Structure-based design of novel HIV protease inhibitors: carboxamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones as potent nonpeptidic inhibitors.

The low oral bioavailability and rapid biliary excretion of peptide-derived HIV protease inhibitors have limited their utility as potential therapeutic agents. Our broad screening program to discover nonpeptidic HIV protease inhibitors had previously identified compound II (phenprocoumon, K(i) = 1 muM) as a lead template. Crystal structures of HIV protease complexes containing the peptide-derived inhibitor I (1-(naphthoxyacetyl)-L-histidyl-5(S)-amino-6-cyclohexyl-3 (R),4(R)-dihydroxy-2(R)-isopropylhexanoyl-L-isoleucine N-(2-pyridylmethyl)amide) and nonpeptidic inhibitors, such as phenprocoumon (compound II), provided a rational basis for the structure-based design of more active analogues. This investigation reports on the important finding of a carboxamide functionally appropriately added to the 4-hydroxycoumarin and the 4-hydroxy-2-pyrone templates which resulted in a new promising series of nonpeptidic HIV protease inhibitors with improved enzyme-binding affinity. The most active diastereomer of the carboxamide-containing compound XXIV inhibited HIV-1 protease with a K(i) value of 0.0014 muM. This research provides a new design direction for the discovery of more potent HIV protease inhibitors as potential therapeutic agents for the treatment of HIV infection.

Structure-based design of nonpeptidic HIV protease inhibitors from a cyclooctylpyranone lead structure.

Recently, the novel cyclooctylpyranone HIV protease inhibitor 1 was identified in our labs, and an X-ray structure of this inhibitor complexed with HIV-2 protease was obtained. This crystal structure was used to develop two strategies for creating derivatives of 1 with enhanced enzyme inhibitory activity. The first strategy, substitution on the cyclooctyl ring, met with limited success, but provided some interesting information about the conformationally-flexible cycloocytyl ring on the inhibitors. The second strategy, substitution at the meta position of the aryl ring, was far more successful and generated compounds, such as the carboxamide derivatives 41 (Ki = 3.0 +/- 0.4 nM) and 36 (Ki = 4.0 +/- 0.8 nM), which were significantly more active than the corresponding unsubstituted cycloocytlpyranone 2 (Ki = 11.7 +/- 4.7 nM). An X-ray crystal structure of 36 complexed with HIV-1 protease indicated the increase in binding affinity is most likely due to the additional interactions between the amide substituent and the S3 region of the protease.

Use of medium-sized cycloalkyl rings to enhance secondary binding: discovery of a new class of human immunodeficiency virus (HIV) protease inhibitors.

A unique strategy for the enhancement of secondary binding of an inhibitor to an enzyme has been demonstrated in the design of new human immunodeficiency virus (HIV) protease inhibitors. When the planar benzene ring of a 4-hydroxycoumarin lead compound (1a, Ki = 0.800 microM) was replaced with medium-sized (i.e., 7-9), conformationally-flexible, alkyl rings, the enzyme inhibitory activity of the resulting compounds was dramatically improved, and inhibitors with more than 50-fold better binding (e.g., 5d, Ki = 0.015 microM) were obtained. X-ray crystal structures of these inhibitors complexed with HIV protease indicated the cycloalkyl rings were able to fold into the S1' pocket of the enzyme and fill it much more effectively than the rigid benzene ring of the 4-hydroxycoumarin compound. This work has resulted in the identification of a promising lead structure for the design of potent, deliverable HIV protease inhibitors. Compound 5d, a small (MW = 324), nonpeptidic structure, has already shown several advantages over peptidic inhibitors, including high oral bioavailability (91-99%), a relatively long half-life (4.9 h), and ease of synthesis (three steps).

Cycloalkylpyranones and cycloalkyldihydropyrones as HIV protease inhibitors: exploring the impact of ring size on structure-activity relationships.

Previously, 3-substituted cycloalkylpyranones, such as 2d, have proven to be effective inhibitors of HIV protease. In an initial series of 3-(1-phenylpropyl) derivatives with various cycloalkyl ring sizes, the cyclooctyl analog was the most potent. We became interested in exploring the influence of other structural changes, such as substitution on the phenyl ring and saturation of the 5,6-double bond, on the cycloalkyl ring size structure-activity relationship (SAR). Saturation of the 5,6-double bond in the pyrone ring significantly impacts the SAR, altering the optimal ring size from eight to six. Substitution of a sulfonamide at the meta position of the phenyl ring dramatically increases the potency of these inhibitors, but it does not change the optimal ring size in either the cycloalkylpyranone or the cycloalkyldihydropyrone series. This work has led to the identification of compounds with superb binding affinity for the HIV protease (Ki values in the 10-50 pM range). In addition, the cycloalkyldihydropyrones showed excellent antiviral activity in cell culture, with ED50 values as low as 1 microM.

