depth of the strained SiGe layer. This technique avoids irradiation damage within the top Si layer that typically results from ion implantation used to create H trapping regions in the conventional ion-cut method. We explain the strain-facilitated layer transfer as being due to preferential vacancy aggregation within the strained layer and subsequent trapping of hydrogen, which lead to cracking in a well controlled manner. © 2005 American Institute of Physics.