Iterative Design Cycle Research Articles

In silico Prediction of Total Human Plasma Clearance

The prediction of the total human plasma clearance of novel chemical entities continues to be of paramount importance in drug design and optimization, because it impacts both dose size and dose regimen. Although many in vivo and in vitro methods have been proposed, a well-constructed, well-validated, and less resource-intensive computational tool would still be very useful in an iterative compound design cycle. A new completely in silico linear PLS (partial least-squares) model to predict the human plasma clearance was built on the basis of a large data set of 754 compounds using physicochemical descriptors and structural fragments, the latter able to better represent biotransformation processes. The model has been validated using the "ELASTICO" approach (Enhanced Leave Analog-Structural, Therapeutic, Ionization Class Out) based on ten therapeutic/structural analog classes. The model yields a geometric mean fold error (GMFE) of 2.1 and a percentage of compounds predicted within 2- and 3-fold error of 59% and 80%, respectively, showing an improved performance when compared with previous published works in predicting clearance of neutral compounds, and a very good performance with ionized molecules at pH 7.5, able to compare favorably with fairly accurate in vivo methods.

Design of Chemistry Teacher Education Course on Nature of Science

To enhance students’ understanding of nature of science (NOS), teachers need adequate pedagogical content knowledge related to NOS. The educational design research study presented here describes the design and development of a pre-service chemistry teacher education course on NOS instruction. The study documents two iterative cycles of problem analysis, design, implementation, and evaluation. The main aims of the study were (1) to create an in-depth and detailed description of the process used in the development of the course and the design solutions produced, and (2) to evaluate how the design solutions affected participants’ commitment to teach NOS. Based on the problem analysis based on challenges recognized from the previous research, three design solutions were produced: (1) definition of central dimensions of domain-specific NOS for chemistry education, (2) teaching cycle for explicit and structured opportunities for reflection and discussion, and (3) design assignments to translate NOS understanding into classroom practice. The major data-sources used in the evaluation of the design solutions were the four in-depth interviews conducted after the course. Based on the evaluation, the design solutions supported internalizing understanding of NOS and transforming the understanding to instruction. Supporting the implementation of new innovative teaching practices such as NOS instruction in pre-service teacher education is a challenge. However, the success of the participants in implementing NOS instruction demonstrates, that a pre-service teacher education course can be successful in producing early adopters of NOS instruction and thus might be one of the first steps in injecting NOS instruction into the curriculum.

A novel research approach to enhance research group‐level science data management

AbstractThis poster will report an alternative approach compared with large‐scale collection, technology development, or discipline informatics projects by exploring the enhancement of science data management at the research group level. An academic‐year‐long project involving both information and technical analysts demonstrated that flexible introduction of IT in the form of a relational database solution presents an opportunity to incorporate data‐related practices, behavior, and schemes into a tool. The project team used cognitive work analysis and participatory design research methods to sensitively analyze research practice and then design and introduce a prototype tool. Iterative analysis and design cycles captured and incorporated the complex practices of researchers who struggle with the accumulation of samples and related digital data files from years of research. Design research processes will be analyzed according to project developments and research group reaction to the proposed data management tool design and preliminary attempt at implementation.

Abstract A235: Structure-guided evolution of potent and selective oral inhibitors of CHK1 through scaffold morphing.

