Essential ingredients for rational drug design.

This short review focuses on three aspects of rational drug design that we consider of utmost importance: the conformation of small molecules in solid form, the conformation of small molecules in solution and lesser studied interactions in protein-ligand complexes. Using examples from recent literature, we will illustrate these different aspects and how they have contributed to the discovery of potent modulators.

N-terminal modification of VEGF-A C terminus-derived peptides delineates structural features involved in neuropilin-1 binding and functional activity.

The interaction between VEGF-A and its neuropilin (NRP) receptors mediates a number of important biological effects. NRP1 and the related molecule NRP2 are widely expressed on multiple tumour types and throughout the tumour vasculature, and are emerging as critical molecules required for the progression of angiogenic diseases. Given the increasing evidence supporting a role for NRP1 in tumour development, there is growing interest in developing inhibitors of NRP1 interactions with VEGF and its other ligands. In order to probe the interaction we synthesised a number of exon 7- and 8-derived bicyclic peptides with N-terminal lipophilic groups and found a simple N-octanoyl derivative (EG00086) to be the most potent and functionally active. Detailed modelling studies indicated that new intramolecular hydrogen bonds were formed, stabilising the structure and possibly contributing to the potency. Removal of a salt bridge between D142 and R164 implicated in VEGF-A binding to neuropilin-1 had a minor effect on potency. Isothermal calorimetry was used to assess binding of EG00086 to NRP1 and NRP2, and the stability of the peptide in serum and in vivo was investigated. EG00086 is a potent blocker of VEGF-promoted cellular adhesion to extracellular matrices, and phosphorylation of p130Cas contributes to this effect.

Preparation and ring-opening reactions of N-diphenylphosphinyl vinyl aziridines.

Predominantly (E)-N-diphenylphosphinyl vinyl aziridines are prepared by a reaction of N-diphenylphosphinyl imines with α-bromoallyllithium in the presence of freshly fused ZnCl2. These aziridines undergo a ring-opening reaction with a variety of carbon and heteronucleophiles, in good yield, and generally with good regioselectivity.

MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo.

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.

Small Molecule Neuropilin-1 Antagonists Combine Antiangiogenic and Antitumor Activity with Immune Modulation through Reduction of Transforming Growth Factor Beta (TGFß) Production in Regulatory T-Cells.

We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound 1 (EG01377) was identified as having properties suitable for further investigation. Compound 1 was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, 1 was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1+, FoxP3+, and CD25+ populations of Tregs from mice, 1 was able to block a glioma-conditioned medium-induced increase in TGFß production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies.

Fortification and health: challenges and opportunities.

Fortification is the process of adding nutrients or non-nutrient bioactive components to edible products (e.g., food, food constituents, or supplements). Fortification can be used to correct or prevent widespread nutrient intake shortfalls and associated deficiencies, to balance the total nutrient profile of a diet, to restore nutrients lost in processing, or to appeal to consumers looking to supplement their diet. Food fortification could be considered as a public health strategy to enhance nutrient intakes of a population. Over the past century, fortification has been effective at reducing the risk of nutrient deficiency diseases such as beriberi, goiter, pellagra, and rickets. However, the world today is very different from when fortification emerged in the 1920s. Although early fortification programs were designed to eliminate deficiency diseases, current fortification programs are based on low dietary intakes rather than a diagnosable condition. Moving forward, we must be diligent in our approach to achieving effective and responsible fortification practices and policies, including responsible marketing of fortified products. Fortification must be applied prudently, its effects monitored diligently, and the public informed effectively about its benefits through consumer education efforts. Clear lines of authority for establishing fortification guidelines should be developed and should take into account changing population demographics, changes in the food supply, and advances in technology. This article is a summary of a symposium presented at the ASN Scientific Sessions and Annual Meeting at Experimental Biology 2014 on current issues involving fortification focusing primarily on the United States and Canada and recommendations for the development of responsible fortification practices to ensure their safety and effectiveness.

Principles for building public-private partnerships to benefit food safety, nutrition, and health research.

The present article articulates principles for effective public-private partnerships (PPPs) in scientific research. Recognizing that PPPs represent one approach for creating research collaborations and that there are other methods outside the scope of this article, PPPs can be useful in leveraging diverse expertise among government, academic, and industry researchers to address public health needs and questions concerned with nutrition, health, food science, and food and ingredient safety. A three-step process was used to identify the principles proposed herein: step 1) review of existing PPP guidelines, both in the peer-reviewed literature and at 16 disparate non-industry organizations; step 2) analysis of relevant successful or promising PPPs; and step 3) formal background interviews of 27 experienced, senior-level individuals from academia, government, industry, foundations, and non-governmental organizations. This process resulted in the articulation of 12 potential principles for establishing and managing successful research PPPs. The review of existing guidelines showed that guidelines for research partnerships currently reside largely within institutions rather than in the peer-reviewed literature. This article aims to introduce these principles into the literature to serve as a framework for dialogue and for future PPPs.

