Crystal structure of dihydrofolate reductase from the filarial nematode W. bancrofti in complex with NADPH and folate.

Lymphatic filariasis is a debilitating illness with an estimated 50 million cases as of 2018. The majority of cases are caused by the parasitic worm W. bancrofti and additional cases by the worms B. malayi and B. timori. Dihydrofolate reductase (DHFR) is an established target in the treatment of cancer, bacterial, and protozoal infections and may be a potential target for drugs targeting parasitic worm infections, including filariasis. Recent studies have shown that known antifolate compounds, including methotrexate, inhibit the activity of W. bancrofti DHFR (WbDHFR). However, the absence of structural information for filarial DHFRs has limited the study of more in-depth structure-function relationships. We report the structure of WbDHFR complexed with NADPH and folate using X-ray diffraction data measured to 2.47 Å resolution. The structure of WbDHFR reveals the usual DHFR fold and is currently only the second nematode DHFR structure in the Protein Data Bank. The equilibrium dissociation constants for NADPH (90 ± 29 nM) and folate (23 ± 4 nM) were determined by equilibrium titrations. The interactions of known antifolates with WbDHFR were analyzed using molecular docking programs and molecular dynamics simulations. Antifolates with a hydrophobic core and extended linker formed favorable interactions with WbDHFR. These combined data should now facilitate the rational design of filarial DHFR inhibitors, which in turn can be used to determine whether DHFR is a viable drug target for filariasis and whether existing antifolates may be repurposed for its treatment.

Development of 3D-Printed Layered PLGA Films for Drug Delivery and Evaluation of Drug Release Behaviors.

3D printing has been widely used to rapidly manufacture a variety of solid dosage forms on-demand, without sacrificing precision. This study used extrusion-based 3D printing to prepare single-layered, tri-layered, and core-in-shell poly(lactic-co-glycolic acid) (PLGA) films carrying paclitaxel and rapamycin in combination or lidocaine alone. Each layer was composed of either low molecular weight (MW) PLGA or high MW PLGA. In vitro drug release kinetics of paclitaxel, rapamycin, and lidocaine for PLGA films were assessed and compared with PLGA-polyethylene glycol (PEG)-PLGA hydrogel discs. Regardless of the structure of PLGA film, paclitaxel (half-time: 54-63 days) was released faster than when compared with rapamycin (half-time: 74-80 days). In contrast, single-layered PLGA-PEG-PLGA discs released rapamycin (half-time 5.7 h) at a more rapid rate than paclitaxel (half-time: 7.3 h). Single-layered PLGA-PEG-PLGA discs enabled a faster drug release than PLGA films, noting that the disc matrices dissolve in water in 24 h. Similarly, lidocaine incorporated in PLGA films (half-time: 13-36 days) exhibited slower release patterns than that in PLGA-PEG-PLGA discs (half-time: 2.6 h). In vitro drug release patterns were explained using molecular models that simulate drug-polymer interactions. Analysis of models suggested that drug-polymer interactions, location of each drug in the polymeric matrix, and solubility of drugs in water were major factors that determine drug release behaviors from the polymeric films and discs.

Limonene-induced activation of A2A adenosine receptors reduces airway inflammation and reactivity in a mouse model of asthma.

Animal models of asthma have shown that limonene, a naturally occurring terpene in citrus fruits, can reduce inflammation and airway reactivity. However, the mechanism of these effects is unknown. We first performed computational and molecular docking analyses that showed limonene could bind to both A2A and A2B receptors. The pharmacological studies were carried out with A2A adenosine receptor knock-out (A2AKO) and wild-type (WT) mice using ovalbumin (OVA) to generate the asthma phenotype. We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) and NECA (nonselective adenosine analog) by administering limonene as an inhalation prior to OVA aerosol challenges in one group of allergic mice for both WT and KO. In whole-body plethysmography studies, we observed that airway responsiveness to MCh in WT SEN group was significantly lowered upon limonene treatment but no effect was observed in A2AKO. Limonene also attenuated NECA-induced airway responsiveness in WT allergic mice with no effect being observed in A2AKO groups. Differential BAL analysis showed that limonene reduced levels of eosinophils in allergic WT mice but not in A2AKO. However, limonene reduced neutrophils in sensitized A2AKO mice, suggesting that it may activate A2B receptors as well. These data indicate that limonene-induced reduction in airway inflammation and airway reactivity occurs mainly via activation of A2AAR but A2B receptors may also play a supporting role.

