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PURPOSE: To determine whether smartphone photographs of children’s eyelids are reliable for diagnosing the presence of chalazia. METHODS: In this prospective cross-sectional study, 60 participants, 7 months to 16.5 years of age, at four sites were enrolled; all participants had a chalazion measuring at least 2 mm on at least one eyelid based on an in-person clinical examination by a pediatric eye care professional. Smartphone photographs taken by the parent during the office visit were uploaded to the study website. A masked reader assessed each photograph for the presence or absence of chalazia; results were compared with the gold standard clinical examination results. Sensitivity and specificity for the presence of chalazion by eyelid were calculated. RESULTS: Photographs were available for 240 eyelids; 85 had at least one chalazion and 155 were without a chalazion based on clinical examination. The masked reader correctly classified 68 of 85 eyelids with at least one chalazion and 151 of 155 eyelids without chalazia for a sensitivity of 80% (95% CI, 72%–86%) and a specificity of 97% (95% CI, 94%–99%). Sensitivity improved to 89% for chalazia 5 mm or larger and 94% when superficially located within the eyelid. CONCLUSIONS: Parental smartphone photographs appear to be useful in assessing chalazia in children as an alternative to in-office follow-up examinations. These photographs may be a valuable outcome measure in future clinical trials of chalazia treatment, especially when assessing larger lesions.

Importance This is the first large-scale randomized clinical trial evaluating the effectiveness and safety of overminus spectacle therapy for treatment of intermittent exotropia (IXT). Objective To evaluate the effectiveness of overminus spectacles to improve distance IXT control. Design, Setting, and Participants This randomized clinical trial conducted at 56 clinical sites between January 2017 and January 2019 associated with the Pediatric Eye Disease Investigator Group enrolled 386 children aged 3 to 10 years with IXT, a mean distance control score of 2 or worse, and a refractive error between 1.00 and −6.00 diopters (D). Data analysis was performed from February to December 2020. Interventions Participants were randomly assigned to overminus spectacle therapy (−2.50 D for 12 months, then −1.25 D for 3 months, followed by nonoverminus spectacles for 3 months) or to nonoverminus spectacle use. Main Outcomes and Measures Primary and secondary outcomes were the mean distance IXT control scores of participants examined after 12 months of treatment (primary outcome) and at 18 months (3 months after treatment ended) assessed by an examiner masked to treatment group. Change in refractive error from baseline to 12 months was compared between groups. Analyses were performed using the intention-to-treat population. Results The mean (SD) age of 196 participants randomized to overminus therapy and 190 participants randomized to nonoverminus treatment was 6.3 (2.1) years, and 226 (59%) were female. Mean distance control at 12 months was better in participants treated with overminus spectacles than with nonoverminus spectacles (1.8 vs 2.8 points; adjusted difference, −0.8; 95% CI, −1.0 to −0.5;P Conclusions and Relevance Children 3 to 10 years of age had improved distance exotropia control when assessed wearing overminus spectacles after 12 months of overminus treatment; however, this treatment was associated with increased myopic shift. The beneficial effect of overminus lens therapy on distance exotropia control was not maintained after treatment was tapered off for 3 months and children were examined 3 months later. Trial Registration ClinicalTrials.gov Identifier:NCT02807350

Veterans Treatment Courts (VTCs) were created to: (1) address issues unique to U.S. Military Veterans; and, (2) where possible, avoid punishing veterans for crimes that may have been committed as a direct result of their mental conditions (e.g., Posttraumatic Stress Disorder (PTSD) and/or Traumatic Brain Injury (TBI)). Little research has been undertaken to ascertain whether VTCs are accomplishing their intended goals. To that end, the present case study examined three operating VTCs in order to determine if they are meeting their objectives. The research provides further information about VTC operations and recidivism rates. The research further reveals that, while the three VTCs examined are arguably benefitting veterans, they are not reaching a greater part of the population due largely to the gatekeeping role of VTC prosecutors, and the rigid eligibility requirements established by the three VTCs for veteran participation. Consequently, it's possible that the low recidivism rates reported by the three VTCs examined are inflated or constitute false positives.

We have designed and synthesized a series of HIV protease inhibitors (PIs) with enamino-oxindole substituents optimized to interact with the S2' subsite of the HIV protease binding pocket. Several of these inhibitors have sub-nanomolar K(i) and antiviral IC(50) in the low nM range against WT HIV and against a panel of multi-drug resistant (MDR) strains.

