Uncoiling collagen: a multidimensional mass spectrometry study.

Mass spectrometry can be used to determine structural information about ions by activating precursors and analysing the resulting series of fragments. Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) is a technique that correlates the mass-to-charge (m/z) ratio of fragment and precursor ions in a single spectrum. 2D FT-ICR MS records the fragmentation of all ions in a sample without the need for isolation. To analyse specific precursors, horizontal cross-sections of the spectrum (fragment ion scans) are taken, providing an alternative to conventional tandem mass spectrometry (MS/MS) experiments. In this work, 2D FT-ICR MS has been used to study the tryptic digest of type I collagen, a large protein. Fragment ion scans have been extracted from the 2D FT-ICR MS spectrum for precursor m/z ratios: 951.81, 850.41, 634.34, and 659.34, and 2D FT-ICR MS spectra are compared with a set of 1D MS/MS spectra using different fragmentation methods. The results show that two-dimensional mass spectrometry excells at MS/MS of complex mixtures, simplifying spectra by eliminating contaminant peaks, and aiding the identification of species in the sample. Currently, with desktop computers, 2D FT-ICR MS is limited by data processing power, a limitation which should be alleviated using cluster parallel computing. In order to explore 2D FT-ICR MS for collagen, with reasonable computing time, the resolution in the fragment ion dimension is limited to 256k data points (compared to 4M data points in 1D MS/MS spectra), but the vertical precursor ion dimension has 4096 lines, so the total data set is 1G data points (4 Gbytes). The fragment ion coverage obtained with a blind, unoptimized 2D FT-ICR MS experiment was lower than conventional MS/MS, but MS/MS information is obtained for all ions in the sample regardless of selection and isolation. Finally, although all 2D FT-ICR MS peak assignments were made with the aid of 1D FT-ICR MS data, these results demonstrate the promise of 2D FT-ICR MS as a technique for studying complex protein digest mixtures.

The potential application of a transcriptionally regulated oncolytic herpes simplex virus for human cancer therapy.

BACKGROUND: Emerging studies have shown the potential benefit of arming oncolytic viruses with therapeutic genes. However, most of these therapeutic genes are placed under the regulation of ubiquitous viral promoters. Our goal is to generate a safer yet potent oncolytic herpes simplex virus type-1 (HSV-1) for cancer therapy. METHODS: Using bacterial artificial chromosome (BAC) recombineering, a cell cycle-regulatable luciferase transgene cassette was replaced with the infected cell protein 6 (ICP6) coding region (encoded for UL39 or large subunit of ribonucleotide reductase) of the HSV-1 genome. These recombinant viruses, YE-PC8, were further tested for its proliferation-dependent luciferase gene expression. RESULTS: The ability of YE-PC8 to confer proliferation-dependent transgene expression was demonstrated by injecting similar amount of viruses into the tumour-bearing region of the brain and the contralateral normal brain parenchyma of the same mouse. The results showed enhanced levels of luciferase activities in the tumour region but not in the normal brain parenchyma. Similar findings were observed in YE-PC8-infected short-term human brain patient-derived glioma cells compared with normal human astrocytes. intratumoural injection of YE-PC8 viruses resulted in 77% and 80% of tumour regression in human glioma and human hepatocellular carcinoma xenografts, respectively. CONCLUSION: YE-PC8 viruses confer tumour selectivity in proliferating cells and may be developed further as a feasible approach to treat human cancers.

Suicidal gene therapy in the effective control of primary human hepatocellular carcinoma as monitored by noninvasive bioimaging.

Hepatocellular carcinoma (HCC) is usually refractory to the available treatments. For cancer gene therapy purposes, real-time imaging of therapeutic gene expression is of great importance because there are multiple factors that modulate the therapeutic gene expression in a complex tumor microenvironment. As a consequence, multiple doses of therapeutic viral vectors may be required for improved efficacy. In the present study, the luciferase reporter gene and the yeast cytosine deaminase (yCD) genes were bicistronically expressed using the foot-and-mouth disease virus 2A peptide under the regulation of the cytomegalovirus (CMV) promoter. The effectiveness of the yCD/5-FC (5-fluorocytosine) killing efficacy mediated by the herpes simplex virus type 1 (HSV-1) amplicon viral vector was shown using HCC and non-HCC cell lines in vitro. In addition, in vivo experiment also showed tumor regression of a primary HCC 26-1004 tumor xenograft in tumor expressing high levels of the yCD gene (as determined by noninvasive imaging) after intratumoral injection of 1.5 × 10(6) TU HGCX-L2C HSV-1 amplicon viral vector and 5-FC administration. The HSV-1 amplicon viral vector coupled with the yCD/5-FC prodrug activated suicide gene could potentially be of use in clinical gene therapy for HCC.

