Synthesis of novel cyclic urea based HIV-1 protease inhibitors

Over a 3-year period, 663 children aged under 13 years were seen with a history of foreign body (FB) ingestion. Seventy-six per cent of the children were less than 6 years old. Coins and chicken or fish bones were the most common objects ingested. In 27 (4%) children, the FB had been expelled or removed before arrival, and in a further 133 (20%), no FB could be identified. Three patients required emergency airway management. Less than 50% of the children presenting with dysphagia or vomiting had identifiable FBs lodged in the oropharynx or proximal oesophagus, whilst 22% of the children with FBs requiring removal from these sites were asymptomatic. Oesophageal FBs were removed under direct vision (six cases), by rigid oesophagoscopy under general anaesthetic (77 cases), or using a balloon catheter under sedation (26 cases). All 224 FBs detected in the stomach or beyond were allowed to pass naturally, and delayed passage occurred in only one case. Passage of 11 alkaline disc batteries occurred without complication. No patient required surgical removal of an FB.     

Design and synthesis of novel conformationally restricted HIV protease inhibitors

A set of HIV protease inhibitors represented by compound 2 has previously been described. Structural and conformational analysis of this compound suggested that conformational restriction of the P1/P2 portion of the molecule could lead to a novel set of potent protease inhibitors. Thus, probe compounds 3-7 were designed, synthesized, and found to be potent inhibitors of HIV protease.     

Nonpeptide cyclic cyanoguanidines as HIV-1 protease inhibitors: synthesis, structure-activity relationships, and X-ray crystal structure studies

Comparison of the high-resolution X-ray structures of the native HIV-1 protease and its complexes with the inhibitors suggested that the enzyme flaps are flexible. The movement at the tip of the flaps could be as large as 7 A. On the basis of this observation, cyclic cyanoguanidines have been designed, synthesized, and evaluated as HIV-1 protease (PR) inhibitors. Cyclic cyanoguanidines were found to be very potent inhibitors of HIV-1 protease. The choice of cyclic cyanoguanidines over cyclic guanidines was based on the reduced basicity of the former. X-ray structure studies of the HIV PR complex with cyclic cyanoguanidine demonstrated that in analogy to cyclic urea, cyclic cyanoguanidines also displace the unique structural water molecule. The structure-activity relationship of the cyclic cyanoguanidines is compared with that of the corresponding cyclic urea analogues. The differences in binding constants of the two series of compounds have been rationalized using high-resolution X-ray structure information.     

Design, synthesis, and conformational analysis of a novel series of HIV protease inhibitors

A combination of structure-based design and both solution, and solid-phase synthesis were utilized to derive a potent (nM) series of HIV-1 protease inhibitors bearing a structurally novel backbone. Detailed structural analysis of several inhibitors prepared in this series has suggested that rigidification of the P1/P2 region of this class of molecules may result in compounds with improved potency.     

Stereoisomers of cyclic urea HIV-1 protease inhibitors: synthesis and binding affinities

We have synthesized stereoisomers of cyclic urea HIV-1 protease inhibitors to study the effect of varying configurations on binding affinities. Four different synthetic approaches were used to prepare the desired cyclic urea stereoisomers. The original cyclic urea synthesis using amino acid starting materials was used to prepare three isomers. Three additional isomers were prepared by synthetic routes utilizing L-tartaric acid and D-sorbitol as chiral starting materials. A stereoselective hydroxyl inversion of the cyclic urea trans-diol was used to prepare three additional isomers. In all 9 of the 10 possible cyclic urea stereoisomers were prepared, and their binding affinities are described.     

Design and synthesis of new potent C2-symmetric HIV-1 protease inhibitors. Use of L-mannaric acid as a peptidomimetic scaffold

A study on the use of derivatized carbohydrates as C2-symmetric HIV-1 protease inhibitors has been undertaken. L-Mannaric acid (6) was bis-O-benzylated at C-2 and C-5 and subsequently coupled with amino acids and amines to give C2-symmetric products based on C-terminal duplication. Potent HIV protease inhibitors, 28 Ki = 0.4 nM and 43 Ki = 0.2 nM, have been discovered, and two synthetic methodologies have been developed, one whereby these inhibitors can be prepared in just three chemical steps from commercially available materials. A remarkable increase in potency going from IC50 = 5000 nM (23) to IC50 = 15 nM (28) was observed upon exchanging -COOMe for -CONHMe in the inhibitor, resulting in the net addition of one hydrogen bond interaction between each of the two -NH- groups and the HIV protease backbone (Gly 48/148). The X-ray crystal structures of 43 and of 48 have been determined (Figures 5 and 6), revealing the binding mode of these inhibitors which will aid further design.     

