IMPACT organizational survey highlighting provision of services for patients with locally advanced and recurrent colorectal cancer across Great Britain and Ireland.

AIM: Locally advanced and recurrent colorectal cancer represents a complex clinical entity, which requires multidisciplinary decision-making and management. The aim of this work is to understand the provision of clinical services in this cohort of patients across Great Britain and Ireland (GB&I) as a key essential step to help facilitate future service development and improvement. METHOD: A cross-sectional, organizational survey was sent to all colorectal cancer multidisciplinary teams (MDTs) across GB&I. It consisted of 12 key questions addressing the provision of specialist services and advanced surgical techniques. Results are reported in line with the CHERRIES guideline. RESULTS: One hundred and seventy-five MDTs across GB&I participated, with 142 English, 13 Welsh, 14 Scottish, 3 Northern Irish and 3 Irish MDTs. The overall response rate was 93.5% (175/187). Ninety (51.4%) hospital sites reported having a specialist dedicated or subsection MDT. Specialist advanced nursing support was available in 46 (26.2%) hospitals, with a dedicated advanced colorectal cancer outpatient clinic available in 31 (17.7%) hospitals. One hundred and thirteen MDTs (64.5%) offered surgery for advanced colonic cancer, 82 (46.8%) for recurrent colonic cancer, 58 (33.1%) for advanced rectal cancer and 39 (22.2%) for recurrent rectal cancer. A variable number of MDTs offered specialist surgical techniques, including distal sacrectomy [33 (18.9%)], high sacrectomy [16 (9.1%)], complex vascular resection ± reconstruction [33 (18.9%)] and extended lymphadenectomy (pelvic sidewall or para-aortic) [44 (25.1%)]. CONCLUSION: The IMPACT organizational survey highlights the current variation in the delivery and provision of clinical services for patients with advanced and recurrent colorectal cancer across Great Britain and Ireland.

Use of 3M-052-AF with Alum adjuvant in HIV trimer vaccine induces human autologous neutralizing antibodies.

Stabilized trimers preserving the native-like HIV envelope structure may be key components of a preventive HIV vaccine regimen to induce broadly neutralizing antibodies (bnAbs). We evaluated trimeric BG505 SOSIP.664 gp140 formulated with a novel TLR7/8 signaling adjuvant, 3M-052-AF/Alum, for safety, adjuvant dose-finding, and immunogenicity in a first-in-healthy adult (n = 17), randomized, and placebo-controlled trial (HVTN 137A). The vaccine regimen appeared safe. Robust, trimer-specific antibody, and B cell and CD4+ T cell responses emerged after vaccination. Five vaccinees developed serum autologous tier 2 nAbs (ID50 titer, 1:28-1:8647) after two to three doses targeting C3/V5 and/or V1/V2/V3 Env regions by electron microscopy and mutated pseudovirus-based neutralization analyses. Trimer-specific, B cell-derived monoclonal antibody activities confirmed these results and showed weak heterologous neutralization in the strongest responder. Our findings demonstrate the clinical utility of the 3M-052-AF/Alum adjuvant and support further improvements of trimer-based Env immunogens to focus responses on multiple broad nAb epitopes.

Structure-Based Identification of Novel Histone Deacetylase 4 (HDAC4) Inhibitors.

Histone deacetylases (HDACs) are important cancer drug targets. Existing FDA-approved drugs target the catalytic pocket of HDACs, which is conserved across subfamilies (classes) of HDAC. However, engineering specificity is an important goal. Herein, we use molecular modeling approaches to identify and target potential novel pockets specific to Class IIA HDAC-HDAC4 at the interface between HDAC4 and the transcriptional corepressor component protein NCoR. These pockets were screened using an ensemble docking approach combined with consensus scoring to identify compounds with a different binding mechanism than the currently known HDAC modulators. Binding was compared in experimental assays between HDAC4 and HDAC3, which belong to a different family of HDACs. HDAC4 was significantly inhibited by compound 88402 but not HDAC3. Two other compounds (67436 and 134199) had IC50 values in the low micromolar range for both HDACs, which is comparable to the known inhibitor of HDAC4, SAHA (Vorinostat). However, both of these compounds were significantly weaker inhibitors of HDAC3 than SAHA and thus more selective, albeit to a limited extent. Five compounds exhibited activity on human breast carcinoma and/or urothelial carcinoma cell lines. The present result suggests potential mechanistic and chemical approaches for developing selective HDAC4 modulators.

