Practice Trends in the Management of Asymptomatic Breast Reconstruction Patients with Textured Implants: A Survey Analysis.

Despite the increasing prevalence of breast implant associated anaplastic large cell lymphoma, there remains a paucity of literature guiding management of asymptomatic patients with textured breast implants. This risk can be anxiety provoking in breast reconstruction patients given their history of cancer or increased future risk. The purpose of this study is to evaluate current practice trends when managing the concerned asymptomatic patient following textured implant-based breast reconstruction. Methods: An electronic survey was distributed to members of the American Society of Plastic Surgeons, regarding management of asymptomatic breast reconstruction patients with textured devices. Anonymous responses were collected, and statistical analysis was performed. Results: A total of 304 responses were received. Of respondents, 237 (92%) have managed asymptomatic patients with textured devices. Historically, the overwhelming majority (89%) used textured devices; however, only 25% report current use. Regarding management of asymptomatic breast reconstruction patients, 87% recommend conservative management, while 13% recommend surgical management. When surgery is performed, 16.3% of respondents elected for implant exchange, 33.8% recommended implant exchange with partial capsulectomy, and 49.8% elected for implant exchange with total capsulectomy. Evaluation of practice patterns based on demographics demonstrated statistically significant differences in current use of textured devices and management of acellular dermal matrix. Conclusions: Despite decreased current use, there is a significant population of asymptomatic breast reconstruction patients with a history of textured devices concerned for risk of breast implant associated anaplastic large cell lymphoma. This survey demonstrates ongoing variability in surgeon recommendations regarding conservative and surgical management of these patients and the need for continued development of evidence-based guidelines.

Efficacy of Demineralized Bone Matrix for Revision Alveolar Bone Grafting in Patients Previously Treated with Bone Morphogenetic Protein 2 (BMP-2).

OBJECTIVE: This study investigates the effectiveness of demineralized bone matrix (DBX) to close alveolar clefts in patients previously treated with bone morphogenic protein-2 (BMP-2) who remained with bone nonunion. DESIGN: This is an IRB-approved retrospective, single-center study. SETTING: This study was conducted at a tertiary academic center. PATIENTS/PARTICIPANTS: We searched for all surgical encounters with the Current Procedural Terminology (CPT) code 42210 from the years 2013-2019. Included patients were diagnosed with cleft alveolus, previous BMP-2 exposure and required revision bone grafting during mixed dentition for persistent alveolar defects. INTERVENTIONS: 17 patients underwent revision alveolar bone grafting (ABG) with either DBX (n = 10) or autograft (n = 7) to repair persistent bony cleft. MAIN OUTCOME MEASURE(S): The primary study outcome measured was alveolar bone graft revision failure described as continued alveolar nonunion. RESULTS: The median age at revision ABG was 13.1 ± 3.3 years, with a mean follow-up time of 4.9 years (1.1-9.2 years). Patients were 53% male, 47% had a unilateral cleft lip and alveolus. 58.8% of patients were treated with DBX in the cleft, 41.2% treated with autograft from iliac crest. Overall, 11.8% (n = 2) of all revisions failed, requiring a second revision. The average time to reoperation was 2.06 years, and both were re-grafted with autograft. There was no statistically significant difference between the type of bone graft source used and the failure rate obtained (P = .1544). CONCLUSIONS: DBX and autologous iliac crest bone grafts achieve similar alveolar union rates during revision ABG in patients treated with previous BMP-2 to the alveolar cleft.

Radioactive and stable isotopes reveal variations in nearshore submarine groundwater discharge composition and magnitude across low inflow northwestern Gulf of Mexico estuaries.