A robust experimental and computational analysis framework at multiple resolutions, modalities and coverages.

The ability to study cancer-immune cell communication across the whole tumor section without tissue dissociation is needed, especially for cancer immunotherapy development, which requires understanding of molecular mechanisms and discovery of more druggable targets. In this work, we assembled and evaluated an integrated experimental framework and analytical process to enable genome-wide scale discovery of ligand-receptors potentially used for cellular crosstalks, followed by targeted validation. We assessed the complementarity of four different technologies: single-cell RNA sequencing and Spatial transcriptomic (measuring over >20,000 genes), RNA In Situ Hybridization (RNAscope, measuring 4-12 genes) and Opal Polaris multiplex protein staining (4-9 proteins). To utilize the multimodal data, we implemented existing methods and also developed STRISH (Spatial TRanscriptomic In Situ Hybridization), a computational method that can automatically scan across the whole tissue section for local expression of gene (e.g. RNAscope data) and/or protein markers (e.g. Polaris data) to recapitulate an interaction landscape across the whole tissue. We evaluated the approach to discover and validate cell-cell interaction in situ through in-depth analysis of two types of cancer, basal cell carcinoma and squamous cell carcinoma, which account for over 70% of cancer cases. We showed that inference of cell-cell interactions using scRNA-seq data can misdetect or detect false positive interactions. Spatial transcriptomics still suffers from misdetecting lowly expressed ligand-receptor interactions, but reduces false discovery. RNAscope and Polaris are sensitive methods for defining the location of potential ligand receptor interactions, and the STRISH program can determine the probability that local gene co-expression reflects true cell-cell interaction. We expect that the approach described here will be widely applied to discover and validate ligand receptor interaction in different types of solid cancer tumors.

Targeting human glioma cells using HSV-1 amplicon peptide display vector.

Targeting cell infection using herpes simplex virus type 1 (HSV-1) vectors is a complicated issue as the process involves multiple interactions of viral envelope glycoproteins and cellular host surface proteins. In this study, we have inserted a human glioma-specific peptide sequence (denoted as MG11) into a peptide display HSV-1 amplicon vector replacing the heparan sulfate-binding domain of glycoprotein C (gC). The modified MG11:gC envelope recombinant vectors were subsequently packaged into virions in the presence of helper virus deleted for gC. Our results showed that the tropism of these HSV-1 recombinant virions was increased for human glioma cells in culture as compared with wild-type virions. The binding of these recombinant virions could also be blocked effectively by pre-incubating the cells with the glioma-specific peptide, indicating that MG11 peptide and the recombinant virions competed for the same or similar receptor-binding sites on the cell surface of human glioma cells. Furthermore, preferential homing of these virions was shown in xenograft glioma mouse model following intravascular delivery. Taken together, these results validated the hypothesis that HSV-1 binding to cells can be redirected to human gliomas through the incorporation of MG11 peptide sequence to the virions.

Rational design of potent, bioavailable, nonpeptide cyclic ureas as HIV protease inhibitors.

Mechanistic information and structure-based design methods have been used to design a series of nonpeptide cyclic ureas that are potent inhibitors of human immunodeficiency virus (HIV) protease and HIV replication. A fundamental feature of these inhibitors is the cyclic urea carbonyl oxygen that mimics the hydrogen-bonding features of a key structural water molecule. The success of the design in both displacing and mimicking the structural water molecule was confirmed by x-ray crystallographic studies. Highly selective, preorganized inhibitors with relatively low molecular weight and high oral bioavailability were synthesized.

Enhancing legal preparedness for the prevention and control of infectious diseases: experience from severe acute respiratory syndrome in Hong Kong.