HIV BG505 SOSIP.664 trimer with 3M-052-AF/alum induces human autologous tier-2 neutralizing antibodies.

Stabilized trimers preserving the native-like HIV envelope structure may be key components of a preventive HIV vaccine regimen to induce broadly neutralizing antibodies (bnAbs). We evaluated trimeric BG505 SOSIP.664 gp140, formulated with a novel TLR7/8 signaling adjuvant, 3M-052-AF/Alum, for safety, adjuvant dose-finding and immunogenicity in a first-in-healthy adult (n=17), randomized, placebo-controlled trial (HVTN 137A). The vaccine regimen appeared safe. Robust, trimer-specific antibody, B-cell and CD4+ T-cell responses emerged post-vaccination. Five vaccinees developed serum autologous tier-2 nAbs (ID50 titer, 1:28-1:8647) after 2-3 doses targeting C3/V5 and/or V1/V2/V3 Env regions by electron microscopy and mutated pseudovirus-based neutralization analyses. Trimer-specific, B-cell-derived monoclonal antibody activities confirmed these results and showed weak heterologous neutralization in the strongest responder. Our findings demonstrate the clinical utility of the 3M-052-AF/alum adjuvant and support further improvements of trimer-based Env immunogens to focus responses on multiple broad nAb epitopes. KEY TAKEAWAY/TAKE-HOME MESSAGES: HIV BG505 SOSIP.664 trimer with novel 3M-052-AF/alum adjuvant in humans appears safe and induces serum neutralizing antibodies to matched clade A, tier 2 virus, that map to diverse Env epitopes with relatively high titers. The novel adjuvant may be an important mediator of vaccine response.

Modulating Enzyme Function via Dynamic Allostery within Biliverdin Reductase B.

The biliverdin reductase B (BLVRB) class of enzymes catalyze the NADPH-dependent reduction of multiple flavin substrates and are emerging as critical players in cellular redox regulation. However, the role of dynamics and allostery have not been addressed, prompting studies here that have revealed a position 15 Å away from the active site within human BLVRB (T164) that is inherently dynamic and can be mutated to control global micro-millisecond motions and function. By comparing the inherent dynamics through nuclear magnetic resonance (NMR) relaxation approaches of evolutionarily distinct BLVRB homologues and by applying our previously developed Relaxation And Single Site Multiple Mutations (RASSMM) approach that monitors both the functional and dynamic effects of multiple mutations to the single T164 site, we have discovered that the most dramatic mutagenic effects coincide with evolutionary changes and these modulate coenzyme binding. Thus, evolutionarily changing sites distal to the active site serve as dynamic "dials" to globally modulate motions and function. Despite the distal dynamic and functional coupling modulated by this site, micro-millisecond motions span an order of magnitude in their apparent kinetic rates of motions. Thus, global dynamics within BLVRB are a collection of partially coupled motions tied to catalytic function.

Pelvic pain: What are the symptoms and predictors for surgery, endometriosis and endometriosis severity.