Structure-enhanced methods in the development of non-nucleoside inhibitors targeting HIV reverse transcriptase variants.

Resistance continues to emerge as a leading cause for antiretroviral treatment failure. Several mutations in HIV reverse transcriptase (RT) confer resistance to non-nucleoside inhibitors (NNRTIs), vital components of antiretroviral combination therapies. Since the majority of mutations are located in the NNRTI binding pocket, crystal structures of RT variants in complex with NNRTIs have provided ideas for new drug design strategies. This article reviews the impact of RT crystal structures on the multidisciplinary design and development of new inhibitors with improved resistance profiles.

Discovery and crystallography of bicyclic arylaminoazines as potent inhibitors of HIV-1 reverse transcriptase.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-indolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility. Crystal structures for two complexes enhance the analysis of the structure-activity data.

Illuminating the molecular mechanisms of tyrosine kinase inhibitor resistance for the FGFR1 gatekeeper mutation: the Achilles' heel of targeted therapy.

Human fibroblast growth factor receptors (FGFRs) 1-4 are a family of receptor tyrosine kinases that can serve as drivers of tumorigenesis. In particular, FGFR1 gene amplification has been implicated in squamous cell lung and breast cancers. Tyrosine kinase inhibitors (TKIs) targeting FGFR1, including AZD4547 and E3810 (Lucitanib), are currently in early phase clinical trials. Unfortunately, drug resistance limits the long-term success of TKIs, with mutations at the "gatekeeper" residue leading to tumor progression. Here we show the first structural and kinetic characterization of the FGFR1 gatekeeper mutation, V561M FGFR1. The V561M mutation confers a 38-fold increase in autophosphorylation achieved at least in part by a network of interacting residues forming a hydrophobic spine to stabilize the active conformation. Moreover, kinetic assays established that the V561M mutation confers significant resistance to E3810, while retaining affinity for AZD4547. Structural analyses of these TKIs with wild type (WT) and gatekeeper mutant forms of FGFR1 offer clues to developing inhibitors that maintain potency against gatekeeper mutations. We show that AZD4547 affinity is preserved by V561M FGFR1 due to a flexible linker that allows multiple inhibitor binding modes. This is the first example of a TKI binding in distinct conformations to WT and gatekeeper mutant forms of FGFR, highlighting adaptable regions in both the inhibitor and binding pocket crucial for drug design. Exploiting inhibitor flexibility to overcome drug resistance has been a successful strategy for combatting diseases such as AIDS and may be an important approach for designing inhibitors effective against kinase gatekeeper mutations.

Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase-dihydrofolate reductase.

Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors.

Substituted pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis thymidylate synthase inhibitors.

Cryptosporidiosis, a gastrointestinal disease caused by a protozoan Cryptosporidium hominis is often fatal in immunocompromised individuals. There is little clinical data to show that the existing treatment by nitazoxanide and paromomycin is effective in immunocompromised individuals. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer and malaria. A novel series of classical antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines have been evaluated as Cryptosporidium hominis thymidylate synthase (ChTS) inhibitors. Crystal structure in complex with the most potent compound, a 2'-chlorophenyl with a sulfur bridge with a Ki of 8.83±0.67 nM is discussed in terms of several Van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate. Of these interactions, two interactions with the non-conserved residues (A287 and S290) offer an opportunity to develop ChTS specific inhibitors. Compound 6 serves as a lead compound for analog design and its crystal structure provides clues for the design of ChTS specific inhibitors.

Pre-steady state kinetic analysis of cyclobutyl derivatives of 2'-deoxyadenosine 5'-triphosphate as inhibitors of HIV-1 reverse transcriptase.

Pre-steady state kinetic analysis was utilized for biochemical evaluation of a series of cyclobutyl adenosine nucleotide analogs with HIV-1 RT(WT). The phosphonyl-diphosphate form of the cyclobutyl nucleotide, 5, was the most efficiently incorporated of the series. Nucleotide 5 was fourfold more efficiently incorporated than the FDA approved TFV-DP by RT(WT). The kinetics of incorporation for 5 using the drug resistant mutant enzyme K65R was also determined. Compound 5 was threefold more efficiently incorporated compared to TFV-DP with RT(K65R). These results demonstrate cyclobutyl adenosine analogs can act as substrates for incorporation by HIV-1 RT and be a potential scaffold for HIV inhibitors.