Abstract The DNA damage response network ensures the fidelity of DNA replication and controls the repair of damage arising during cellular replication or from exogenous agents such as genotoxic drugs. Checkpoint Kinase 1 (CHK1) is a serine/threonine kinase occupying a central position in this complex network of cell regulatory and DNA repair mechanisms. G1/S, S or G2/M cell cycle checkpoints are activated in response to genotoxic antitumor drugs to provide an opportunity for repair of damaged DNA or to activate apoptotic pathways. Unlike normal cells, human cancer cells frequently have functional defects in the tumor suppressor p53 with consequent loss of G1/S checkpoint control and greater reliance on S and G2/M checkpoints. Thus CHK1 inhibitors which abrogate the S and G2/M checkpoints will selectively sensitize p53 deficient cancer cells to DNA damaging agents. CHK1 inhibition by siRNA and several small molecule inhibitors have confirmed this in preclinical studies. The challenges of improving the CHK1 potency and selectivity of our initial, fragment derived pyrazolopyridine inhibitors, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps. Initial hit compounds were optimised into potent inhibitors of CHK1 using iterative cycles of design, synthesis, assay and crystallography, progressing through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and isoquinolines. The potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, and potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts when dosed i.p. in nude mice. Further lead optimisation led to orally bioavailable analogues with good in vitro ADME and in vivo pharmacokinetic properties, exemplified by CCT244747. CCT244747 has demonstrated both in vivo pharmacodynamic modulation of signaling through CHK1 and potentiation of cytotoxic drugs in human tumor xenografts. In summary, we show how a fragment derived compound with weak, micromolar activity against CHK1 evolved through a scaffold hopping strategy to give the selective CHK1 isoquinoline inhibitor SAR-020106, from which optimisation of pharmacokinetic properties led to potent, selective and orally bioavailable CHK1 inhibitors such as CCT244747. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A235.

Tricyclic Series of Heat Shock Protein 90 (Hsp90) Inhibitors Part I: Discovery of Tricyclic Imidazo[4,5-c]pyridines as Potent Inhibitors of the Hsp90 Molecular Chaperone

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed after a low throughput screen (LTS) of a focused library containing approximately 21K compounds selected by virtual screening. The initial [1-{3-H-imidazo[4-5-c]pyridin-2-yl}-3,4-dihydro-2H-pyrido[2,1-a]isoindole-6-one] (1) compound showed moderate activity (IC(50) = 7.6 μM on Hsp82, the yeast homologue of Hsp90). A high-resolution X-ray structure shows that compound 1 binds into an "induced" hydrophobic pocket, 10-15 Å away from the ATP/resorcinol binding site. Iterative cycles of structure-based drug design (SBDD) and chemical synthesis led to the design and preparation of analogues with improved affinity. These optimized molecules make productive interactions within the ATP binding site as reported by other Hsp90 inhibitors. This resulted in compound 8, which is a highly potent inhibitor in biochemical and cellular assays (K(d) = 0.35 nM on Hsp90; IC(50) = 30 nM on SKBr3 mammary carcinoma cells) and in an in vivo leukemia model.

Optimisation of ITK inhibitors through successive iterative design cycles

Based on a hit cluster of compounds inhibiting interleukin-2 inducible T-cell kinase (ITK) in the submicromolar range a series of ITK inhibitor libraries were synthesized. Through iterative design cycles including kinase crystal structure information, indolylindazole libraries were identified which showed low nanomolar activity in enzymatic and cellular assays. The potential of these novel lead series was confirmed through in vivo tests in an anti-CD3-IL2 mouse model. The intravenous administration of highly potent ITK inhibitor 11o resulted in dose-dependent, efficient suppression of IL-2.

Identification of potent ITK inhibitors through focused compound library design including structural information

A series of novel compound libraries inhibiting interleukin-2 inducible T cell kinase (ITK) were designed, synthesized and evaluated. In the first design cycle two library scaffolds were identified showing low micromolar inhibition of ITK. Further iterative design cycles including crystal structure information of ITK and structurally related kinases led to the identification of indolylindazole and indolylpyrazolopyridine compounds with low nanomolar ITK inhibition.

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.

Modeling and simulation to assist context aware system design

In this paper we present a modelling and visualization design approach for context-aware systems, specifically focusing on applications that support location tracking and that exhibit their behaviour as actuations in the deployment environment. The design tool addresses two key challenges in the design cycle for ubiquitous computing systems. Firstly, facilitating cost-effective, repeatable and easily (re-)configurable experiments that support thorough testing of the target deployment space, including outlier test scenarios. Secondly, supporting an iterative design cycle, using traceability of the causal relationships between context and exhibited system behaviour. The aim is to identify potentially problematic behaviour in the system under test, which can be provided as feedback to lead informed redesign of the prototype system. In this paper we present initial experiments using this tool to test a simple automatic lighting application. We discuss the results and the implications of the findings from these experiments for the models used to drive this design tool.