Four novel linear non-peptidic HIV-1 protease inhibitors derived from 2,5-diamino-1,6-diphenyl-3-hexanol were synthesized and characterized. All of them exhibit tight binding to HIV-1 protease, with inhibition constants Ki in the range 20 pm–5 nm. The investigated inhibitors were crystallized, and their crystal structures were determined by X-ray diffraction. In all cases, the conformations found in the crystalline state differ significantly from the conformations obtained by computational docking of the inhibitor in the binding cleft of native HIV-1 protease. Owing to the prevalence of hydrophobic substituents in all these inhibitors, the conformational mobility in water solution is restricted to their compact forms. The spectrum of low-energy conformations in solution dramatically changes during the formation of inhibitor crystals (phenyl ring stacking as a leading motif) or during the formation of a complex with HIV-1 protease (elongated conformation suitable to fit the enzyme pockets as a factor responsible for tight binding). High conformational flexibility and low conformational stress in the molecules of these inhibitors most likely increase their biological activity in comparison with more rigid compounds.

We have developed a novel procedure to monitor the real-time cleavage of natural unmodified peptides (dark substrates). In the competition-based assay, the initial cleavage rate of a fluorogenic peptide substrate is measured in the presence of a second substrate that is not required to exhibit any optical property change upon cleavage. Using a unique experimental design and steady-state enzyme kinetics for a two-substrate system, we were able to determine both Km and k(cat) values for cleavage of the dark substrate. The method was applied to HIV-1 protease and to the V82F/I84V drug resistant mutant enzyme. Using two different substrates, we showed that the kinetic parameters derived from the competition assay are in good agreement with those determined independently using standard direct assay. This method can be applied to other enzyme systems as long as they have one substrate for which catalysis can be conveniently monitored in real time.

Plasmepsin II is one of the four catalytically active plasmepsins found in the food vacuole of Plasmodium falciparum. These enzymes initiate hemoglobin degradation by cleavage at the alpha-chain between Phe33 and Leu34. The crystal structures of Ser205 mutant plasmepsin II from P. falciparum in complex with two inhibitors have been refined at a resolution of 1.8 A in the space group I222 and to R factors of 19.9 and 19.5%. Each crystal contains one monomer in the asymmetric unit. Both inhibitors have a Phe-Leu core and incorporate tetrahedral transition-state mimetic hydroxypropylamine. The inhibitor rs367 possesses a 2,6-dimethylphenyloxyacetyl group at the P2 position and 3-aminobenzamide at the P2' position, while rs370 has the same P2 group but 4-aminobenzamide in the P2' position. These complexes reveal key conserved hydrogen bonds between the inhibitor and the binding-cavity residues, notably with the flap residues Val78 and Ser79, the catalytic dyad Asp34 and Asp214 and the residues Ser218 and Gly36 that are in proximity to the catalytic dyad. The structures also show unexpected conformational variability of the binding cavity of plasmepsin II and may reflect the mode of binding of the hemoglobin alpha-chain for cleavage.

Plasmepsin-2 is a malarial aspartic proteinase that has been implicated in the initial steps of hemoglobin degradation in parasites and thus represents an attractive antimalarial target. Escherichia coli expressed proplasmepsin-2 is capable of activation at acidic pH by autocatalytic cleavage of the pro part region, which results in products of different length. We designed a 10-amino-acid deletion in the pro part region that allows faster generation of homogeneous enzyme upon activation. Incorporation of a (His)6 tag onto the N-terminus of the pro part enables on-column refolding of proplasmepsin-2 and simplifies proenzyme purification and pro part separation after activation. The proposed purification procedure results in highly pure and easily crystallizable enzyme.

We identified UIC-94003, a nonpeptidic human immunodeficiency virus (HIV) protease inhibitor (PI), containing 3( R ),3a( S ),6a( R )- bis -tetrahydrofuranyl urethane ( bis -THF) and a sulfonamide isostere, which is extremely potent against a wide spectrum of HIV (50% inhibitory concentration, 0.0003 to 0.0005 μM). UIC-94003 was also potent against multi-PI-resistant HIV-1 strains isolated from patients who had no response to any existing antiviral regimens after having received a variety of antiviral agents (50% inhibitory concentration, 0.0005 to 0.0055 μM). Upon selection of HIV-1 in the presence of UIC-94003, mutants carrying a novel active-site mutation, A28S, in the presence of L10F, M46I, I50V, A71V, and N88D appeared. Modeling analysis revealed that the close contact of UIC-94003 with the main chains of the protease active-site amino acids (Asp29 and Asp30) differed from that of other PIs and may be important for its potency and wide-spectrum activity against a variety of drug-resistant HIV-1 variants. Thus, introduction of inhibitor interactions with the main chains of key amino acids and seeking a unique inhibitor-enzyme contact profile should provide a framework for developing novel PIs for treating patients harboring multi-PI-resistant HIV-1.

