Cost-Utility Analysis Compared Between Radiotherapy Alone and Combined Surgery and Radiotherapy for Symptomatic Spinal Metastases in Thailand

Objective To investigate the patient quality of life and cost-utility compared between radiotherapy alone and combined surgery and radiotherapy for spinal metastasis (SM) in Thailand. Methods Patients with SM with an indication for surgery during 2018–2020 were prospectively recruited. Patients were assigned to either the combination surgery and radiotherapy group or the radiotherapy alone group. Quality of life was assessed by EuroQol-5D-5L (EQ-5D-5L) questionnaire, and relevant healthcare costs were collected pretreatment, and at 3-month and 6-month posttreatment. Total lifetime cost and quality-adjusted life-years (QALYs) were estimated for each group. Results Twenty-four SM patients (18 females, 6 males) were included. Of those, 12 patients underwent combination treatment, and 12 underwent radiotherapy alone. At 6-month posttreatment, 10 patients in the surgery group, and 11 patients in the nonsurgery group remained alive for a survival rate of 83.3% and 91.7%, retrospectively. At 6-month posttreatment, the mean utility in the combination treatment group was significantly better than in the radiotherapy alone group (0.804 ± 0.264 vs. 0.518 ± 0.282, respectively; p = 0.011). Total lifetime costs were 59,863.14 United States dollar (USD) in the combination treatment group and 24,526.97 USD in the radiation-only group. The incremental cost-effectiveness ratio using 6-month follow-up data was 57,074.01 USD per QALY gained. Conclusion Surgical treatment combined with radiotherapy to treat SM significantly improved patient quality of life compared to radiotherapy alone during the 6-month posttreatment period. However, combination treatment was found not to be cost-effective compared to radiotherapy alone for SM at the Thailand willingness-to-pay threshold of 5,113 USD/QALY

Revealing the sequence and resulting cellular morphology of receptor-ligand interactions during plasmodium falciparum invasion of erythrocytes

During blood stage Plasmodium falciparum infection, merozoites invade uninfected erythrocytes via a complex, multistep process involving a series of distinct receptor-ligand binding events. Understanding each element in this process increases the potential to block the parasite's life cycle via drugs or vaccines. To investigate specific receptor-ligand interactions, they were systematically blocked using a combination of genetic deletion, enzymatic receptor cleavage and inhibition of binding via antibodies, peptides and small molecules, and the resulting temporal changes in invasion and morphological effects on erythrocytes were filmed using live cell imaging. Analysis of the videos have shown receptor-ligand interactions occur in the following sequence with the following cellular morphologies; 1) an early heparin-blockable interaction which weakly deforms the erythrocyte, 2) EBA and PfRh ligands which strongly deform the erythrocyte, a process dependant on the merozoite's actin-myosin motor, 3) a PfRh5-basigin binding step which results in a pore or opening between parasite and host through which it appears small molecules and possibly invasion components can flow and 4) an AMA1-RON2 interaction that mediates tight junction formation, which acts as an anchor point for internalization. In addition to enhancing general knowledge of apicomplexan biology, this work provides a rational basis to combine sequentially acting merozoite vaccine candidates in a single multi-receptor-blocking vaccine

Analysis of 6-cys proteins and calcium fluxes during erythrocyte invasion by Plasmodium falciparum parasites