To determine how submarine groundwater discharge (SGD) magnitudes and composition (fresh or saline/recirculated) vary in nearshore low inflow estuaries across ⁓125 km of a semiarid coastline, this study assessed three south Texas estuaries, using radon [222Rn], radium [226Ra and 224Ra], and water isotopes [δ18O and δD]. Mass balance models of time-series 222Rn, found to be representative of total SGD in this study, revealed much higher SGD inputs to the Nueces Estuary (average [x̅] Nueces, Corpus Christi and Oso Bays: 120, 83, and 44 cm·d-1, respectively), attributed to anthropogenically-disturbed substrates and potentially surfacing growth-faults. The lowest 222Rn-derived SGD occurred in the Upper Laguna Madre Estuary (x̅: Upper Laguna Madre and Baffin Bay: 21 and 18 cm·d-1, respectively), explained by the drier climate, lower anthropogenic disturbance, and neighboring groundwater cone of depression. Aransas Bay in the Mission Aransas Estuary received greater average annual precipitation but exhibited low total SGD rates (x̅: 23 cm·d-1). Seasonally, average 222Rn-derived SGD rates increased following Hurricane Harvey (43 cm·d-1 in spring to 64 cm·d-1 in summer). In the Nueces Estuary, the overall 222Rn-derived SGDs were substantially higher than SGDs from 224Ra and 226Ra. The closer agreement between 224Ra and 222Rn-derived SGD and larger 224Ra rates in the Upper Laguna Madre Estuary, Aransas Bay and Oso Bay indicate that saline/recirculated SGD contributions were significant. Values of δ18O and δD confirm these types of inputs, with effects of evaporation/salinization more pronounced where recirculation was predominant and the opposite where terrestrial/222Rn-derived SGD inputs dominate. 226Ra-derived SGDs were lower than the 224Ra due to different behavior of the two isotopes while released into water following transport through saline and fine-grained estuarine sediments or due to wind-driven disturbances.

Parental Rejecting Behaviors: Validating a Behaviorally Based Youth-Report Measure of Parental Emotional Abuse.

Positive and warm parental attitudes are associated with better social and emotional child functioning, whereas negative or rejecting parental attitudes are associated with poor outcomes, such as aggression, impaired self-esteem, and emotional instability. The current study investigated the reliability and validity of scores on an interview adaptation of a measure of parental rejecting behavior (PRB) in a sample of detained adolescents. Participants (N = 198) completed a measure assessing their memories of the frequency of specific parental behaviors associated with rejection and self-report measures of exposure to abuse/neglect and community violence, and internalizing and externalizing psychopathology. PRB scores were internally consistent and associated with several kinds of child maltreatment. PRB scores correlated uniquely with indices of internalizing and externalizing symptomatology, even after controlling for indices of overall child maltreatment or a specific index of emotional abuse. The pattern of correlations suggests that the measure provides a valid index of parental emotional abuse, which may help identify youth at risk for both internalizing and externalizing disorders.

Emergency Medical Services resource capacity and competency amid COVID-19 in the United States: Preliminary findings from a national survey

OBJECTIVEThis study aimed to investigate available resources, Personal Protective Equipment (PPE) availability, sanitation practices, institutional policies, and opinions among EMS professionals in the United States amid the COVID-19 pandemic using a self-report survey questionnaire. METHODSAn online 42-question multiple choice survey was randomly distributed between April 1, 2020, and April 12, 2020 to various active Emergency Medical Services (EMS) paid personnel in all 50 U.S. states including the District of Columbia (n=165). We approximate a 95% confidence interval ({+/-} 0.0755). RESULTSAn overwhelming number of EMS providers report having limited access to N95 respirators, receiving little or no benefits from COVID-19 related work, and report no institutional policy on social distancing practices despite CDC recommendations. For providers who do have access to N95 respirators, 31% report having to use the same mask for 1 week or longer. Approximately [1/3] of the surveyed participants were unsure of when a COVID-19 patient is infectious. The data suggests regular decontamination of EMS equipment after each patient contact is not a regular practice. DISCUSSIONCurrent practices to educate EMS providers on appropriate response to the novel coronavirus may not be sufficient, and future patients may benefit from a nationally established COVID-19 EMS response protocol. Further investigation on whether current EMS practices are contributing to the spread of infection is warranted. The data reveals concerning deficits in COVID-19 related education and administrative protocols which pose as a serious public health concern that should be urgently addressed. Key MessagesO_ST_ABSWhat is already known on this subjectC_ST_ABSO_LICOVID-19 presents as a national emergency in the United States, and all efforts to mitigate the spread of disease should be considered C_LIO_LIEmergency Medical Services personnel play a pivotal role in patient outcomes and are often the first healthcare providers to make contact with COVID-19 patients C_LIO_LIThe CDC has provided an Interim guidance for EMS professionals amid the COVID-19 pandemic C_LI What this study addsO_LIDue to varied decontamination practices and administrative protocols that are non-compliant with CDC recommendations, EMS providers may inadvertently contribute to the spread of infection C_LIO_LIDue to varied knowledge and opinions of EMS providers on COVID-19, current pandemic education approaches may need to be revisited C_LI

Microfabrication, separations, and detection by mass spectrometry on ultrathin-layer chromatography plates prepared via the low-pressure chemical vapor deposition of silicon nitride onto carbon nanotube templates.