Recent experimental observations support the assumption that all families of polynucleotide polymerases have a universal “two-metal-ion” mechanism of nucleotide addition. This mechanism provides a general picture of the nucleotidyl transfer reaction. However, the detailed reaction pathway is still a matter of debate. We investigated two potential reaction pathways for DNA polymerase β using density-functional theory. Our model consists of 67 atoms of the polymerase active site and includes all major features thought to be important for catalysis. The first mechanism we investigated involves the formation of a PO3 intermediate. This intermediate is thought to be involved in phosphate reactions in solution and could be accommodated in the polymerase β active site. However, the barrier to formation of this intermediate is 37.0 kcal/mol, and we do not expect that this mechanism is the one that occurs in the enzyme. The second mechanism that leads to a pentacoordinated intermediate appears to be feasible. This...

Abstract: Structural Biochemistry Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, P.O Box B, Frederick, MD 21702-1201. Present Address: Tibotec Inc., 1330 Piccard Dr., Rockville, MD, 20850. 1Present Address: Gilford Pharmaceuticals Inc., 6611 Tributary Street, Baltimore, MD, 21224. Plasmepsin 2 is a malarial aspartic proteinase that has been implicated in initial steps of hemoglobin degradation in parasite and thus represents an attractive antimalarial target. We designed and synthesized a number of quenched fluorogenic substrates and measured the kinetic constants of their cleavage by plasmepsin-2. High sensitivity and superior kinetic properties make the substrates suitable for different application including fluorescence plate reader.

We have investigated the influence of different substitutions and solvent effects on the pKa values of the hydroxy group for a set of pyrone and dihydropyrone HIV-1 protease inhibitors. Absolute and relative pKa values were calculated for model compounds using a combination of density functional theory (DFT) and continuum solvation methods and were compared to experimental data for related compounds. The theoretical results shed light on the unusual pH dependence of the inhibition constants for these compounds and may lead to the design of new, improved pyrone and dihydropyrone inhibitors of HIV-1 protease.

Objective It is thought as a consequence of continuous replication, HIV-1 has acquired an optimal fitness state and that suboptimal antiretroviral therapy selects for drug resistant variants which show impaired fitness in the absence of the drug. In this paper we studied the evolution and fitness of viral populations appearing in a patient who received protease monotherapy. Methods Two factors contributing to fitness, drug resistance and protease catalytic activity, were studied at the enzymatic and virological level. Results The first drug resistant viral variants that were selected in vivo harboured one to three protease substitutions. These mutants showed reduced protease activity and consequently a reduction in viral replication capacity. During continued in vivo replication of these viruses in the presence of the drug, novel variants harbouring additional substitutions in the viral protease appeared. These variants did not display any further increase in drug resistance but demonstrated clearly increased protease activity. Consequently the replication capacity of these viruses was raised to a level at which they replicated better than the original wild-type virus. Conclusion This study indicates that the viral population in the patient does not have to represent the fittest possible variants, and thus antiretroviral therapy may drive the viral population first through a lower fitness level and then to a higher fitness level.

We designed, synthesized, and identified JE-2147, an allophenylnorstatine-containing dipeptide HIV protease inhibitor (PI), which is potent against a wide spectrum of HIV-1, HIV-2, simian immunodeficiency virus, and various clinical HIV-1 strains in vitro . Drug-resistant clinical HIV-1 strains, isolated from seven patients who had failed 9–11 different anti-HIV therapeutics after 32–83 months, had a variety of drug-resistance-related amino acid substitutions and were highly and invariably resistant to all of the currently available anti-HIV agents. JE-2147 was, however, extremely potent against all such drug-resistant strains, with IC 50 values ranging from 13–41 nM (50 compared with that of wild-type HIV-1). The emergence of JE-2147-resistant HIV-1 variants in vitro was substantially delayed compared with that of HIV-1 resistant to another allophenylnorstatine-containing compound, KNI-272, and other related PIs. Structural analysis revealed that the presence of a flexible P2′ moiety is important for the potency of JE-2147 toward wild-type and mutant viruses. These data suggest that the use of flexible components may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1. Further development of JE-2147 for treating patients harboring multi-PI-resistant HIV-1 is warranted.

A series of novel unsymmetrical anthranilamide-containing HIV protease inhibitors was designed. The structure-activity studies revealed a series of potent P2–P3′ inhibitors that incorporate an anthranilamide group at the P2′ position. A reduction in molecular weight and lipophilicity is achieved by a judicious choice of P2 ligands (i.e., aromatic, heteroaromatic, carbamate, and peptidic). A systematic investigation led to the 5-thiazolyl carbamate analog 8m, which exhibited a favorable C max EC 50 ratio (>30), plasma half-life (>8 h), and potent in vitro antiviral activity (EC50 = 0.2 uM).