A synthetic low-frequency mammalian oscillator

Circadian clocks have long been known to be essential for the maintenance of physiological and behavioral processes in a variety of organisms ranging from plants to humans. Dysfunctions that subvert gene expression of oscillatory circadian-clock components may result in severe pathologies, including tumors and metabolic disorders. While the underlying molecular mechanisms and dynamics of complex gene behavior are not fully understood, synthetic approaches have provided substantial insight into the operation of complex control circuits, including that of oscillatory networks. Using iterative cycles of mathematical model-guided design and experimental analyses, we have developed a novel low-frequency mammalian oscillator. It incorporates intronically encoded siRNA-based silencing of the tetracycline-dependent transactivator to enable the autonomous and robust expression of a fluorescent transgene with periods of 26 h, a circadian clock-like oscillatory behavior. Using fluorescence-based time-lapse microscopy of engineered CHO-K1 cells, we profiled expression dynamics of a destabilized yellow fluorescent protein variant in single cells and real time. The novel oscillator design may enable further insights into the system dynamics of natural periodic processes as well as into siRNA-mediated transcription silencing. It may foster advances in design, analysis and application of complex synthetic systems in future gene therapy initiatives.

Albumin-based nanoparticles as magnetic resonance contrast agents: I. Concept, first syntheses and characterisation

To develop a platform for molecular magnetic resonance imaging, we prepared gadolinium-bearing albumin-polylactic acid nanoparticles in the size range 20-40 nm diameter. Iterative cycles of design and testing upscaled the synthesis procedures to gram amounts for physicochemical characterisation and for pharmacokinetic testing. Morphological analyses showed that the nanoparticles were spheroidal with rough surfaces. Particle sizes were measured by direct transmission electron microscopical measurements from negatively contrasted preparations, and by use of photon correlation spectroscopy; the two methods each documented nanoparticle sizes less than 100 nm and generally 10-40 nm diameter, though with significant intrabatch and interbatch variability. The particles' charge sufficed to hold them in suspension. HSA retained its tertiary structure in the particles. The nanoparticles were stable against turbulent flow conditions and against heat, though not against detergents. MRI imaging of liquid columns was possible at nanoparticle concentrations below 10 mg/ml. The particles were non-cytotoxic, non-thrombogenic and non-immunogenic in a range of assay systems developed for toxicity testing of nanoparticles. They were micellar prior to lyophilisation, but loosely structured aggregated masses after lyophilisation and subsequent resuspension. These nanoparticles provide a platform for further development, based on non-toxic materials of low immunogenicity already in clinical use, not expensive, and synthesized using methods which can be upscaled for industrial production.

Glass-composite prototyping for flow PCR with in situ DNA analysis

In this article, low cost microfluidic devices have been used for simultaneous amplification and analysis of DNA. Temperature gradient flow PCR was performed, during which the unique fluorescence signature of the amplifying product was determined. The devices were fabricated using xurography, a fast and highly flexible prototype manufacturing method. Each complete iterative design cycle, from concept to prototype, was completed in less than 1 h. The resulting devices were of a 96% glass composition, thereby possessing a high thermal stability during continuous-flow PCR. Volumetric flow rates up to 4 microl/min induced no measurable change in the temperature distribution within the microchannel. By incorporating a preliminary channel passivation protocol, even the first microliters through the system exhibited a high amplification efficiency, thereby demonstrating the biocompatibility of this fabrication technique for DNA amplification microfluidics. The serpentine microchannel induced 23 temperature gradient cycles in 15 min at a 2 microl/min flow rate. Fluorescent images of the device were acquired while and/or after the PCR mixture filled the microchannel. Because of the relatively high initial concentration of the phage DNA template (PhiX174), images taken after 10 min (less than 15 PCR cycles) could be used to positively identify the PCR product. A single fluorescent image of a full device provided the amplification curve for the entire reaction as well as multiple high resolution melting curves of the amplifying sample. In addition, the signal-to-noise ratio associated with the spatial fluorescence was characterized as a function of spatial redundancy and acquisition time.