The use of legislation as a health protection tool forms an important and distinct aspect in the arena of public health. A review of Hong Kong's infectious disease legislation was conducted with a view to updating the legal framework for the prevention of infectious diseases, in order to strengthen the capacity of law to support strategy in the control of infectious diseases. This article shares Hong Kong's experience in reforming its public health legislation to: (1) update terminology and re-organize provisions in accordance with modern public health disease control principles and control mechanisms for disease; (2) enhance responsiveness for better preparedness and flexibility in handling emergent infections; (3) ensure appropriate checks and balances to coercive powers; and (4) introduce emergency powers for the handling of public health emergencies.

Oral Health Indicators for Risk of Malnutrition in Elders.

OBJECTIVES: Using both clinical parameters and subjective measures of oral health, this study aimed to identify useful oral health indicators for the risk of malnutrition in elders. DESIGN: Cross-sectional study. SETTING: Five community centers run by non-government organizations (NGOs). PARTICIPANTS: 195 community dwelling elders (65 or above). MEASUREMENTS: An interviewer-administered questionnaire was completed to collect information on elders' socio-demographic background and oral health perception and practice. Their number of teeth, number of occluding tooth pairs, dental caries, and periodontal condition were examined. General Oral Health Assessment Index (GOHAI), an instrument for assessing oral health related quality of life (OHQoL), was used as a subjective measure of oral health. The elders' nutritional status was evaluated by using the Mini-Nutritional Assessment (MNA). RESULTS: The mean (SD) DFT was 3.3 (3.1). Over 60% of elders had periodontal pockets; 33% had fewer than 20 teeth and 6% were edentulous. The mean (SD) of occluding tooth pairs was 7.1 (4.8). The mean (SD) total GOHAI score was 56.4 (8.0); 60% reported negative impact of oral health on their quality of life. The mean (SD) MNA score was 25.0 (2.9); 30% had malnutrition or were at risk. After controlling for socio-demographic factors, none of the clinical indicators (dental caries, periodontal status, number of teeth, and number of occluding tooth pairs) were associated with risk of malnutrition (all p>0.05). Poorer OHQoL indicated a higher chance for malnutrition in both adjusted models (OR of 0.914; 95% CI of 0.850-0.982; p=0.014 and OR of 0.915; 95% CI of 0.852-0.984; p=0.017). Tooth loss and untreated decayed teeth (DT) were significant/marginally significant determinants of poor OHQoL. CONCLUSION: Elders' tooth loss and unmet treatment need for dental caries were associated with compromised quality of life, which indicated increased likelihood for malnutrition.

The oncogenic potential of the Pax3-FKHR fusion protein requires the Pax3 homeodomain recognition helix but not the Pax3 paired-box DNA binding domain.

The chimeric transcription factor Pax3-FKHR, produced by the t(2;13)(q35;q14) chromosomal translocation in alveolar rhabdomyosarcoma, consists of the two Pax3 DNA binding domains (paired box and homeodomain) fused to the C-terminal forkhead (FKHR) sequences that contain a potent transcriptional activation domain. To determine which of these domains are required for cellular transformation, Pax3, Pax3-FKHR, and selected mutants were retrovirally expressed in NIH 3T3 cells and evaluated for their capacity to promote anchorage-independent cell growth. Mutational analysis revealed that both the third alpha-helix of the homeodomain and a small region of the FKHR transactivation domain are absolutely required for efficient transformation by the Pax3-FKHR fusion protein. Surprisingly, point mutations in the paired domain that abrogate sequence-specific DNA binding retained transformation potential equivalent to that of the wild-type protein. This finding suggests that DNA binding mediated through the Pax3 paired box is not required for transformation. Our results demonstrate that the integrity of the Pax3 homeodomain recognition helix and the FKHR transactivation domain is necessary for efficient cellular transformation by the Pax3-FKHR fusion protein.

Targeting proliferating tumor cells via the transcriptional control of therapeutic genes.