A methodology is presented for calculating relative binding free energies of enzyme−inhibitor associations in aqueous solvent. The methodology uses synthesis of semiempirical quantum chemistry to determine the protonation state of important residues in the enzyme active site, molecular mechanics to determine the gas-phase energetic contributions to the relative binding free energy, and dielectric continuum solvation to calculate electrostatic hydration contributions. The methodology is then applied to the calculation of the relative binding free energy of the inhibitors KNI-272, Ro31-8959, L-735,524, and A-77003 to HIV-1 protease and its I84V mutant. The calculated relative binding free energy is sensitive to the active-site protonation state of the aspartic acid residues of HIV-1 protease. The protonation state is inhibitor dependent. Given a particular protonation state, it was found that quantitatively accurate relative binding free energies could only be achieved when solvent effects were included. Th...

The changes in the inhibitor binding constants due to the mutation of isoleucine to valine at position 84 of HIV-1 protease are calculated using molecular dynamics simulations. The calculations are done for three potent inhibitors--KNI-272, L-735,524 (indinavir or MK-639), and Ro 31-8959 (saquinavir). The calculations agree with the experimental data both in terms of an overall trend and in the magnitude of the resulting free energy change. HIV-1 protease is a homodimer, so each mutation causes two changes in the enzyme. The decrease in the binding free energy from each mutated side chain differs among the three inhibitors and correlates well with the size of the cavities induced in the protein interior near the mutated residue. The cavities are created as a result of a mutation to a smaller side chain, but the cavities are less than would be predicted from the wild-type structures, indicating that there is significant relaxation to partially fill the cavities.

Two different structures of ligand-free HIV protease have been determined by X-ray crystallography. These structures differ in the position of two 12 residue, β-hairpin regions (or “flaps”) which cap the active site. The movements of the flaps must be involved in the binding of substrates since, in either conformation, the flaps block the binding site. One of these structures is similar to structures of the ligand-bound enzyme; however, the importance of both structures to enzyme function is unclear. This transformation takes place on a time scale too long for conventional molecular dynamics simulations, so the process was studied by first identifying a reaction path between the two structures and then calculating the free energy along this path using umbrella sampling. For the ligand-free enzyme, it is found that the two structures are nearly equally stable, with the ligand-bound-type structure being less stable, consistent with X-ray crystallography data. The more stable open structure does not have a lower potential energy, but is stabilized by entropy. The transition occurs through a collapse and reformation of the β-sheet structure of the conformationally flexible, glycine-rich flap ends. Additionally, some problems in studying conformational changes in proteins through the use of a single reaction path are addressed. Proteins 32:7–16, 1998. © 1998 Wiley-Liss, Inc.

Density functional theory (DFT) methods were used to calculate geometric and electronic properties for the metal binding sites belonging to three distinct zinc finger families, as well as for that of a binuclear M2Cys6 sequence. DFT-derived structural parameters such as bond distances and angles for ZnII and CdII binding sites were in agreement with experimental values obtained from EXAFS, X-ray crystallography, and multidimensional NMR spectroscopy for related systems. Calculations of the electronic properties of these peptides indicate that the chelating residues alone are responsible for the majority of the free energy of metal binding, with only a small contribution attributable to neighboring residues. Our calculations also predict that the binding affinity for a given metal ion increases with the number of thiolate ligands, and is consistent with experimental observations of the relative metal ion affinities for peptides from different zinc finger families. For all zinc finger families, calculated ionization potentials for CdII-bound peptides were larger than those for the ZnII-bound species, leading to the prediction of faster redox reaction rates for the latter. We discuss the implications of these results for the structure and function of zinc fingers, as well as for the design of compounds affecting their chemical integrity.

The catalytic activity and inhibitor binding energetics of enzymes are often pH-dependent properties. Aspartic proteases comprise an important class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasmepsin II, an aspartic proteinase found in Plasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynamic parameters (DeltaG, DeltaH, DeltaCp, and DeltaS) were measured as functions of both pH and temperature. In the pH range from 4.5 to 7.0, pepstatin binding is accompanied by proton transfer between the solvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and the number and pKa values of ionizable residues whose pKa values change during ligand binding. These residues were identified as the two catalytic aspartates, with pKas of 6.5 and 3.0, and His 164, with a pKa of 7.5, based on the three-dimensional structure of the pepstatin-plasmepsin II complex. At pH 5.0, where the protease has optimum activity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site aspartic acid residues is -1. These experimental results provide direct measurement for the protonation states of the catalytic aspartates in the presence of bound ligands. Comparison of the thermodynamic and structural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin II, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.