© 2015 Dr. Tana TaechalertpaisarnPlasmodium parasites amplify their population within the human host by invading, growing and replicating within the body’s erythrocytes. When the population becomes high enough, the damage caused produces symptomatic malaria disease. To develop new drugs and vaccines against malaria it is therefore important to know as much possible about how parasites grow within the human host and particularly about how the extracellular merozoite stage invades erythrocytes, since this short-lived stage is highly vulnerable. This thesis provides new information from the most deadly human malaria pathogen P. falciparum, on the biochemical characteristics of a little known family of merozoite surface proteins which were thought to facilitate erythrocyte invasion as well revealing with unprecedented resolution, new details about how merozoites enter erythrocytes. P12, P38, P41, and P92 comprise a group of blood-stage merozoite surface proteins that belong to the 6-cys family and all except P41 are predicted to have membrane anchors. To functionally characterize the proteins, specific antibodies were made and were then employed to block merozoite invasion by interfering with the binding of 6-cys to erythrocytes. The effect of the antibodies was very weak and therefore not indicative of a major role for 6-cys in invasion. The antibodies were then used as localization probes and indicated that P12 and P41 were at the merozoite periphery with some concentrated towards the apex. In addition, the non-anchored P41 was held on the merozoite surface through heterodimerization with the membrane anchored P12. Despite the P12/P41 heterodimer being in prime position to bind erythrocytes during invasion no evidence for binding could be established. Characterisation of P92 was next conducted and revealed that like the P12/P41 heterodimer, it was tightly associated with the parasite membrane and later cleaved off possibly during invasion. On the other hand, P38 did not shed from the merozoite surface, and it was carried into the erythrocyte. P92 was strictly localised to the apical end of the merozoite while P38 displayed both apical and surface localisation. Similar to the P12/P41 heterodimer, P92 does not appear to bind erythrocytes. In a final attempt to derive a function for the blood stage 6-cys, their genes were individually knocked out but none of the mutants produced any defective growth or invasion phenotypes suggestive of function. To further study invasion, the morphology and kinetics of this process in P. falciparum merozoites was examined with high-speed live-cell microscopy. With greater temporal resolution, novel cellular actions of the merozoites were observed. For example, during the 7.5 s pre-invasion phase the merozoite deforms the erythrocyte plasma membrane multiple times whilst re-orientating. After a brief rest, the merozoite invaded over a ~17 s period forming a vacuole mainly from wrapping the erythrocyte’s membrane around itself. About 18.5 s after entry, the merozoite began spinning in a clockwise direction to possibly to help disconnect itself from the erythrocyte membrane. After spinning had commenced the host erythrocyte began to develop a spiculated appearance called echinocytosis. Suspecting that calcium influx into the erythrocyte during invasion might be responsible for the echinocytosis, the appearance of these fluxes was monitored during invasion by live cell imaging. These observations confirmed for the first time, that a calcium flux originated as an intense spot emanating from the area of contact between the merozoite and erythrocyte suggestive of pore formation between the cells. Further experiments with modified levels of calcium indicated the ion is required for efficient invasion and may play role in causing echinocytosis. Other work using the calcium flux as a visual marker indicated that pore formation coincided with the deployment of tight adhesive proteins from the merozoite that commit it to invasion. The live cell imaging work presented therefore sheds considerable light on many details of merozoite invasion that could inform future drug and vaccine development. Supplementary Videos: Video 1. High-speed time-lapse acquisition of 3D7 merozoite invading the erythrocyte (40 fps). Video 2. The 3D7 merozoite invading the BODIPY FL C12-sphingomyelin labelled erythrocyte (2 fps, 2× real speed). Video 3. The 3D7 merozoite invading the erythrocyte in the presence of Fluo-4 AM showing the punctate apical calcium and calcium influx in the infected erythrocyte (3 fps, 8× real speed) Video 4. Fluo-4-stained 3D7 parasite culture showing merozoites attempting to invade in the presence of R1 peptide (3 fps, 8× real speed). The punctate apical calcium and influx in the attached erythrocyte were detectable. Video 5. Fluo-4-stained 3D7 parasite culture showing merozoites attempting to invade in the presence of R1 peptide (variable speed). The echinocytotic erythrocyte had not recovered after ~20 min of recording. Video 6. CytD-treated 3D7 merozoite attempting to invade the Fluo-4 labelled erythrocyte (variable speed). The punctate apical calcium was visible but the calcium influx was difficult to observe. The echinocytotic erythrocyte had not recovered after ~20 min of recording

Design, Optimization and Syntheses of Small Molecules to Disrupt Protein-Protein Interactions

Protein-protein interactions (PPIs) are one of the basic mechanisms in cellular biology, but also involve in diseases if they are dysregulation. Disrupting aberrant PPI activities is useful in medicinal chemistry. One approach to inhibit PPIs is to design small molecule peptidomimetics bearing side-chain orientations similar to protein ligands, in which those mimics might displace or interfere the native PPIs. Previous research in our group developed Exploring Key Orientations (EKO) program that matches Cɑ-Cß coordinates of virtual small molecules to the side-chain vectors of proteins at PPI interfaces. Similar Cɑ-Cß orientations between mimics and protein ligands indicate that small molecules might be suitable to displace protein ligands, i.e. those compounds might interfere PPIs. We used EKO to deduce small molecules that might disrupt medicinally-relevant PPIs. Herein, EKO implicated our designed mimics, hydantoin-oxazoline, triazole-oxazole and triazole-oxazoline derivatives, might disrupt Nef•MHC-I•AP1 and NEDD8•NAE interactions, in which they are relevant to HIV-1 and cancer diseases respectively. After learning from these projects, we designed hydantoin-piperazine analogues to disrupt PCSK9•LDLR interaction that causes hypercholesterolemia disease. Although the firstgeneration hydantoin-piperazine derivatives did not show good PCSK9•LDLR inhibition, we modified chemotype structures by cooperating with a docking program, Glide, to improve inhibitory potencies. As a result, we successfully obtained lead compounds that significantly disrupt PCSK9•LDLR interaction with the measurable binding affinities. Besides these protein targets, we synthesized another minimalist mimic, oxazoline piperidine-2,4-dione, that has conformational biases toward helical and sheet-turn-sheet motifs. This structure potentially has favorable cellular- and oral-permeability calculated by QikProp. We are also interested in how to design molecules suitable for PPI inhibition. A concept of secondary structure mimicry is widely applied to design molecules that resemble a secondary structure at an PPI interface, hence possibly disrupt protein-protein interaction. However, there is no direct study to prove a correlation between secondary structure mimicry and interface mimicry. To respond this issue, we used EKO to match several new chemotypes on the ideal secondary structures and PPIs database, and then compared the frequencies of secondary structures that chemotypes matched at PPI interfaces to the ideal secondary structure biases of each chemotype. We found that, in general, good secondary structure mimics tend to match frequently at PPI interfaces; however, they mostly match on non-ideal secondary structure motifs