Discovery of the imidazo[1,5- a][1,2,4]-triazolo[1,5- d][1,4]benzodiazepine scaffold as a novel, potent and selective GABA A α5 inverse agonist series

Through iterative design cycles we have discovered a number of novel new classes where the imidazo[1,5- a][1,2,4]-triazolo[1,5- d][1,4]benzodiazepine was deemed the most promising GABA A α5 inverse agonist class with potential for cognitive enhancement. This class combines a modest subtype binding selectivity with inverse agonism and has the most favourable molecular properties for further lead optimisation towards a central nervous system (CNS) acting medicine.

Design of a Catheter-Based Device for Performing Percutaneous Chordal-Cutting Procedures

In this paper we detail the rapid design, fabrication and testing of a percutaneous catheterbased device that is envisioned to enable externally controlled manipulation and cutting of specific chordae tendinae within the heart. The importance of this work is that it (a) provides a means that surgeons may use to alleviate problems associated with some forms of mitral valve regurgitation and (b) demonstrates how a deterministic design process may be used to drive design innovation in medical devices while lowering development cost/time/resources. In the United States alone, approximately 500,000 people develop ischemic or functional MR per year. A chordal cutting procedure and device could allow many patients, who would otherwise be unable to survive open-heart surgery, to undergo a potentially life-saving operation at reduced risk. The design process has enabled us to generate a solution to this problem in a relatively short time. A deterministic design process was used to generate several design concepts and then evaluate and compare each concept based on a set of functional requirements. A final concept to be alpha prototyped was then chosen, optimized, and fabricated. The design process made it possible to make rapid progress during the project and to achieve a device design that worked the first time. This approach is important to medical device design as it reduces engineering effort, cost, and the amount of time spent in iterative design cycles. An overview of the design process will be presented and discussed within the context of a specific case study–the rapid design/fabrication of a chordal cutting device. Experimental results will be used to assess: (i) The performance of the catheter in maneuvering into the heart and grasping various structures. (ii) The effectiveness of the catheter's RF ablation tip at cutting chordae inside of a heart. In the first experiment, the catheter was guided to the basal chordae under direct visualization, which showed that the catheter is capable of successfully grasping a chord. During the second experiment, ultrasound was shown to be a viable method of visualizing the catheter within the heart. During this experiment, once contact between the chord and RF ablator tip was confirmed, the chord was successfully ablated. We will also discuss experiments that are currently underway to visualize the catheter utilizing a Trans-Esophageal Echo probe, as well as imaging the mitral valve from the apex of the heart with a laparoscope so that video of the basal chord being grasped and cut can be acquired on a heart whose anatomical structures are intact. A brief synopsis will then be given of how the design process has been used in research and educational collaborations between MIT and local hospitals.

Discovery of Antibacterial Biotin Carboxylase Inhibitors by Virtual Screening and Fragment-Based Approaches

As part of our effort to inhibit bacterial fatty acid biosynthesis through the recently validated target biotin carboxylase, we employed a unique combination of two emergent lead discovery strategies. We used both de novo fragment-based drug discovery and virtual screening, which employs 3D shape and electrostatic property similarity searching. We screened a collection of unbiased low-molecular-weight molecules and identified a structurally diverse collection of weak-binding but ligand-efficient fragments as potential building blocks for biotin carboxylase ATP-competitive inhibitors. Through iterative cycles of structure-based drug design relying on successive fragment costructures, we improved the potency of the initial hits by up to 3000-fold while maintaining their ligand-efficiency and desirable physicochemical properties. In one example, hit-expansion efforts resulted in a series of amino-oxazoles with antibacterial activity. These results successfully demonstrate that virtual screening approaches can substantially augment fragment-based screening approaches to identify novel antibacterial agents.

Sequencing of design tasks based on the degree of permissible concurrency

Rapid product design and development are crucial factors to maintaining a competitive advantage in today's intense market. Time-consuming iterative design cycles cause critical delays within the design development process. One widely applied method to identify such iterations is the design structure matrix (DSM). Due to its distinctive ability in consistently rearranging factors of a complex problem using subjective measures, the analytic hierarchy process (AHP) was requisitely applied to quantify DSM binary relations. Despite its advantages, AHP becomes intractable for large design problems, where substantial pairwise comparisons take place. The core contribution of this paper is overcome this drawback, thus facilitate faster sequencing of design tasks within a quantified DSM while utilising the benefits of AHP. This will lead to reducing time spent in iterating within design subcycles and eventually reduce time-to-market. The developed methodology is illustrated on a design of a low emission vehicle (LEV).