The 4-hydroxy-5,6-dihydropyrone template was utilized as a flexible scaffolding from which to build potent active site inhibitors of HIV protease. Dihydropyrone 1c (5,6-dihydro-4-hydroxy-6-phenyl-3-[(2-phenylethyl)thio]-2H-pyran-2-one) was modeled in the active site of HIV protease utilizing a similar binding mode found for the previously reported 4-hydroxybenzopyran-2-ones. Our model led us to pursue the synthesis of 6,6-disubstituted dihydropyrones with the aim of filling S1 and S2 and thereby increasing the potency of the parent dihydropyrone 1c which did not fill S2. Toward this end we attached various hydrophobic and hydrophilic side chains at the 6-position of the dihydropyrone to mimic the natural and unnatural amino acids known to be effective substrates at P2 and P2'. Parent dihydropyrone 1c (IC50 = 2100 nM) was elaborated into compounds with greater than a 100-fold increase in potency [18c, IC50 = 5 nM, 5-(3,6-dihydro-4-hydroxy-6-oxo-2-phenyl-5-[2-phenylethyl)thio] -2H-pyran-2-yl)pentanoic acid and 12c, IC50 = 51 nM, 5,6-dihydro-4-hydroxy-6-phenyl-6-(2-phenylethyl)-3- [(2-phenyl-ethyl)thio]-2H-pyran-2-one]. Optimization of the 3-position fragment to fill S1' and S2' afforded potent HIV protease inhibitor 49 [IC50 = 10 nM, 3-[(2-tert-butyl-5-methylphenyl)sulfanyl]-5,6-dihydro-4 -hydroxy-6-phenyl-6-(2-phenylethyl)-2H-pyran-2-one]. The resulting low molecular weight compounds (475) have one or no chiral centers and are readily synthesized.

Tumor suppressor protein p53 is a tetrameric phosphoprotein that activates transcription from several cell cycle regulating genes in response to DNA damage. Tetramer formation is critical to p53's ability to activate transcription; however, posttranslational modifications and protein stabilization also contribute to p53's ability to activate transcription. To determine if phosphorylation affects tetramer formation, we synthesized phosphopeptides corresponding to residues 303-393 of human p53, which includes the domain responsible for tetramer formation. Phosphate was chemically incorporated at Ser315, Ser378, or Ser392 and also at both Ser315 and Ser392. Equilibrium ultracentrifugal analyses showed that phosphorylation at Ser392 increased the association constant for reversible tetramer formation nearly 10-fold. Phosphorylation of either Ser315 or Ser378 had little effect on tetramer formation, but phosphorylation of Ser315 largely reversed the effect of phosphorylation at Ser392. Analyses by calorimetry demonstrated that phosphorylation may influence subunit affinity (and, in turn, DNA binding) by an enthalpy-driven process, possibly between the C-terminal residues and the region immediately adjacent to Ser315. The Kd for the tetramer-monomer transition of the unphosphorylated p53 C-terminal domain was determined to be approximately 1-10 microM. Thus, in normal, undamaged cells p53 may be largely monomeric. Enhancement of tetramer formation through phosphorylation of Ser392, coupled with a DNA-damage-induced increase in its nuclear concentration, could provide a switch that activates p53 as a transcription factor in response to DNA damage.

Human tumor suppressor protein p53 is a 393-amino acid phosphoprotein that enhances transcription in response to DNA damage from several genes that regulate cell cycle progression. The tetrameric state of p53 is critical to wild-type function; the p53 tetramerization element is located in the C-terminal region of the protein. This region is phosphorylated at several evolutionarily conserved serines, suggesting that phosphorylation may be an important regulator of p53 function. In order to determine the effect of phosphorylation on tetramer formation, we synthesized phosphopeptides corresponding to p53(Ser303-Asp393) with phosphate incorporated at Ser315, Ser378, or Ser392, and at both Ser315 and Ser392. Equilibrium ultracentrifugation analysis showed that phosphorylation at Ser392 increased the association constant for tetramer formation nearly ten-fold. By itself, phosphorylation at Ser315 or Ser378 had little effect on tetramer formation, but Ser315 largely reversed the effect of phosphorylation at Ser392. Analysis by calorimetry suggests that phosphorylation may influence subunit affinity by an enthalpy driven process.

Human cathepsin D is a lysosomal aspartic protease that has been implicated in breast cancer metastasis and Alzheimer's disease. Based on a crystal structure of a human cathepsin D-pepstatin A complex, a series of statine-containing inhibitors was designed, synthesized, and tested for inhibitory activity toward the enzyme in vitro. The compounds were modified systematically at individual positions (P4, P3, P2, P1, and P2t) with the aim of mapping the cathepsin D subsite preferences. The experimentally obtained SAR data were correlated on the basis of molecular modeling. Side-chain preferences for the peptidomimetic inhibitors differed from those found previously using peptide substrates (Scarborough PE et al., 1993, Protein Sci 2:264-276). In addition, the effects of single side-chain modifications were often nonadditive. Structure-activity relationships, modeling, and thermodynamic analysis indicated that entropy plays a major stabilizing role in inhibitor binding to cathepsin D.