BACKGROUND: Chronic pelvic pain (CPP) is a common condition which significantly impacts the quality of life and wellbeing of many women. Laparoscopy with histopathology is recommended for investigation of pelvic pain and identification of endometriosis with concurrent removal. Never-the-less, the association between endometriosis and pelvic pain is challenging, with endometriosis identified in only 30-50% of women with pain. AIMS: To explore the predictors for undergoing surgery, for identifying endometriosis and endometriosis severity in a cohort of women with CPP. MATERIALS AND METHODS: This study forms part of the Persistent Pelvic Pain project, a prospective observational cohort study (ANZCTR:ACTRN12616000150448). Women referred to a public gynaecology clinic with pain were randomised to one of two gynaecology units for routine care and followed for 36 months with 6-monthly surveys assessing demographics, medical history, quality of life, and pain symptoms measured on a Likert scale. Operative notes were reviewed and endometriosis staged. RESULTS: Of 471 women recruited, 102 women underwent laparoscopy or laparotomy, of whom 52 had endometriosis (n = 37 stage I-II; n = 15 Stage III-IV). Gynaecology unit, pelvic pain intensity and lower parity were all predictors of surgery (odds ratio (OR) 0.342; 95% CI 0.209-0.561; OR 1.303; 95% CI: 1.079-1.573; OR 0.767; 95% CI: 0.620-0.949, respectively). There were no predictors identified for endometriosis diagnosis and the only predictor of severity was increasing age (OR 1.155; 95% CI: 1.047-1.310). CONCLUSIONS: Gynaecology unit and pain intensity were key predictors of undergoing laparoscopy; however, pain severity did not predict endometriosis diagnosis or staging. These findings indicate the need to review current frameworks guiding practice toward surgery for pelvic pain.

Bat influenza vectored NS1-truncated live vaccine protects pigs against heterologous virus challenge.

Swine influenza is an important disease for the swine industry. Currently used whole inactivated virus (WIV) vaccines can induce vaccine-associated enhanced respiratory disease (VAERD) in pigs when the vaccine strains mismatch with the infected viruses. Live attenuated influenza virus vaccine (LAIV) is effective to protect pigs against homologous and heterologous swine influenza virus infections without inducing VAERD but has safety concerns due to potential reassortment with circulating viruses. Herein, we used a chimeric bat influenza Bat09:mH3mN2 virus, which contains both surface HA and NA gene open reading frames of the A/swine/Texas/4199-2/1998 (H3N2) and six internal genes from the novel bat H17N10 virus, to develop modified live-attenuated viruses (MLVs) as vaccine candidates which cannot reassort with canonical influenza A viruses by co-infection. Two attenuated MLV vaccine candidates including the virus that expresses a truncated NS1 (Bat09:mH3mN2-NS1-128, MLV1) or expresses both a truncated NS1 and the swine IL-18 (Bat09:mH3mN2-NS1-128-IL-18, MLV2) were generated and evaluated in pigs against a heterologous H3N2 virus using the WIV vaccine as a control. Compared to the WIV vaccine, both MLV vaccines were able to reduce lesions and virus replication in lungs and limit nasal virus shedding without VAERD, also induced significantly higher levels of mucosal IgA response in lungs and significantly increased numbers of antigen-specific IFN-γ secreting cells against the challenge virus. However, no significant difference was observed in efficacy between the MLV1 and MLV2. These results indicate that bat influenza vectored MLV vaccines can be used as a safe live vaccine to prevent swine influenza.

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19 F NMR.

R67 dihydrofolate reductase (R67 DHFR) is a plasmid-encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry-related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N-termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N-terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4- and 5-fluoroindole labeled protein and severe line broadening for 6- and 7-fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the 19 F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.

Impaired lipid metabolism in astrocytes underlies degeneration of cortical projection neurons in hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSPs) are caused by a length-dependent axonopathy of long corticospinal neurons, but how axons of these cortical projection neurons (PNs) degenerate remains elusive. We generated isogenic human pluripotent stem cell (hPSC) lines for two ATL1 missense mutations associated with SPG3A, the most common early-onset autosomal dominant HSP. In hPSC-derived cortical PNs, ATL1 mutations resulted in reduced axonal outgrowth, impaired axonal transport, and accumulated axonal swellings, recapitulating disease-specific phenotypes. Importantly, ATL1 mutations dysregulated proteolipid gene expression, reduced lipid droplet size in astrocytes, and unexpectedly disrupted cholesterol transfer from glia to neurons, leading to cholesterol deficiency in SPG3A cortical PNs. Applying cholesterol or conditioned medium from control astrocytes, a major source of cholesterol in the brain, rescued aberrant axonal transport and swellings in SPG3A cortical PNs. Furthermore, treatment with the NR1H2 agonist GW3965 corrected lipid droplet defects in SPG3A astrocytes and promoted cholesterol efflux from astrocytes, leading to restoration of cholesterol levels and rescue of axonal degeneration in SPG3A cortical PNs. These results reveal a non-cell autonomous mechanism underlying axonal degeneration of cortical PNs mediated by impaired cholesterol homeostasis in glia.