Microfabrication of ultrathin-layer chromatography (UTLC) plates via conformal deposition of silicon nitride by low-pressure chemical vapor deposition onto patterned carbon nanotube (CNT) scaffolds was demonstrated. After removal of the CNTs and hydroxylation, the resulting UTLC phase showed no expansion or distortion of their microfeatures and the absence/reduction of remaining nitrogenic species. Developing time of a mixture of lipophilic dyes on this UTLC plates was 86% shorter than on high-performance thin-layer chromatography (HPTLC) plates. A water-soluble food dye mixture was also separated resulting in low band broadening and reduced developing time compared to HPTLC. For the latter example, mobile phase optimization on a single UTLC plate consisted of 14 developments with different mobile phases, each preceded by a plate prewashing step. The same plate was again reused for additional 11 separations under varying conditions resulting in a development procedure with a mean separation efficiency of 233,000theoretical plates/m and a reduced mobile phase consumption of only 400µL. This repeated use proved the physical robustness of the ultrathin layer and its resistance to damage. The layer was highly suited for hyphenation to ambient mass spectrometry, including desorption electrospray ionization (DESI) mass spectrometry imaging and direct analysis in real time (DART) mass spectrometry.

A phenomenological model for the photocurrent transient relaxation observed in ZnO-based photodetector devices.

We present a phenomenological model for the photocurrent transient relaxation observed in ZnO-based metal-semiconductor-metal (MSM) planar photodetector devices based on time-resolved surface band bending. Surface band bending decreases during illumination, due to migration of photogenerated holes to the surface. Immediately after turning off illumination, conduction-band electrons must overcome a relatively low energy barrier to recombine with photogenerated holes at the surface; however, with increasing time, the adsorption of oxygen at the surface or electron trapping in the depletion region increases band bending, resulting in an increased bulk/surface energy barrier that slows the transport of photogenerated electrons. We present a complex rate equation based on thermionic transition of charge carriers to and from the surface and numerically fit this model to transient photocurrent measurements of several MSM planar ZnO photodetectors at variable temperature. Fitting parameters are found to be consistent with measured values in the literature. An understanding of the mechanism for persistent photoconductivity could lead to mitigation in future device applications.

Trimethoprim resistance of dihydrofolate reductase variants from clinical isolates of Pneumocystis jirovecii.

Pneumocystis jirovecii is an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants of P. jirovecii DHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, with Ki values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increased Km values for dihydrofolic acid (DHFA). The catalytic rate constant (kcat) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of kcat, as well as a Km for NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis.

Polymer nanoparticles for immunotherapy from encapsulated tumor-associated antigens and whole tumor cells.

Encapsulation of tumor-associated antigens (TAA) in polymer nanoparticles is a promising approach to increasing the efficiency of antigen (Ag) delivery for antitumor vaccines. We optimized a polymer preparation method to deliver both defined tumor-associated proteins and the complex mixtures of tumor Ags present in tumors. Tumor Ags were encapsulated in a biodegradable, 50:50 poly(D,L-lactide co-glycolide) copolymer (PLGA) by emulsification and solvent extraction. Two particular Ags were studied, gp100 (a melanoma-associated antigen) and ovalbumin (OVA), as well as mixtures of proteins and lysates of tumor cells. The efficiency of encapsulation was measured by protein assays of dissolved nanoparticles. Ag stability after release from nanoparticles was verified by SDS-acrylamide gel electrophoresis and Western blot analysis. Molecular weight and protein loading interact to define the encapsulation efficiency and release rate of nanoparticles formulated from 50:50 PLGA. A midrange molecular weight polymer had more desirable release properties at 100 mg/mL than at 50 mg/mL protein loading, indicating the need for optimization of nanoparticle formulation for preparations with different particle loadings. Mixtures of proteins derived from cell lysates were reliably encapsulated into nanoparticles, which released the spectrum of proteins contained in lysates. Antigenic proteins were co-encapsulated with cell lysate and released from nanoparticles; these Ags retained their antigenicity and functioned better than soluble Ags when tested in in vitro assays of T cell cytokine formation and in vivo tumor vaccination challenge.