Plasmodium falciparum is the major causative agent of malaria, a disease of worldwide importance. Resistance to current drugs such as chloroquine and mefloquine is spreading at an alarming rate, and our antimalarial armamentarium is almost depleted. The malarial parasite encodes two homologous aspartic proteases, plasmepsins I and II, which are essential components of its hemoglobin-degradation pathway and are novel targets for antimalarial drug development. We have determined the crystal structure of recombinant plasmepsin II complexed with pepstatin A. This represents the first reported crystal structure of a protein from P. falciparum. The crystals contain molecules in two different conformations, revealing a remarkable degree of interdomain flexibility of the enzyme. The structure was used to design a series of selective low molecular weight compounds that inhibit both plasmepsin II and the growth of P. falciparum in culture.

The structure of the HIV-1 protease in complex with a pseudo-C2 symmetric inhibitor, which contains a central difluoroketone motif, has been determined with X-ray diffraction data extending to 1.7 A resolution. The electron density map clearly indicates that the inhibitor is bound in a symmetric fashion as the hydrated, or gemdiol, form of the difluoroketone. Refinement of the complex reveals a unique, and almost symmetric, set of interactions between the geminal hydroxyl groups, the geminal fluorine atoms, and the active-site aspartate residues. Several hydrogen bonding patterns are consistent with that conformation. The lowest energy hydrogen disposition, as determined by semiempirical energy calculations, shows only one active site aspartate protonated. A comparison between the corresponding dihedral angles of the difluorodiol core and those of a hydrated peptide bond analog, calculated ab-initio, shows that the inhibitor core is a mimic of a hydrated peptide bond in a gauche conformation. The feasibility of an anti-gauche transition for a peptide bond after hydration is verified by extensive molecular dynamics simulations. The simulations suggest that rotation about the C-N scissile bond would readily occur after hydration and would be driven by the optimization of the interactions of peptide side-chains with the enzyme. These results, together with the characterization of a transition state leading to bond breakage via a concerted exchange of two protons, suggest a proteolysis mechanism whereby only one active site aspartate is initially protonated. The steps of this mechanism are: asymmetric binding of the substrate; hydration of the peptidic carbonyl by an active site water; proton translocation between the active site aspartate residues simultaneously with carbonyl hydration; optimization of the binding of the entire substrate facilitated by the flexible structure of the hydrated peptide bond, which, in turn, forces the hydrated peptide bond to assume a gauche conformation; simultaneous proton exchange whereby one hydroxyl donates a proton to the charged aspartate, and, at the same time, the nitrogen lone pair accepts a proton from the other aspartate; and, bond breakage and regeneration of the initial protonation state of the aspartate residues.

KNI-272 is a peptidomimetic transition state analog inhibitor, having very high specificity and binding affinity for the HIV-1 protease. In order to understand the interactions that enhance drug bi...

Inhibitors of HIV-1 protease represent a new class of antiretroviral compounds. Here, we report the design and synthesis of two novel C2 symmetry-based inhibitors, MP-134 and MP-167, specifically targeted against HIV-1 variants with reduced sensitivity to another related protease inhibitor, A-77003. In addition, we describe the in vitro selection of viral variants with reduced sensitivity to these two protease inhibitors. An isoleucine-to-valine substitution at residue 84 (I84V) of the HIV-1 protease confers resistance to MP-134, whereas a glycine-to-valine substitution at residue 48 (G48V) confers resistance to MP-167. Testing other protease inhibitors against these variants has revealed specific overlapping patterns of resistance among these agents. These findings have important implications in the design of combination regimens using multiple protease inhibitors and underscore the need to develop non-cross-resistant compounds to be used toward this goal.

Several molecular modeling techniques were used to generate an all-atom molecular model of a receptor binding site starting only from Ca atom coordinates. The model consists of 48 noncontiguous residues of the non-nucleoside binding site of HIV-1 reverse transcriptase and was generated using a congeneric series of nevirapine analogs as structural probes. On the basis of the receptor-ligand atom contacts, the program HINT was used to develop a 3D quantitative structure activity relationship that predicted the rank order of binding affinities for the series of inhibitors. Electronic profiles of the ligands in their docked conformations were characterized using electrostatic potential maps and frontier orbital calculations. These results led to the development of a 3D stereoelectronic pharmacophore which was used to construct 3D queries for database searches. A search of the National Cancer Institute's open database identified a lead compound that exhibited moderate antiviral activity.