Antimalarial target vulnerability of the putative Plasmodium falciparum methionine synthase

Background Plasmodium falciparum possesses a cobalamin-dependent methionine synthase (MS). MS is putatively encoded by the PF3D7_1233700 gene, which is orthologous and syntenic in Plasmodium. However, its vulnerability as an antimalarial target has not been assessed. Methods We edited the PF3D7_1233700 and PF3D7_0417200 (dihydrofolate reductase-thymidylate synthase, DHFR-TS) genes and obtained transgenic P. falciparum parasites expressing epitope-tagged target proteins under the control of the glmS ribozyme. Conditional loss-of-function mutants were obtained by treating transgenic parasites with glucosamine. Results DHFR-TS, but not MS mutants showed a significant proliferation defect over 96 h, suggesting that P. falciparum MS is not a vulnerable antimalarial target

Discriminant Analysis PCA-LDA Assisted Surface-Enhanced Raman Spectroscopy for Direct Identification of Malaria-Infected Red Blood Cells

Various methods for detecting malaria have been developed in recent years, each with its own set of advantages. These methods include microscopic, antigen-based, and molecular-based analysis of blood samples. This study aimed to develop a new, alternative procedure for clinical use by using a large data set of surface-enhanced Raman spectra to distinguish normal and infected red blood cells. PCA-LDA algorithms were used to produce models for separating P. falciparum (3D7)-infected red blood cells and normal red blood cells based on their Raman spectra. Both average normalized spectra and spectral imaging were considered. However, these initial spectra could hardly differentiate normal cells from the infected cells. Then, discrimination analysis was applied to assist in the classification and visualization of the different spectral data sets. The results showed a clear separation in the PCA-LDA coordinate. A blind test was also carried out to evaluate the efficiency of the PCA-LDA separation model and achieved a prediction accuracy of up to 80%. Considering that the PCA-LDA separation accuracy will improve when a larger set of training data is incorporated into the existing database, the proposed method could be highly effective for the identification of malaria-infected red blood cells

Small Molecule Inhibitors of the PCSK9·LDLR Interaction

The protein–protein interaction between proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) is a relatively new, and extremely important, validated therapeutic target for treatment and prevention of heart disease. Experts in the area agree that the first small molecules to disrupt PCSK9·LDLR would represent a milestone in this field, yet few credible leads have been reported. This paper describes how side-chain orientations in preferred conformations of carefully designed chemotypes were compared with LDLR side chains at the PCSK9·LDLR interface to find molecules that would mimic interface regions of LDLR. This approach is an example of the procedure called EKO (Exploring Key Orientations). The guiding hypothesis on which EKO is based is that good matches indicate the chemotypes bearing the same side chains as the protein at the sites of overlay have the potential to disrupt the parent protein–protein interaction. In the event, the EKO procedure and one round of combinatorial fragment-based virtual docking led to the discovery of seven compounds that bound PCSK9 (SPR and ELISA) and had a favorable outcome in a cellular assay (hepatocyte uptake of fluorescently labeled low-density lipoprotein particles) and increased the expression LDLR on hepatocytes in culture. Three promising hit compounds in this series had dissociation constants for PCSK9 binding in the 20–40 μM range, and one of these was modified with a photoaffinity label and shown to form a covalent conjugate with PCSK9 on photolysis

A New Amino Acid for Improving Permeability and Solubility in Macrocyclic Peptides through Side Chain-to-Backbone Hydrogen Bonding

Despite the notoriously poor membrane permeability of peptides, many cyclic peptide natural products show high passive membrane permeability and potently inhibit a variety of "undruggable" intracellular targets. A major impediment to the design of cyclic peptides with good permeability is the high desolvation energy associated with the peptide backbone amide NH groups. While several strategies have been proposed to mitigate this deleterious effect, only few studies have used polar side chains to sequester backbone NH groups. We investigated the ability of N,N-pyrrolidinylglutamine (Pye), whose side chain contains a powerful hydrogen-bond-accepting C═O amide group but no hydrogen-bond donors, to sequester exposed backbone NH groups in a series of cyclic hexapeptide diastereomers. Analyses revealed that specific Leu-to-Pye substitutions conferred dramatic improvements in aqueous solubility and permeability in a scaffold- and position-dependent manner. Therefore, this approach offers a complementary tool for improving membrane permeability and solubility in cyclic peptides