We have previously reported the construction of a cell cycle-regulated HSV-1 amplicon vector (denoted as pC8-36) that confers luciferase reporter gene activities dependent on cellular divisions. However, luciferase reporter gene is well known for its relatively high sensitivity, thus, it is crucial to evaluate the therapeutic efficacy of a transcriptional targeted vector. In this report, we have engineered the FasL and FADD genes into pC8-36 and demonstrated their efficacy for the treatment of human gliomas in vitro and in vivo. Using trypan blue dye exclusion and TUNEL assay, FasL expression mediated by pC8-36 was shown to induce a significantly higher percentage of cell death in proliferating cells than those observed in the G(1)-arrested cells. The observed cell killing effect correlated well with the level of FasL protein expression when analyzed by ELISA assay. Furthermore, the incorporation of both FasL and FADD into pC8-36 resulted in the enhancement of apoptosis in the target glioma cells both in vitro and in vivo. Targeting proliferating tumor cells via the transcriptional control of therapeutic genes could potentially improve the safety and efficacy of cancer gene therapy, and thus would allow the development of strategies for more effective anticancer therapies.

Improved cyclic urea inhibitors of the HIV-1 protease: synthesis, potency, resistance profile, human pharmacokinetics and X-ray crystal structure of DMP 450.

BACKGROUND: Effective HIV protease inhibitors must combine potency towards wild-type and mutant variants of HIV with oral bioavailability such that drug levels in relevant tissues continuously exceed that required for inhibition of virus replication. Computer-aided design led to the discovery of cyclic urea inhibitors of the HIV protease. We set out to improve the physical properties and oral bioavailability of these compounds. RESULTS: We have synthesized DMP 450 (bis-methanesulfonic acid salt), a water-soluble cyclic urea compound and a potent inhibitor of HIV replication in cell culture that also inhibits variants of HIV with single amino acid substitutions in the protease. DMP 450 is highly selective for HIV protease, consistent with displacement of the retrovirus-specific structural water molecule. Single doses of 10 mg kg-1 DMP 450 result in plasma levels in man in excess of that required to inhibit wild-type and several mutant HIVs. A plasmid-based, in vivo assay model suggests that maintenance of plasma levels of DMP 450 near the antiviral IC90 suppresses HIV protease activity in the animal. We did identify mutants that are resistant to DMP 450, however; multiple mutations within the protease gene caused a significant reduction in the antiviral response. CONCLUSIONS: DMP 450 is a significant advance within the cyclic urea class of HIV protease inhibitors due to its exceptional oral bioavailability. The data presented here suggest that an optimal cyclic urea will provide clinical benefit in treating AIDS if it combines favorable pharmacokinetics with potent activity against not only single mutants of HIV, but also multiply-mutant variants.

Design and selection of DMP 850 and DMP 851: the next generation of cyclic urea HIV protease inhibitors.

BACKGROUND: Recent clinical trials have demonstrated that HIV protease inhibitors are useful in the treatment of AIDS. It is necessary, however, to use HIV protease inhibitors in combination with other antiviral agents to inhibit the development of resistance. The daunting ability of the virus to rapidly generate resistant mutants suggests that there is an ongoing need for new HIV protease inhibitors with superior pharmacokinetic and efficacy profiles. In our attempts to design and select improved cyclic urea HIV protease inhibitors, we have simultaneously optimized potency, resistance profile, protein binding and oral bioavailability. RESULTS: We have discovered that nonsymmetrical cyclic ureas containing a 3-aminoindazole P2 group are potent inhibitors of HIV protease with excellent oral bioavailability. Furthermore, the 3-aminoindazole group forms four hydrogen bonds with the enzyme and imparts a good resistance profile. The nonsymmetrical 3-aminoindazoles DMP 850 and DMP 851 were selected as our next generation of cyclic urea HIV protease inhibitors because they achieve 8 h trough blood levels in dog, with a 10 mg/kg dose, at or above the protein-binding-adjusted IC90 value for the worst single mutant--that containing the Ile84-->Val mutation. CONCLUSIONS: In selecting our next generation of cyclic urea HIV protease inhibitors, we established a rigorous set of criteria designed to maximize chances for a sustained antiviral effect in HIV-infected individuals. As DMP 850 and DMP 851 provide plasma levels of free drug that are sufficient to inhibit wild-type HIV and several mutant forms of HIV, they could show improved ability to decrease viral load for clinically significant time periods. The ultimate success of DMP 850 and DMP 851 in clinical trials might depend on achieving or exceeding the oral bioavailability seen in dog.

Three-dimensional solution structure of the HIV-1 protease complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of the HIV-1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported. This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5 degrees, respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A. As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.