Structure-based design of novel HIV protease inhibitors: sulfonamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones as potent non-peptidic inhibitors.

The low oral bioavailability and rapid biliary excretion of peptide-derived HIV protease inhibitors have limited their utility as potential therapeutic agents. Our broad screening program to discover non-peptidic HIV protease inhibitors previously identified compound I (phenprocoumon, Ki = 1 microM) as a lead template. Structure-based design of potent non-peptidic inhibitors, utilizing crystal structures of HIV protease/inhibitor complexes, provided a rational basis for the previously reported carboxamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones. The amino acid containing compound V (Ki = 4 nM) provided an example of a promising new series of HIV protease inhibitors with significantly improved enzymatic binding affinity. In this report, further structure-activity relationship studies, in which the carboxamide is replaced by a sulfonamide functionality, led to the identification of another series of nonamino acid containing promising inhibitors with significantly enhanced enzyme binding affinity and in vitro antiviral activity. The most active diastereomer of the sulfonamide-containing pyrone XVIII (Ki = 0.5 nM) shows improved antiviral activity (IC50 = 0.6 nM) and represents an example of a new design direction for the discovery of more potent non-peptidic HIV protease inhibitors as potential therapeutic agents for the treatment of HIV infection.

Structure-based design of HIV protease inhibitors: 5,6-dihydro-4-hydroxy-2-pyrones as effective, nonpeptidic inhibitors.

From a broad screening program, the 4-hydroxycoumarin phenprocoumon (I) was previously identified as a lead template with HIV protease inhibitory activity. The crystal structure of phenprocoumon/HIV protease complex initiated a structure-based design effort that initially identified the 4-hydroxy-2-pyrone U-96988 (II) as a first-generation clinical candidate for the potential treatment of HIV infection. Based upon the crystal structure of the 4-hydroxy-2-pyrone III/HIV protease complex, a series of analogues incorporating a 5,6-dihydro-4-hydroxy-2-pyrone template were studied. It was recognized that in addition to having the required pharmacophore (the 4-hydroxy group with hydrogen-bonding interaction with the two catalytic aspartic acid residues and the lactone moiety replacing the ubiquitous water molecule in the active site), these 5,6-dihydro-4-hydroxy-2-pyrones incorporated side chains at the C-6 position that appropriately extended into the S1' and S2' subsites of the enzyme active site. The crystal structures of a number of representative 5,6-dihydro-4-hydroxy-2-pyrones complexed with the HIV protease were also determined to provide better understanding of the interaction between the enzyme and these inhibitors to aid the structure-based drug design effort. The crystal structures of the ligands in the enzyme active site did not always agree with the conformations expected from experience with previous pyrone inhibitors. This is likely due to the increased flexibility of the dihydropyrone ring. From this study, compound XIX exhibited reasonably high enzyme inhibitory activity (Ki = 15 nM) and showed antiviral activity (IC50 = 5 microM) in the cell-culture assay. This result provided a research direction which led to the discovery of active 5,6-dihydro-4-hydroxy-2-pyrones as potential agents for the treatment of HIV infection.

Tipranavir (PNU-140690): a potent, orally bioavailable nonpeptidic HIV protease inhibitor of the 5,6-dihydro-4-hydroxy-2-pyrone sulfonamide class.

A broad screening program previously identified phenprocoumon (1) as a small molecule template for inhibition of HIV protease. Subsequent modification of this lead through iterative cycles of structure-based design led to the activity enhancements of pyrone and dihydropyrone ring systems (II and V) and amide-based substitution (III). Incorporation of sulfonamide substitution within the dihydropyrone template provided a series of highly potent HIV protease inhibitors, with structure-activity relationships described in this paper. Crystallographic studies provided further information on important binding interactions responsible for high enzymatic binding. These studies culminated in compound VI, which inhibits HIV protease with a Ki value of 8 pM and shows an IC90 value of 100 nM in antiviral cell culture. Clinical trials of this compound (PNU-140690, Tipranavir) for treatment of HIV infection are currently underway.

Structure-based design of nonpeptidic HIV protease inhibitors: the sulfonamide-substituted cyclooctylpyramones.