Guided by the structure of HIV PR complexed with 2S,3R,4S,5S-2,5-bis[N,N'-((3-hydroxy-2-methylphenyl)carbonyl)amino]-3,4-dihydroxy-1,6-diphenyl hexane (1), a novel, achiral, non-peptidic anthranil (Ant) group was designed as a P2/P2′ ligand. Symmetry-based inhibitors containing N-(2-pyridinylmethoxy-carbonyl)anthranil group are potent anti viral agents. Compounds 12 and 14 exhibited protease inhibitory activity of 60 and 70 pM, anti viral activity of 13 and 56 nM and cellular toxicity of > 10 uM respectively. We describe a HIV PR/1 structure-guided design of the novel, nonpeptidic, achiral anthranilamide group as a P2/P2′ ligand for the symmetry-based HIV PR inhibitors. X-ray crystallographic analysis of HIV PR/12 complex reveal that the anthranilamide group participates in both the polar and hydrophobic interactions that are commonly observed between the enzyme and peptidic inhibitor in the vicinity of the S2/S2′ subsites. The antiviral activity of compound 12 compares favorably with other potent HIV PR inhibitors currently undergoing clinical trials.

Readily accessible, non-peptidic, achiral 2-methylbenzamides were designed to serve as P2/P2′ ligands for symmetry-based inhibitors of HIV-1 Protease. Introduction of 3-hydroxy substituent provided a potent inhibitor 7 ( K i = 0.8 nM).

Structural analysis reveals mechanisms of drug resistance to HIV-1 protease inhibitors. These results have implications for the design of drugs and therapeutic strategies to combat drug resistance in AIDS.

Background: HIV-1 protease (HIV PR), an aspartic protease, cleaves Phe–Pro bonds in the Gag and Gag–Pol viral polyproteins. Substrate-based peptide mimics constitute a major class of inhibitors of HIV PR presently being developed for AIDS treatment. One such compound, KNI-272, which incorporates allophenylnorstatine (Apns)–thioproline (Thp) in place of Phe–Pro, has potent antiviral activity and is undergoing clinical trials. The structure of the enzyme–inhibitor complex should lead to an understanding of the structural basis for its tight binding properties and provide a framework for interpreting the emerging resistance to this drug. Results The three-dimensional crystal structure of KNI-272 bound to HIV PR has been determined to 2.0 a resolution and used to analyze structure–activity data and drug resistance for the Arg8→Gln and Ile84→Val mutations in HIV PR. The conformationally constrained Apns–Thp linkage is favorably recognized in its low energy trans conformation, which results in a symmetric mode of binding to the active-site aspartic acids and also explains the unusual preference of HIV PR for the S, or syn, hydroxyl group of the Apns residue. The inhibitor recognizes the enzyme via hydrogen bonds to three bridging water molecules, including one that is coordinated directly to the catalytic Asp 125 residue. Conclusion The structure of the HIV PR/KNI-272 complex illustrates the importance of limiting the conformational degrees of freedom and of using protein-bound water molecules for building potent inhibitors. The binding mode of HIV PR inhibitors can be predicted from the stereochemical relationship between adjacent hydroxyl-bearing and side chain bearing carbon atoms of the P1 substituent. Our structure also provides a framework for designing analogs targeted to drug-resistant mutant enzymes.

A major problem in the development of antiviral therapies for AIDS has been the emergence of drug resistance. We report an analysis of the structure of a Val 82 to Ala mutant of HIV-1 proteinase complexed to A-77003, a C2 symmetry-based inhibitor. Modelling studies predicted that the V82A mutation would result in decreased van der Waals' interactions with the phenyl rings of A-77003 in both S1 and S1' subsites. Unexpected rearrangements of the protein backbone, however, resulted in favourable re-packing of inhibitor and enzyme atoms in the S1 but not the S1' subsite. This analysis reveals the importance of enzyme flexibility in accommodating alternate packing arrangements, and can be applied to the re-design of inhibitors targeted to drug resistant variants which emerge in the clinic.

Four novel linear non-peptidic HIV-1 protease inhibitors derived from 2,5-diamino-1,6-diphenyl-3-hexanol were synthesized and characterized. All of them exhibit tight binding to HIV-1 protease, with inhibition constants K(i) in the range 20 pm-5 nm. The investigated inhibitors were crystallized, and their crystal structures were determined by X-ray diffraction. In all cases, the conformations found in the crystalline state differ significantly from the conformations obtained by computational docking of the inhibitor in the binding cleft of native HIV-1 protease. Owing to the prevalence of hydrophobic substituents in all these inhibitors, the conformational mobility in water solution is restricted to their compact forms. The spectrum of low-energy conformations in solution dramatically changes during the formation of inhibitor crystals (phenyl ring stacking as a leading motif) or during the formation of a complex with HIV-1 protease (elongated conformation suitable to fit the enzyme pockets as a factor responsible for tight binding). High conformational flexibility and low conformational stress in the molecules of these inhibitors most likely increase their biological activity in comparison with more rigid compounds.