Isolation of rat dihydrofolate reductase gene and characterization of recombinant enzyme.

While assays of many antifolate inhibitors for dihydrofolate reductase (DHFR) have been performed using rat DHFR as a target, neither the sequence nor the structure of rat DHFR is known. Here, we report the isolation of the rat DHFR gene through screening of a rat liver cDNA library. The rat liver DHFR gene has an open reading frame of 561 bp encoding a protein of 187 amino acids. Comparisons of the rat enzyme with those from other species indicate a high level of conservation at the primary sequence level and more so for the amino acid residues comprising the active site of the enzyme. Expression of the rat DHFR gene in bacteria produced a recombinant protein with high enzymatic activity. The recombinant protein also paralleled the human enzyme with respect to the inhibition by most of the antifolates tested with PT652 and PT653 showing a reversal in their patterns. Our results indicated that rat DHFR can be used as a model to study antifolate compounds as potential drug candidates. However, variations between rat and human DHFR enzymes, coupled with unique features in the inhibitors, could lead to the observed differences in enzyme sensitivity and selectivity.

Structure of rat transthyretin (rTTR) complex with thyroxine at 2.5 A resolution: first non-biased insight into thyroxine binding reveals different hormone orientation in two binding sites.

The first observation of the unique environment for thyroxine (T(4)) binding in tetrameric rat transthyretin (rTTR) is reported as determined by X-ray diffraction. These data revealed different modes of hormone binding in the two unique hormone-binding sites in the rat TTR tetramer channel. Differences in the orientation of thyroxine and the position of water molecules in the two binding sites further suggest a mechanism for the docking pathway of the hormone into the channel of TTR. Crystals of the rat transthyretin-thyroxine complex are isomorphous with those reported for apo rTTR and crystallized in the tetragonal space group P4(3)2(1)2 with four independent TTR monomeric subunits in the asymmetric part of the crystal lattice. Data were collected to 2.5 A resolution and the structure was refined to R = 20.9% for 15 384 data in the resolution range 12-2.5 A. Similar to human TTR, the rat protein is also a 54 000 Da tetramer with four identical polypeptide chains of 127 amino-acid residues. Of the 22 amino-acid residues which differ between the human and rat sequences, none are in the thyroxine-binding domains. Analysis of these structural data reveals that the tertiary structure is similar to that of hTTR, with only small differences in the flexible loop regions on the surface of the structure. Conformational changes of the amino acids in the channel result in a hydrogen-bonded network that connects the two binding domains, in contrast to the hydrogen bonds formed along the tetramer interface in the apo transthyretin structure. These changes suggest a mechanism for the signal transmission between thyroxine-binding domains.

Structure of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis.

The coleopteran-active delta-endotoxin Cry3Bb1 from Bacillus thuringiensis (Bt) strain EG7231 is uniquely toxic to Diabrotica undecimpunctata, the Southern corn rootworm, while retaining activity against Leptinotarsa decemlineata, the Colorado potato beetle. The crystal structure of the delta-endotoxin Cry3Bb1 has been refined using data collected to 2.4 A resolution, with a residual R factor of 17.5% and an R(free) of 25.3%. The structure is made up of three domains: I, a seven-helix bundle (residues 64-294); II, a three-sheet domain (residues 295-502); and III, a beta-sandwich domain (residues 503-652). The monomers in the orthorhombic C222(1) crystal lattice form a dimeric quaternary structure across a crystallographic twofold axis, with a channel formed involving interactions between domains I and III. There are 23 hydrogen bonds between the two monomers conferring structural stability on the dimer. It has been demonstrated that Cry3Bb1 and the similar toxin Cry3A form oligomers in solution. The structural results presented here indicate that the interactions between domains I and III could be responsible for the initial higher order structure and have implications for the biological activity of these toxins. There are seven additional single amino-acid residues in the sequence of Cry3Bb1 compared with that of Cry3A; one in domain I, two in domain II and four in domain III, which also shows the largest conformational difference between the two proteins. These changes can be implicated in the selectivity differences noted for these two delta-endotoxins.

Structure of a new polymorphic monoclinic form of human transthyretin at 3 A resolution reveals a mixed complex between unliganded and T4-bound tetramers of TTR.