Protein engineering of improved prolyl endopeptidases for celiac sprue therapy

Due to their unique ability to cleave immunotoxic gluten peptides endoproteolytically, prolyl endopeptidases (PEPs) are attractive oral therapeutic candidates for protecting celiac sprue patients from the toxic effects of dietary gluten. Enhancing the activity and stability of PEPs under gastric conditions (low pH, high pepsin concentration) is a challenge for protein engineers. Using a combination of sequence- and structure-based approaches together with machine learning algorithms, we have identified improved variants of the Sphingomonas capsulata PEP, a target of clinical relevance. Through two rounds of iterative mutagenesis and analysis, variants with as much as 20% enhanced specific activity at pH 4.5 and 200-fold greater resistance to pepsin were identified. Our results vividly reinforce the concept that conservative changes in proteins, especially in hydrophobic residues within tightly packed regions, can profoundly influence protein structure and function in ways that are difficult to predict entirely from first principles and must therefore be optimized through iterative design and analytical cycles. Incubation with whole wheat bread under simulated gastric conditions also suggests that some variants have pharmacologically significant improvements in gluten detoxification activity.

A small molecular scaffold for selective inhibition of Wip1 phosphatase.

Using a pyrrole-based scaffold, we developed a series of small molecules that mimic the three-dimensional arrangement of the polar and hydrophobic functional groups of the best cyclic-peptide inhibitor. Iterative optimization cycles of design, synthesis and kinetic testing has lead to an effective inhibitor of Wip1, that is selective for this phosphatase over others. The picture shows the structure of the best inhibitor bound to the active site of the enzyme.

Bound To Shock: Protection from Lethal Endotoxemic Shock by a Novel, Nontoxic, Alkylpolyamine Lipopolysaccharide Sequestrant

Lipopolysaccharide (LPS), or endotoxin, a structural component of gram-negative bacterial outer membranes, plays a key role in the pathogenesis of septic shock, a syndrome of severe systemic inflammation which leads to multiple-system organ failure. Despite advances in antimicrobial chemotherapy, sepsis continues to be the commonest cause of death in the critically ill patient. This is attributable to the lack of therapeutic options that aim at limiting the exposure to the toxin and the prevention of subsequent downstream inflammatory processes. Polymyxin B (PMB), a peptide antibiotic, is a prototype small molecule that binds and neutralizes LPS toxicity. However, the antibiotic is too toxic for systemic use as an LPS sequestrant. Based on a nuclear magnetic resonance-derived model of polymyxin B-LPS complex, we had earlier identified the pharmacophore necessary for optimal recognition and neutralization of the toxin. Iterative cycles of pharmacophore-based ligand design and evaluation have yielded a synthetically easily accessible N(1),mono-alkyl-mono-homologated spermine derivative, DS-96. We have found that DS-96 binds LPS and neutralizes its toxicity with a potency indistinguishable from that of PMB in a wide range of in vitro assays, affords complete protection in a murine model of LPS-induced lethality, and is apparently nontoxic in vertebrate animal models.

Fragment Based Drug Discovery using rational design

Fragment-based discovery has recently emerged as a new approach for the generation of novel small molecule therapeutic agents. The use of high throughput X-ray crystallography, as well as NMR, in fragment-based discovery approaches will be exemplified in this talk. Methodology that utilizes high throughput X-ray crystallography and NMR to screen fragment libraries will be described. This approach for lead generation has distinct advantages over conventional bioassay-based screening in that very low-affinity fragments with novel structures can be identified. These “fragment hits” can then be rapidly optimized for potency and DMPK properties using iterative cycles of medicinal chemistry and structure-based drug design. The development of novel lead compounds using this approach will be described for targets such as the cyclin-dependant kinases and aurora kinase, key proteins involved in cancer. These compounds, AT7519 and AT9283, were identified using fragment-discovery and are now being tested in clinical trials as potential anti-cancer therapies.