A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

The Structural Basis for Nonsteroidal Anti-Inflammatory Drug Inhibition of Human Dihydrofolate Reductase.

Although nonsteroidal anti-inflammatory drugs (NSAIDs) target primarily cyclooxygenase enzymes, a subset of NSAIDs containing carboxylate groups also has been reported to competitively inhibit dihydrofolate reductase (DHFR). In this study, we have characterized NSAID interactions with human DHFR based on kinetic, NMR, and X-ray crystallographic methods. The NSAIDs target a region of the folate binding site that interacts with the p-aminobenzoyl-l-glutamate (pABG) moiety of folate and inhibit cooperatively with ligands that target the adjacent pteridine-recognition subsite. NSAIDs containing benzoate or salicylate groups were identified as having the highest potency. Among those tested, diflunisal, a salicylate derivative not previously identified to have anti-folate activity, was found to have a Ki of 34 µM, well below peak plasma diflunisal levels reached at typical dosage levels. The potential of these drugs to interfere with the inflammatory process by multiple pathways introduces the possibility of further optimization to design dual-targeted analogs.

Effects of Osmolytes on Ligand Binding to Dihydropteroate Synthase from Bacillus anthracis.

Osmolyte interactions with ligands can affect their affinity for proteins and are dependent upon the cosolute and the functional groups of the ligand. Here, we explored ligand binding to Bacillus anthracis dihydropteroate synthase (BaDHPS) under osmotic stress conditions. Osmolyte effects were specific to the cosolute and ligand, suggesting interaction of the osmolytes with the free ligands in solution. The association rates of pterin pyrophosphate were mostly unaffected by the osmolytes, except for a 2-fold decrease in the presence of 1 M trehalose, while the dissociation rates decreased in most osmolyte solutions. The viscosity and dielectric constant of the solution did not correlate with the effects of the osmolytes. Experimental results were compared with predicted preferential interaction coefficients (Δµ23/RT) between the osmolytes and ligands. The Δµ23/RT were able to predict the experimental data for most of the osmolytes. Trehalose and proline effects did not correlate with the predicted values, indicating that these two osmolytes may affect binding in more complex ways than simple preferential interactions. Additionally, osmolytes weakly interacted with the sulfa drug sulfathiazole, which altered its affinity for BaDHPS, suggesting that these types of weak interactions can also impact drug binding. As osmolytes affect ligands binding to two different folate cycle enzymes (DHFRs and DHPS), we predicted how ligand binding to other folate cycle enzymes will be altered by the presence of osmolytes.

Catalytic activity and stabilization of phenyl-modified glucose oxidase at high hydrostatic pressure.

Glucose oxidase (GOx) was modified by attaching phenyl groups to either carboxyl or amino side chains on the enzyme. High hydrostatic pressure (HHP) stabilized the aniline-, and benzoate-modified GOx at 69.1-80 °C compared to atmospheric pressure. At 240 MPa and 80.0 °C, the first order rate constant of inactivation kinact. of aniline-modified GOx was 20 × 10-2 min-1, or 3.7 times smaller than for the native GOx, while the kinact for benzoate-modified GOx was 26 × 10-2 min-1, or 2.8 times smaller than for the native GOx at the same temperature. Furthermore, at 240 MPa and 80.0 °C, the kinact of the aniline-modified GOx was 69 times smaller than the kinact of native GOx (1530 × 10-2 min-1) at 0.1 MPa and 80.0 °C. Similar results were obtained for benzoate-modified GOx. At each temperature in this study (25-69.1 °C), the catalytic activity of the native, aniline-, or benzoate-modified GOx increased with HHP, and reached a maximum at around 180 MPa. At 180 MPa and 69.1 °C, aniline-modified GOx produced the fastest catalytic rate, followed by benzoate-modified GOx, and then native GOx. An increase in temperature increased the activation volume of the reaction. Similarly, the activation energy increased with pressure. The combination of HHP and hydrophobic modification made GOx more thermostable and increased the effect of temperature in enzyme activity.

Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family.

Biliverdin reductase B (BLVRB) family members are general flavin reductases critical in maintaining cellular redox with recent findings revealing that BLVRB alone can dictate cellular fate. However, as opposed to most enzymes, the BLVRB family remains enigmatic with an evolutionarily changing active site and unknown structural and functional consequences. Here, we applied a multi-faceted approach that combines X-ray crystallography, NMR and kinetics methods to elucidate the structural and functional basis of the evolutionarily changing BLVRB active site. Using a panel of three BLVRB isoforms (human, lemur and hyrax) and multiple human BLVRB mutants, our studies reveal a novel evolutionary mechanism where coenzyme 'clamps' formed by arginine side chains at two co-evolving positions within the active site serve to slow coenzyme release (Positions 14 and 78). We find that coenzyme release is further slowed by the weaker binding substrate, resulting in relatively slow turnover numbers. However, different BLVRB active sites imposed by either evolution or mutagenesis exhibit a surprising inverse relationship between coenzyme release and substrate turnover that is independent of the faster chemical step of hydride transfer also measured here. Collectively, our studies have elucidated the role of the evolutionarily changing BLVRB active site that serves to modulate coenzyme release and has revealed that coenzyme release is coupled to substrate turnover.

Metagenomic Next-Generation Sequencing Reveal Presence of a Novel Ungulate Bocaparvovirus in Alpacas.

Viruses belonging to the genus Bocaparvovirus(BoV) are a genetically diverse group of DNA viruses known to cause respiratory, enteric, and neurological diseases in animals, including humans. An intestinal sample from an alpaca (Vicugnapacos) herd with reoccurring diarrhea and respiratory disease was submitted for next-generation sequencing, revealing the presence of a BoV strain. The alpaca BoV strain (AlBoV) had a 58.58% whole genome nucleotide percent identity to a camel BoV from Dubai, belonging to a tentative ungulate BoV 8 species (UBoV8). Recombination events were lacking with other UBoV strains. The AlBoV genome was comprised of the NS1, NP1, and VP1 proteins. The NS1 protein had the highest amino acid percent identity range (57.89-67.85%) to the members of UBoV8, which was below the 85% cut-off set by the International Committee on Taxonomy of Viruses. The low NS1 amino acid identity suggests that AlBoV is a tentative new species. The whole genome, NS1, NP1, and VP1 phylogenetic trees illustrated distinct branching of AlBoV, sharing a common ancestor with UBoV8. Walker loop and Phospholipase A2 (PLA2) motifs that are vital for virus infectivity were identified in NS1 and VP1 proteins, respectively. Our study reports a novel BoV strain in an alpaca intestinal sample and highlights the need for additional BoV research.

Five-year oncological outcomes after selective neoadjuvant radiotherapy for resectable rectal cancer.

Introduction: There is considerable variation in selection of patients for and type of neoadjuvant radiotherapy administered in the treatment of resectable rectal cancer. The aim of this study was to report outcomes for patients with resected rectal cancer from a unit with step-wise selection for surgery alone, short course radiotherapy (SCRT) or downstaging long course chemoradiotherapy (LCCRT). Material and methods: Cohort analysis of patients with rectal adenocarcinoma resected with curative intent between 2008 and 2012 at a specialist regional colorectal surgery center. The primary endpoints were local recurrence, metastatic recurrence, disease-free survival and overall survival. Exploratory uni- and multi-variable regression analyses were performed to identify predictive factors. Results: About 240 patients were treated by surgery alone, 90 patients received SCRT and 91 patients received LCCRT. Five-year local recurrence was 10.8% in the surgery alone group, 3.3% with SCRT and 18.7% with LCCRT. Metachronous distant metastasis was highest in the SCRT group (13.8% surgery alone, 25.6% SCRT, 15.4% LCCRT). Uni- and multi-variable regression analysis found that local and distant recurrence was attributable predominantly to adverse tumor biology. Conclusions: Patients selected for SCRT had a lower rate of local recurrence than patients selected for surgery alone, but were more likely to develop distant metastasis. There was no difference in overall survival. With low local recurrence rates, distant metastasis is the predominant risk for patients with resectable rectal cancer.