The crystal structure of a new polymorphic form of human transthyretin (hTTR) with a lattice containing a unique assembly of apo hTTR and TTR-T(4) complex has been determined to 3 A resolution. The monoclinic form of human TTR reported here crystallizes in space group P2(1), with unit-cell parameters a = 76.7 (6), b = 96.7 (8), c = 81.7 (4) A, beta = 106.8 (4) degrees. The asymmetric unit contains two tetramers of transthyretin related by the non-crystallographic symmetry (NCS) operation of a 90.28 degrees rotation between two hTTR molecules around an axis close to crystallographic z. The r.m.s. difference between the two tetramers calculated from their C(alpha) positions is 0.48 A. The structure was refined using 15.0-3.0 A resolution data to R = 22.9% and R(free) = 28.9% for reflections F > 0.0sigma(F), and R = 19.7% and R(free) = 25.8% for reflections F > 3.0sigma(F). The intermolecular interactions involve the tips of alpha-helices and loops around Arg21, Glu61 and Ser100 of all monomers. The electron-density maps revealed residual thyroxine (T(4)) bound in only one of the two unique tetrameric TTR molecules, with an occupancy of 53%, while the second tetramer is unliganded. One thyroxine ligand is bound in a way similar to the orientations described for the orthorhombic form of the hTTR-T(4) complex. The T(4) bound in the second site is positioned similar to 3',5'-dinitro-N-acetyl-L-thyronine in its hTTR complex. Differences in the size of the central channel defined by the D, A, G and H beta-strands of two monomeric subunits are observed between the apo TTR and T(4)-bound tetramer. The averaged distances between Ala108 C(alpha) and its equivalent measured across each binding site are 12.34 A for the T(4)-bound and 10.96 A for the unliganded TTR tetramer, respectively. The observed differences might reflect the mechanics of the ligand binding in the channel and possibly explain the observed negative cooperativity effect for ligand binding.

Structural basis of negative cooperativity in transthyretin.

A comparison of the AC and BD binding sites of transthyretin (TTR) was made in terms of the interatomic distances between the Ca atoms of equivalent amino acids, measured across the tetramer channel in each binding site. The comparison of the channel diameter for apo TTR from different sources revealed that in the unliganded transthyretin tetramers the distances between the A, D and H beta-strands are consistently larger, while the distances between the G beta-strands are smaller in one site than in the other. These differences might be described to have a 'wave' character. An analogous analysis performed for transthyretin complexes reveals that the shape of the plot is similar, although the amplitudes of the changes are smaller. The analysis leads us to a model of the changes in the binding sites caused by ligand binding. The sequence of events includes ligand binding in the first site, followed by a slight collapse of this site and concomitant opening of the second site, binding of the second molecule and collapse of the second site. The following opening of the first, already occupied site upon ligand binding in the second site is smaller because of the bridging interactions already formed by the first ligand. This explains the negative cooperativity (NC) effect observed for many ligands in transthyretin.

Complex of rat transthyretin with tetraiodothyroacetic acid refined at 2.1 and 1.8 A resolution.

The crystal structure of rat transthyretin (rTTR) complex with 3,5,3',5'-tetraiodothyroacetic acid (T4Ac) was determined at 1.8 A resolution with low temperature synchrotron data collected at CHESS. The structure was refined to R = 0.207 and Rfree = 0.24 with the use of 8-1.8 A data. The additional 8000 reflections from the incomplete 2.1-1.8 data shell, included in the refinement, reduced the Rfree index by 1.3%. Structure comparison with the model refined against the complete 8-2.1 A data revealed no differences in the ligand orientation and the conformation of the polypeptide chain in the core regions. However, the high-resolution data included in the refinement improved the model in the flexible regions poorly defined with the lower resolution data. Also additional sixteen water molecules were found in the difference map calculated with the extended data. The structure revealed both forward and reverse binding of tetraiodothyroacetic acid in one binding site and two modes of forward ligand binding in the second site, with the phenolic iodine atoms occupying different sets of the halogen binding pockets.

Comparison of binding interactions of dibromoflavonoids with transthyretin.