HIV-1 proteinase (HIV PR) is a dimeric enzyme composed of two identical polypeptide chains that associate with twofold symmetry. We have determined to 1.8 A the crystal structure of a covalently tethered dimer of HIV PR. The tethered dimer:inhibitor complex is identical in nearly every respect to the complex of the same inhibitor with the wild type dimeric molecule, except for the linker region. Our results suggest that the tethered dimer may be a useful surrogate enzyme for studying the effects of single site mutations on substrate and inhibitor binding as well as on enzyme asymmetry, and for simulating independent mutational drift of the two domains which has been proposed to have led to the evolution of modern day, single-chain aspartic proteinases.

Inhibitors of the human immunodeficiency virus type 1 (HIV-1) protease represent a promising addition to the available agents used to inhibit virus replication in a therapeutic setting. HIV-1 is capable of generating phenotypic variants in the face of a variety of selective pressures. The potential to generate variants with reduced sensitivity to a protease inhibitor was examined by selecting for virus growth in cell culture in the presence of the protease inhibitor A-77003. Virus variants grew out in the presence of the inhibitor, and these variants encoded proteases with reduced sensitivity to the inhibitor. Variants were identified that encoded changes in each of the three subsites of the protease that interact with the inhibitor. HIV-1 displays significant potential for altering its interaction with this protease inhibitor, suggesting the need for multiple protease inhibitors with varying specificities.

The increasing availability of high-performance com puters makes it possible to treat larger systems in shorter computing times. However, a more challeng ing aspect of the availability of larger and faster com puters is the exploration of alternative algorithms. This is especially important in view of new computer archi tectures. In this work we describe a novel technique for feedback-scaling individual NOE distance restraints during molecular-dynamics-simulated annealing ex periments.

The incorporation of C 2 symmetry has become a useful paradigm in the design of active site inhibitors for HIV-1 protease (HIV PR) and has led to the design of a series of highly potent, C 2 symmetry-based, diol-containing inhibitors of HIV PR, one of which, A-77003, has reached clinical trials. However, the stereochemistry of the diol core influences protease inhibition and antiviral activity in a manner that is not well understood. We analyzed the crystal structures of a diastereomeric series of C 2 symmetry-based diol inhibitors, along with a deshydroxy analogue, bound to HIV PR and found that the stereochemistry of the diol core influences the mode of binding to the active site aspartic acids

The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.

Glutathione S-transferases (GST) are a major class of phase II detoxifying enzymes that conjugate glutathione to electrophiles. Their involvement in the degradation of chemotherapeutic agents, which contributes to drug resistance, makes this family of enzymes potential targets for therapeutic agents. This study generates, by homology modeling, a 3-D structure of three GST human isozymes of the Mu class, M1b-1b, M2-2 and M3-3, using the Rat3-3 GST structure as a template. The high percentage of identity among these enzymes and the lack of insertions and deletions make the system ideally suited to the technique of homology modeling. A novel technique for the modeling of protein structures was applied. The structure of the template was used to generate a low-resolution crystallographic map in which the initial coordinates of the structure to be modeled were placed. The structure was then annealed within this envelope. In addition, a feedback-restrained molecular dynamics procedure was adopted to scale the template restraints during the simulations. Three independent validation procedures were applied. To assess the reliability of the methods, an identical series of simulation steps to those used in the refinement were applied to the template structure (self modeling). Further, a homology structure for the Rat3-3 template was generated, starting from the modeled M1b-1b structure (reverse modeling). To assess the reasonableness of the modeled structures, two recently developed methodologies to verify protein structures based on statistics of the nonbonded interactions were applied. Overall, the structures appear to be consistent.

The concept of active-site symmetry along with structural and mechanistic considerations of aspartic proteases has been used to design novel,C 2-symmetry-based inhibitors of HIV PR. A comparative molecular-modeling strategy was used in the design of a pseudo-C 2-symmetric diaminoalcohol series and later a diastereomeric series ofC 2-symmetric diaminodiols. The structure of the pseudosymmetric alcohol A-74704, complexed with HIV PR, confirmed the proposed symmetric mode of binding, and also proved useful in subsequent efforts to improve the solubility of the more potent diol series. The crystal-structure analysis of a pseudosymmetricR,S-diol provided insights into the relative contributions of the two hydroxyl groups to the stability of the complex. A comparison of the binding modes for symmetric and asymmetric, substrate-based inhibitors indicates that the influence of symmetry on binding is mainly expressed at the protein level, since the overall subsite symmetry is maintained even with highly asymmetric inhibitors. Thus, while the incorporation ofC 2 symmetry does not necessarily lead to compounds with improved potency over asymmetric compounds, this strategy has led to the design of structurally novel and perhaps pharmacologically desirable inhibitors. The growing number of examples of symmetry-based inhibitors is evidence that symmetry has become a useful paradigm for HIV protease inhibitor design and that structure-based approaches to inhibitor design can contribute in unique and critical ways to the conceptualization of medicinal chemistry strategies that can lead to useful clinical candidates for AIDS, cancer and other diseases.