Stereoisomers of cyclic urea HIV-1 protease inhibitors: synthesis and binding affinities.

We have synthesized stereoisomers of cyclic urea HIV-1 protease inhibitors to study the effect of varying configurations on binding affinities. Four different synthetic approaches were used to prepare the desired cyclic urea stereoisomers. The original cyclic urea synthesis using amino acid starting materials was used to prepare three isomers. Three additional isomers were prepared by synthetic routes utilizing L-tartaric acid and D-sorbitol as chiral starting materials. A stereoselective hydroxyl inversion of the cyclic urea trans-diol was used to prepare three additional isomers. In all 9 of the 10 possible cyclic urea stereoisomers were prepared, and their binding affinities are described.

Cyclic HIV protease inhibitors: design and synthesis of orally bioavailable, pyrazole P2/P2' cyclic ureas with improved potency.

Highly potent HIV-1 protease (HIVPR) inhibitors have been designed and synthesized by introducing bidentate hydrogen-bonding oxime and pyrazole groups at the meta-position of the phenyl ring on the P2/P2' substituents of cyclic ureas. Nonsymmetrical cyclic ureas incorporating 3(1H)-pyrazolylbenzyl as P2 and hydrophilic functionalities as P2' show potent protease inhibition and antiviral activities against HIV and have good oral bioavailabilities. The X-ray structure of HIVPR.10A complex confirms that the two pyrazole rings of 10A form bidentate hydrogen bonds with the side-chain oxygen (C=O) and backbone nitrogen (N-H) of Asp30/30' of HIVPR.

HIV protease inhibitory bis-benzamide cyclic ureas: a quantitative structure-activity relationship analysis.

A series of N,N'-disubstituted cyclic urea 3-benzamides has been synthesized and evaluated for HIV protease inhibition and antiviral activity. Some of these benzamides have been shown to be potent inhibitors of HIV protease with Ki < 0.050 nM and IC90 < 20 nM for viral replication and, as such, may be useful in the treatment of AIDS. The synthesis and quantitative structure-activity relationship for this benzamide series will be discussed.

Nonpeptide cyclic cyanoguanidines as HIV-1 protease inhibitors: synthesis, structure-activity relationships, and X-ray crystal structure studies.

Comparison of the high-resolution X-ray structures of the native HIV-1 protease and its complexes with the inhibitors suggested that the enzyme flaps are flexible. The movement at the tip of the flaps could be as large as 7 A. On the basis of this observation, cyclic cyanoguanidines have been designed, synthesized, and evaluated as HIV-1 protease (PR) inhibitors. Cyclic cyanoguanidines were found to be very potent inhibitors of HIV-1 protease. The choice of cyclic cyanoguanidines over cyclic guanidines was based on the reduced basicity of the former. X-ray structure studies of the HIV PR complex with cyclic cyanoguanidine demonstrated that in analogy to cyclic urea, cyclic cyanoguanidines also displace the unique structural water molecule. The structure-activity relationship of the cyclic cyanoguanidines is compared with that of the corresponding cyclic urea analogues. The differences in binding constants of the two series of compounds have been rationalized using high-resolution X-ray structure information.

Discovery of 1-[3-(aminomethyl)phenyl]-N-3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa.

Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va and Ca2+ on the phospholipid surface of platelets or endothelial cells, factor Xa forms the prothrombinase complex, which is responsible for the proteolysis of prothrombin to catalytically active thrombin. Thrombin, in turn, catalyzes the cleavage of fibrinogen to fibrin, thus initiating a process that ultimately leads to clot formation. Recently, we reported on a series of isoxazoline and isoxazole monobasic noncovalent inhibitors of factor Xa which show good potency in animal models of thrombosis. In this paper, we wish to report on the optimization of the heterocyclic core, which ultimately led to the discovery of a novel pyrazole SN429 (2b; fXa K(i) = 13 pM). We also report on our efforts to improve the oral bioavailability and pharmacokinetic profile of this series while maintaining subnanomolar potency and in vitro selectivity. This was achieved by replacing the highly basic benzamidine P1 with a less basic benzylamine moiety. Further optimization of the pyrazole core substitution and the biphenyl P4 culminated in the discovery of DPC423 (17h), a highly potent, selective, and orally active factor Xa inhibitor which was chosen for clinical development.