The crystal structure of rat transthyretin (rTTR) complex with the dibromoflavone EMD21388 was determined to 2.3 A resolution and refined to R = 0.203 and Rfree = 0.288. Two different orientations of EMD21388, which differ in the channel penetration by 1.6 A, were found in the A/C binding site of rTTR. The single ligand position observed in the BID site is intermediate between the two positions found in the A/C site. The position of the dibromoflavone in the B/D site is similar to that reported for dibromoaurone in human TTR. The bromine atoms of EMD21388 form strong interactions in the P3 and P3' pockets of rTTR. Due to the different molecular architectures of both ligands, dibromoflavone forms only one interaction with Lys-15 near the channel entrance, while direct interactions with the pair of Lys-15 were reported for dibromoaurone. The C3* methyl group of EMD21388 mediates the bridging interactions between two TTR subunits in the P2 pockets. The interactions of the O2* hydroxyl group of dibromoaurone with the Thr-119 side chain in the P3 pockets are not matched by similar interactions in EMD21388. Both these alternative interactions can explain the competitive binding of 3',5'-dibromoflavonoids to transthyretin.

Computer modeling studies of the structural role of NADPH binding to active site mutants of human dihydrofolate reductase in complex with piritrexim.

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is one of the enzymes active in the folate cycle which plays an important role in DNA synthesis. Inhibition of DHFR is a key element in the treatment of many diseases, including cancer and AIDS related infections. A search for new selective inhibitors is motivated by the resistance to common drugs observed in the course of treatment. In this paper, results of a detailed computer analysis of human DHFR interactions with the lipophilic inhibitor piritrexim (PTX) are presented. It was found that the NADPH cofactor contributes 30% of the total PTX-enzyme interaction energy. Substitution of the highly conserved Glu30 with alanine does not lead to the release of the inhibitor from the hDHFR pocket. The important L22F point mutation does affect PTX orientation but does not changethe binding energy. Simulations of the dynamics of binary hDHFR-PTX complexes were performed with the use of Extensible Systematic Force Field (ESFF) and the results indicate structural changes in the enzyme induced by NADPH binding.

Design, synthesis, and X-ray crystal structure of a potent dual inhibitor of thymidylate synthase and dihydrofolate reductase as an antitumor agent.

A novel N-¿2-amino-4-methyl[(pyrrolo[2, 3-d]pyrimidin-5-yl)ethyl]benzoyl¿-L-glutamic acid (3a) was designed and synthesized as a potent dual inhibitor of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as an antitumor agent. Compound 3b, the N7-benzylated analogue of 3a, was also synthesized as an antitumor agent. The synthesis of 3a was accomplished via a 12-step sequence which involved the synthesis of 2-amino-4-methylpyrrolo[2,3-d]pyrimidine (10) in 5 steps from 2-acetylbutyrolactone. Protection of the 2-amino group of 10 and regioselective iodination at the 5-position followed by palladium-catalyzed coupling afforded intermediate 14 which was converted to 3a by reduction and saponification. Similar synthetic methodology was used for 3b. X-ray crystal structure of the ternary complex of 3a, DHFR, and NADPH showed that the pyrrolo[2, 3-d]pyrimidine ring binds in a "2,4-diamino mode" in which the pyrrole nitrogen mimics the 4-amino moiety of 2,4-diaminopyrimidines. This is the first example of a classical pyrrolo[2,3-d]pyrimidine antifolate shown to have this alternate mode of binding to DHFR. Compounds 3a and 3b were more inhibitory than LY231514 against TS from Lactobacillus casei and Escherichia coli. Analogue 3a was also more inhibitory against DHFR from human, Toxoplasma gondii, and Pneumocystis carinii. Evaluation of 3a against methotrexate (MTX)-resistant cell lines with defined mechanisms indicated that cross-resistance of 3a was much lower than that of MTX. Metabolite protection studies and folylpoly-gamma-glutamate synthetase studies suggest that the antitumor activity of 3a against the growth of tumor cells in culture is a result of dual inhibition of TS and DHFR. Compound 3a inhibited the growth of CCRF-CEM and FaDu cells in culture at ED(50) values of 12.5 and 7.0 nM, respectively, and was more active against FaDu cells than MTX. In contrast, compound 3b was inactive against both cell lines. Compound 3a was evaluated in the National Cancer Institute in vitro preclinical antitumor screening program and afforded IG(50) values in the nanomolar range against a number of tumor cell lines.

Structural studies on bioactive compounds. 30. Crystal structure and molecular modeling studies on the Pneumocystis carinii dihydrofolate reductase cofactor complex with TAB, a highly selective antifolate.