Recently, cyclooctylpyranone derivatives with m-carboxamide substituents (e.g. 2c) were identified as potent, nonpeptidic HIV protease inhibitors, but these compounds lacked significant antiviral activity in cell culture. Substitution of a sulfonamide group at the meta position, however, produces compounds with excellent HIV protease binding affinity and antiviral activity. Guided by an iterative structure-based drug design process, we have prepared and evaluated a number of these derivatives, which are readily available via a seven-step synthesis. A few of the most potent compounds were further evaluated for such characteristics as pharmacokinetics and toxicity in rats and dogs. From this work, the p-cyanophenyl sulfonamide derivative 35k emerged as a promising inhibitor, was selected for further development, and entered phase I clinical trials.

Properties of erythromycin-inducible transposon Tn917 in Streptococcus faecalis.

Streptococcus faecalis strain DS16 harbors two plasmids, a conjugative plasmid, pAD1, which encodes hemolysin and bacteriocin activities, and a nonconjugative plasmid, pAD2, encoding resistance to streptomycin, kanamycin, and erythromycin, the latter of which is inducible. The erythromycin resistance determinant is located on a 3.3-megadalton transposable element designated Tn917, which could be transposed to pAD1 as well as to two other plasmids, pAm gamma 1 and pAM alpha 1. When strain DS16 was exposed to low (inducing) concentrations of erythromycin for a few hours, the frequency of Tn917 transposition from pAD2 to pAD1 increased by an order of magnitude. This induction paralleled induction of erythromycin resistance and was prevented by exposing the cells to inhibitors of deoxyribonucleic acid, ribonucleic acid or protein synthesis. The exposure of strain DS16 to inducing concentrations of erythromycin also enhanced the frequency of erythromycin-resistant transconjugants appearing during mating. Initially, cointegrate molecules, whose molecular weights were approximately the sum of pAD1 and pAD2, accounted for these transconjugants; however, as the induction time increased, pAD1::Tn917 became increasingly prominent.

Electron microscopic mapping of deletions on a streptococcal plasmid carrying extraordinarily long inverted repeats.

Deletions delta 101, delta 102, and delta 103 which occurred within the extraordinarily long inverted repeats of the self-ligated large EcoRI fragment of the streptococcal MLS (macrolides, lincosamides, streptogramin B)-resistance plasmid pSM19035 led to the formation of plasmids pDB101, pDB102, and pDB103. Their molecular lengths were determined by contour length measurements to be 17.8, 17.4, and 13.9 kb, respectively. Electron microscopic examination of self-annealed molecules revealed stem-loop structures with inverted repeats comprising 41 to 91% of the mass of plasmids. Two unique sequences (US1 and US2) separated the inverted repeats in the case of pDB101 and pDB103, while in pDB102 the repeats were joined at one end and separated at the other by a unique sequence (US2). The size of the unique sequence US2 was identical for all three plasmids, and the location of the resistance determinant was determined by electron microscopic examination of self-annealed molecules of the recombinant plasmid pDB201. Mapping of the deletion termini, accomplished by combining electron microscopic and HindIII restriction data, suggested that deletions may occur at preferential sites.

Simultaneous initiation of synthesis of bacteriophage T4 DNA and of deoxyribonucleotides.

In earlier reports we have suggested that bacteriophate T4 DNA replication occurs in a complex composed of the proteins required for polymerization and the system of enzymes synthesizing the deoxyribonucleoside triphosphate precursors of DNA. T4-induced dCMP hydroxymethylase and dTMP synthetase, though demonstrable in extracts soon after infection, are not active in vivo until about 5 min. The in vivo activities increase exponentially for approximately 15 min and then become constant. We have suggested that the exponential period represents the formation of the complexes. This paper shows that the initiation of DNA synthesis and of the two deoxyribonucleotide-synthesizing activities occurs simultaneously and with coinciding exponential kinetics. The in vivo activities of the two enzymes were tested after infection by a number of T4 amber Dna- mutants. Their activities were essentially unchanged compared to the wild-type phage, except on infection by mutants of gene 43 (T4 DNA nucleotidyltransferase or DNA polymerase). With these mutants the rate of increase of dTMP synthetase and dCMP hydroxymethylase activities was always substantially lower than after infection by wild-type phage. It is proposed that an intimate interaction occurs between T4-induced DNA polymerase and the complex of enzymes forming 5-hydroxymethyl-dCMP and dTMP.

Plasmid-related transmissibility and multiple drug resistance in Streptococcus faecalis subsp. zymogenes strain DS16.