Virus survival and fitness when multiple genotypes and subtypes of influenza A viruses exist and circulate in swine.

We performed swine influenza virus (SIV) surveillance in Midwest USA and isolated 100 SIVs including endemic and reassortant H1 and H3 viruses with 2009 pandemic H1N1 genes. To determine virus evolution when different genotypes and subtypes of influenza A viruses circulating in the same swine herd, a virus survival experiment was conducted in pigs mimicking field situations. Five different SIVs were used to infect five pigs individually, then two groups of sentinel pigs were introduced to investigate virus transmission. Results showed that each virus replicated efficiently in lungs of each infected pig, but only reassortant H3N2 and H1N2v viruses transmitted to the primary contact pigs. Interestingly, the parental H1N2v was the majority of virus detected in the second group of sentinel pigs. These data indicate that the H1N2v seems to be more viable in swine herds than other SIV genotypes, and reassortment can enhance viral fitness and transmission.

Identification and evaluation of antivirals for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is the causative agent of Rift Valley fever (RVF) that affects both livestock and humans. There are neither fully licensed RVF vaccines available for human or animal use, nor effective antiviral drugs approved for human use in the U.S. To identify antiviral compounds effective for RVF, we developed and employed a cell-based high-throughput assay using a recombinant RVFV MP-12 strain, which expresses Renilla luciferase in place of the NSs protein, to screen 727 small compounds purchased from the National Institutes of Health. Twenty-three compounds were initially identified using the screening assay. Two compounds, 6-azauridine and mitoxantrone, also inhibited the replication of the parental MP-12 strain encoding the NSs gene, with limited cytotoxic effects. The respective 50% inhibitory concentrations were 29.07 µM and 79.85 µM when tested with the parental MP-12 strain at a multiplicity of infection of 2. The compounds were further evaluated using the STAT-1 KO mouse model. At one hour post intranasal inoculation of MP-12 strain, mice were intranasally treated with each indicated compound twice daily. Mice treated with either placebo or 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurologic symptoms. Onset of disease was, however, delayed in mice treated with either ribavirin or mitoxantrone. The results indicated that mitoxantrone can reduce the severity of diseases in RVFV-infected mice. Our studies build the foundation for the initial screening and efficacy studies of RVF antivirals in a BSL-2 environment, avoiding the higher risks of BSL-3 exposure with wild-type virus.

Crowders Steal Dihydrofolate Reductase Ligands through Quinary Interactions.

Dihydrofolate reductase (DHFR) reduces dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. Due to its role in one carbon metabolism, chromosomal DHFR is the target of the antibacterial drug, trimethoprim. Resistance to trimethoprim has resulted in a type II DHFR that is not structurally related to the chromosomal enzyme target. Because of its metabolic significance, understanding DHFR kinetics and ligand binding behavior in more cell-like conditions, where the total macromolecule concentration can be as great as 300 mg/mL, is important. The progress-curve kinetics and ligand binding properties of the drug target (chromosomal E. coli DHFR) and the drug resistant (R67 DHFR) enzymes were studied in the presence of macromolecular cosolutes. There were varied effects on NADPH oxidation and binding to the two DHFRs, with some cosolutes increasing affinity and others weakening binding. However, DHF binding and reduction in both DHFRs decreased in the presence of all cosolutes. The decreased binding of ligands is mostly attributed to weak associations with the macromolecules, as opposed to crowder effects on the DHFRs. Computer simulations found weak, transient interactions for both ligands with several proteins. The net charge of protein cosolutes correlated with effects on NADP+ binding, with near neutral and positively charged proteins having more detrimental effects on binding. For DHF binding, effects correlated more with the size of binding pockets on the protein crowders. These nonspecific interactions between DHFR ligands and proteins predict that the in vivo efficiency of DHFRs may be much lower than expected from their in vitro rates.