The crystal structure of the ternary complex of NADPH, the potent antifolate [2, 4-diamino-5-¿3-[3-(2-acetyloxyethyl)-3-benzyltriazen-1-yl]-4 -chloroph enyl¿-6-ethylpyrimidine] (TAB, 1) and Pneumocystis carinii dihydrofolate reductase (pcDHFR), refined to 2.1 A resolution, reveals that TAB binds similar to the antifolates trimethoprim and methotrexate. These data also reveal multiple conformations for the binding geometry of TAB with two preferred orientations of the acetyloxy and benzyl groups that results from a 180 degrees rotation about the N2-N3 triazenyl bond. The methyl of the acetyloxy and benzyl ring of TAB probes large hydrophobic regions of the p-aminobenzoyl folate binding pocket of the active site, in particular the region near Phe69, which is unique to the pcDHFR sequence. These results confirm prior molecular modeling investigations of the binding of TAB to pcDHFR that identified four low-energy binding geometries, two involving rotations about the terminal N(2)-N(3) triazenyl linkage and two involving atropisomerism about the pivotal pyrimethamine-phenyl bond. The primary differences in the molecular dynamics (MD) models and those observed in this crystal complex result from small conformational changes in active-site residues on energy minimization. However, two MD models place the acetyloxy and benzyl ring groups in a region of the active site between the cofactor-binding region and the p-aminobenzoyl folate pocket; an orientation never observed in any DHFR crystal structure to date. These conformers interact with solvent near the enzyme surface and are probably not observed due to the loss of specific hydrogen bonds with the enzyme. The high species pcDHFR selectivity of TAB could be the result of ligand flexibility that enables multiple binding orientations at the enzyme active site. Further modification of the acetyloxy region of TAB could increase its potency and selectivity for pcDHFR.

Structure-based design of selective inhibitors of dihydrofolate reductase: synthesis and antiparasitic activity of 2, 4-diaminopteridine analogues with a bridged diarylamine side chain.

As part of a larger search for potent as well as selective inhibitors of dihydrofolate reductase (DHFR) enzymes from opportunistic pathogens found in patients with AIDS and other immune disorders, N-[(2,4-diaminopteridin-6-yl)methyl]dibenz[b,f]azepine (4a) and the corresponding dihydrodibenz[b,f]azepine, dihydroacridine, phenoxazine, phenothiazine, carbazole, and diphenylamine analogues were synthesized from 2, 4-diamino-6-(bromomethyl)pteridine in 50-75% yield by reaction with the sodium salts of the amines in dry tetrahydrofuran at room temperature. The products were tested for the ability to inhibit DHFR from Pneumocystis carinii (pcDHFR), Toxoplasma gondii (tgDHFR), Mycobacterium avium (maDHFR), and rat liver (rlDHFR). The member of the series with the best combination of potency and species selectivity was 4a, with IC(50) values against the four enzymes of 0. 21, 0.043, 0.012, and 4.4 microM, respectively. The dihydroacridine, phenothiazine, and carbazole analogues were also potent, but nonselective. Of the compounds tested, 4a was the only one to successfully combine the potency of trimetrexate with the selectivity of trimethoprim. Molecular docking simulations using published 3D structural coordinates for the crystalline ternary complexes of pcDHFR and hDHFR suggested a possible structural interpretation for the binding selectivity of 4a and the lack of selectivity of the other compounds. According to this model, 4a is selective because of a unique propensity of the seven-membered ring in the dibenz[b,f]azepine moiety to adopt a puckered orientation that allows it to fit more comfortably into the active site of the P. carinii enzyme than into the active site of the human enzyme. Compound 4a was also evaluated for the ability to be taken up into, and retard the growth of, P. carinii and T. gondii in culture. The IC(50) of 4a against P. carinii trophozoites after 7 days of continuous drug treatment was 1.9 microM as compared with previously observed IC(50) values of >340 microM for trimethoprim and 0.27 microM for trimetrexate. In an assay involving [(3)H]uracil incorporation into the nuclear DNA of T. gondii tachyzoites as the surrogate endpoint for growth, the IC(50) of 4a after 5 h of drug exposure was 0.077 microM. The favorable combination of potency and enzyme selectivity shown by 4a suggests that this novel structure may be an interesting lead for structure-activity optimization.