National prevalence of methicillin-resistant Staphylococcus aureus in inpatients at United States health care facilities, 2010.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the most prevalent multidrug-resistant organisms causing health care-associated infections. Limited data are available about how the prevalence of MRSA has changed over the past several years and what MRSA prevention practices have been implemented since the 2006 Association for Professionals in Infection Control and Epidemiology, Inc, MRSA survey. METHODS: We conducted a national prevalence survey of MRSA colonization or infection in inpatients at US health care facilities. The survey was developed, received institutional review board approval, and then was distributed to all US Association for Professionals in Infection Control and Epidemiology, Inc, members. Members were asked to complete the survey on 1 day during the period August 1 to December 30, 2010, reporting the number of inpatients with MRSA infection or colonization and facility- and patient-specific information. RESULTS: Personnel at 590 facilities indicated a state and responded to the survey. All states were represented, except for Alaska and Washington, DC (mean, 12 facilities per state; range, 1-38). Respondents reported 4,476 MRSA-colonized/infected patients in 67,412 inpatients; the overall MRSA prevalence rate was 66.4 per 1,000 inpatients (25.3 infections and 41.1 colonizations per 1,000 inpatients). Active surveillance testing was conducted by 75.7% of the respondents; 39.6% used nonselective media, 37.2% used selective media, and 23.3% used polymerase chain reaction. Detailed data were provided on 3,176 MRSA-colonized/infected patients. Of those in whom colonization/infection status was reported (1,908/3,086 [61.8%] were MRSA colonized and 1,778/3,086 [38.2%] were MRSA infected), most MRSA-colonized or infected patients (78.3%) were detected within 48 hours of admission; the most common site of infection was skin and soft tissue (42.9%); and, using the Centers for Disease Control and Prevention's definitions, approximately 50% would be classified as health care-associated infections. CONCLUSION: Our survey documents that the MRSA prevalence in 2010 is higher than that reported in our 2006 survey. However, the majority of facilities currently are performing active surveillance testing, and, compared with 2006, the rate of MRSA infection has decreased while the rate of MRSA colonization has increased. In addition, compared with 2006, the proportion of MRSA strains recovered from MRSA-colonized/infected patients that are health care-associated strains has deceased, and community-associated strains have increased.

Small molecule inhibitors of the neuropilin-1 vascular endothelial growth factor A (VEGF-A) interaction.

We report the molecular design and synthesis of EG00229, 2, the first small molecule ligand for the VEGF-A receptor neuropilin 1 (NRP1) and the structural characterization of NRP1-ligand complexes by NMR spectroscopy and X-ray crystallography. Mutagenesis studies localized VEGF-A binding in the NRP1 b1 domain and a peptide fragment of VEGF-A was shown to bind at the same site by NMR, providing the basis for small molecule design. Compound 2 demonstrated inhibition of VEGF-A binding to NRP1 and attenuated VEGFR2 phosphorylation in endothelial cells. Inhibition of migration of endothelial cells was also observed. The viability of A549 lung carcinoma cells was reduced by 2, and it increased the potency of the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination. These studies provide the basis for design of specific small molecule inhibitors of ligand binding to NRP1.

National point prevalence of Clostridium difficile in US health care facility inpatients, 2008.

BACKGROUND: Recent published estimates of Clostridium difficile infection (CDI) incidence have been based on small numbers of hospitals or national hospital discharge data. These data suggest that CDI incidence is increasing. METHODS: We conducted a point prevalence survey of C difficile in inpatients at US health care facilities. The survey was developed, received Institutional Review Board approval, and was then distributed to all Association for Professionals in Infection Control and Epidemiology, Inc (APIC) members. They were asked to complete the survey on 1 day between May 7 and August 29, 2008, reporting the number of inpatients with CDI or colonization and facility-specific information. RESULTS: Personnel at 648 hospitals completed the survey; this represents approximately 12.5% of all US acute care facilities. All but 3 states and the District of Columbia were represented (mean, 14 facilities per state; range, 2-43). Eighty-two percent reported that their CDI rate had not decreased in the past 3 years. Respondents reported 1443 C difficile-colonized/infected patients among 110,550 inpatients; the overall C difficile prevalence rate was 13.1 per 1000 inpatients (94.4% infection). Detailed data were provided on 1062 (73.6%) patients. Of these, 55.5% were female, 69.2% were >60 years of age, 67.6% had selected comorbid conditions, 79% had received antimicrobials within 30 days, and 94.4% were detected by enzyme immunoassay. The majority of patients (54.4%) were diagnosed < or =48 hours of hospitalization, but 35% had been admitted to a long-term care facility within 30 days, and 47% had been hospitalized within 90 days; 73% met Centers for Disease Control and Prevention criteria for health care-associated CDI. Most facilities (>90%) used contact isolation for CDI patients. Bleach was used for environmental disinfection more commonly during CDI outbreaks than during nonoutbreak periods. CONCLUSION: Our survey documents a higher C difficile prevalence rate than previous estimates using different methodologies. The majority of inpatient CDI appears to be health care associated. Given that not all patients with diarrhea are tested for CDI and that most facilities use enzyme immunoassays with limited sensitivity to detect C difficile, these are minimum estimates of the US health care facility C difficile burden.

Aurora A and B kinases as targets for cancer: will they be selective for tumors?

Aurora A and B kinases are closely related kinases involved in regulating separate points in the cell cycle. This review highlights the rationale for Aurora kinases as cancer targets and examines the currently known Aurora kinase inhibitors in the patent and scientific literature. The known crystal structures of the Aurora kinases are described with relevance to bound ligand interactions and the prospect of the generation of drug-resistant mutant forms. The potential for selectivity versus primary cells will also be discussed. The status of the inhibitors in clinical development is described.

Characterization of a bicyclic peptide neuropilin-1 (NP-1) antagonist (EG3287) reveals importance of vascular endothelial growth factor exon 8 for NP-1 binding and role of NP-1 in KDR signaling.

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (K(i) 1.2 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-gamma tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1.