Streptococcus faecalis subsp. zymogenes strain DS16 was found to harbor two plasmids, designated pAD1 (35 megadaltons) and pAD2 (15 megadaltons). pAD1 is transmissible and determines a hemolysin-bacteriocin, whereas pAD2 is non-conjugative and determines resistance to erythromycin, streptomycin, and kanamycin. pAD2 could be mobilized by pAD1, but usually involved formation of a pAD1-pAD2 cointegrate.

Direct participation of dCMP hydroxymethylase in synthesis of bacteriophage T4 DNA.

In order to retain in an in situ system the control mechanisms involved in synthesis of bacteriophage T4 DNA, infected cells were made permeable to nucleotides by plasmolysis with concentrated sucrose. Such preparations use exogenous deoxyribonucleotides to synthesize T4 phage DNA. As has been observed with in vivo studies, DNA synthesis was drastically reduced in plasmolyzed preparations from cells infected by amber mutants of genes 1, 32, 41, 42, 43, 44, or 45. Added 5-hydroxymethyl dCTP did not bypass either a mutant of gene 42 (dCMP hydroxymethylase) or of gene 1 (phage-induced deoxyribonucleotide kinase). In a phage system lacking deoxycytidine triphosphatase (gene 56) and the gene-46 product, and therefore incorporating dCTP into DNA, dCTP incorporation did not require dCMP hydroxymethylase, in keeping with in vivo results. With a triple amber mutant of genes 1, 46, and 56 only slight incorporation of dCTP occurred. By contrast, in experiments performed in vivo the synthesis of cytosine-containing DNA was unaffected by an amber mutation in gene 1. These studies provide evidence that dCMP hydroxymethylase, in addition to its known catalytic function, has a second, more direct, role in phage T4 DNA synthesis, apparently in recognition of hydroxymethyl dCTP. The role of the phage-induced deoxyribonucleotide kinase in T4 DNA synthesis in the plasmolyzed system remains unresolved.

Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA.

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.

The ligand affinity of proteins measured by isothermal denaturation kinetics.

An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the presence of potential specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfolding of the proteins. The experimental procedure was developed using thymidylate kinase and stromelysin as target proteins. The kinetics of thermal unfolding of both of these enzymes were consistent with a pathway of two consecutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased the value of the rate constants of both steps in a dose-dependent manner. The dependence of the rate constants on ligand concentration obeyed a simple binding isotherm, the analysis of which yielded an accurate equilibrium constant for ligand binding. The method was validated by comparing its results with those obtained under the same conditions by steady-state fluorescence spectroscopy, circular dichroism, and uv spectrophotometry: The corresponding rate constants were comparable for each of the analytical detection methods.

Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917.

Plasmids pAD1 (37.8 megadaltons) and pAD2 (17.1 megadaltons) of Streptococcus faecalis strain DS16 have been mapped with restriction enzymes. The location of a hemolysin-bacteriocin determinant on the conjugative pAD1 plasmid was derived from analyses of transposon insertions. Electron microscope and hybridization analyses located Tn917(Em) and the streptomycin (Sm) and kanamycin (Km) resistance determinants on the nonconjugative pAD2 plasmid. It was shown previously that the erythromycin (Em) resistance associated with Tn917 is inducible and that transposition from pAD2 to pAD1 is also stimulated by exposure of cells to low concentrations of Em. Here we show that inducing concentrations of Em also increase the conjugative transfer potential of pAD1; this is possibly related to a mild and short-lived inhibitory stress placed on the cells before full induction of resistance. Selection of Em-resistant transconjugants arising from matings between DS16 and a plasmid-free recipient gave rise to transconjugants which primarily harbor stable pAD1::pAD2 cointegrates. A 30-min exposure of donors to Em (0.5 microgram/ml) before mating resulted in a severalfold increase in the number of such transconjugants. However, a small fraction (e.g., 3 of 40) of these Emr Smr Kmr transconjugants harbored pAD1::Tn917 and pAD2 molecules. Since we believe pAD2 is incapable of being mobilized by pAD1 without being covalently linked, it is likely that transfer in these cases involved cointegrates representing structural intermediates in the transposition of Tn917 from pAD2 to pAD1. It follows that such intermediates probably had two copies of Tn917 and readily resolved after transfer. (These cointegrates are different from the stable cointegrates which were shown to have only a single copy of Tn917; the latter are assumed not to be related to transposition.) Two variants with altered Tn917 transposition properties were derived. One of them transposed at an elevated frequency, whereas the other showed no detectabel transposition. In neither case was transposition influenced by Em exposure; however, both remained inducible for Em resistance.