Three-dimensional structure of a mutant HIV-1 protease displaying cross-resistance to all protease inhibitors in clinical trials

Analysis of mutational effects in the human immunodeficiency virus type-1 (HIV-1) provirus has revealed that as few as four amino acid side-chain substitutions in the HIV-1 protease (M46I/L63P/V82T/I84V) suffice to yield viral variants cross-resistant to a panel of protease inhibitors either in or being considered for clinical trials (Condra, J. H., Schleif, W. A., Blahy, O. M., Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). As an initial effort toward elucidation of the molecular mechanism of drug resistance in AIDS therapies, the three-dimensional structure of the HIV-1 protease mutant containing the four substitutions has been determined to 2.4-A resolution with an R factor of 17.1%. The structure of its complex with MK639, a protease inhibitor of the hydroxyaminopentane amide class of peptidomimetics currently in Phase III clinical trials, has been resolved at 2.0 A with an R factor of 17.0%. These structures are compared with those of the wild-type enzyme and its complex with MK639 (Chen, Z., Li, Y., Chen, E., Hall, D. L., Darke, P. L., Culberson, C., Shafer, J., and Kuo, L. C. (1994) J. Biol. Chem. 269, 26344-26348). There is no gross structural alteration of the protease due to the site-specific mutations. The C alpha tracings of the two native structures are identical with a root-mean-square deviation of 0.5 A, and the four substituted side chains are clearly revealed in the electron density map. In the MK639-bound form, the V82T substitution introduces an unfavorable hydrophilic moiety for binding in the active site and the I84V substitution creates a cavity (unoccupied by water) that should lead to a decrease in van der Waals contacts with the inhibitor. These changes are consistent with the observed 70-fold increase in the Ki value (approximately 2.5 kcal/mol) for MK639 as a result of the mutations in the HIV-1 protease. The role of the M46I and L63P substitutions in drug resistance is not obvious from the crystallographic data, but they induce conformational perturbations (0.9-1.1 A) in the flap domain of the native enzyme and may affect the stability and/or activity of the enzyme unrelated directly to binding.     

Structure-based design, synthesis and evaluation of conformationally constrained cysteine protease inhibitors

The inhibition of cysteine proteases is being studied as a strategy to combat parasitic diseases such as Chagas' disease, leishmaniasis, and malaria. Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiologic agent of Chagas' disease. A crystal structure of cruzain, covalently inactivated by fluoromethyl ketone inhibitor 1 (Cbz-Phe-Ala-FMK), was used as a template to design potential inhibitors. Conformationally constrained gamma-lactams containing electrophilic aldehyde (12, 17, 18, 25, 26, and 29) or vinyl sulfone (43, 44, and 46) units were synthesized. Constrained lactam 26 had IC50 values of ca. 20 nM against the Leishmania major protease and ca. 50 nM versus falcipain, an important cysteine protease isolated from Plasmodium falciparum. However, all of the conformationally constrained inhibitors were weak inhibitors of cruzain, compared to unconstrained peptide aldehyde (e.g. 5 ) and vinyl sulfone inhibitors (e.g. 48, which proved to be an excellent inhibitor of cruzain with an apparent second order inhibition rate constant (k(inact)/Ki) of 634,000s(-1)M(-1). A significant reduction in activity was also observed with acyclic inhibitors 30 and 51 containing alpha-methyl phenylalanine residues at the P2 position. These data indicate that the pyrrolidinone ring, especially the quarternary center at P2, interferes with the normal substrate binding mode with cruzain, but not with falcipain or the leishmania protease.     

Molecular recognition of cyclic urea HIV-1 protease inhibitors

As long as the threat of human immunodeficiency virus (HIV) protease drug resistance still exists, there will be a need for more potent antiretroviral agents. We have therefore determined the crystal structures of HIV-1 protease in complex with six cyclic urea inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146, in an attempt to identify 1) the key interactions responsible for their high potency and 2) new interactions that might improve their therapeutic benefit. The structures reveal that the preorganized, C2 symmetric scaffolds of the inhibitors are anchored in the active site of the protease by six hydrogen bonds and that their P1 and P2 substituents participate in extensive van der Waals interactions and hydrogen bonds. Because all of our inhibitors possess benzyl groups at P1 and P1', their relative binding affinities are modulated by the extent of their P2 interactions, e.g. XK216, the least potent inhibitor (Ki (inhibition constant) = 4.70 nM), possesses the smallest P2 and the lowest number of P2-S2 interactions; whereas SD146, the most potent inhibitor (Ki = 0.02 nM), contains a benzimidazolylbenzamide at P2 and participates in fourteen hydrogen bonds and approximately 200 van der Waals interactions. This analysis identifies the strongest interactions between the protease and the inhibitors, suggests ways to improve potency by building into the S2 subsite, and reveals how conformational changes and unique features of the viral protease increase the binding affinity of HIV protease inhibitors.     

Cyclic HIV-1 protease inhibitors derived from mannitol: synthesis, inhibitory potencies, and computational predictions of binding affinities

The molecular characteristics of midbrain dopamine (DA) neurons have been extensively studied in Parkinson's disease (PD). No such studies of the characteristics of midbrain DA neurons in Alzheimer's disease (AD) or Alzheimer's disease with parkinsonism (AD/Park) have been published. We examined the levels of tyrosine hydroxylase (TH) protein, and the expression of TH and dopamine transporter (DAT) mRNAs, in midbrain neurons of PD, AD, and AD/Park cases. In PD, the loss of TH protein in the ventral tier of the substantia nigra pars compacta (SNpc) of the PD group in accompanied by severe losses in the number of neurons that express TH mRNA and DAT mRNA (74% loss). Remaining neurons show a shift to higher concentrations of TH mRNA but a shift to lower concentrations of DAT mRNA per cell. Hence, there is evidence that compensation in the remaining neurons can elevate concentrations of TH mRNA and lower DAT mRNA. Alternatively, there may be a predilection for a loss of neurons with high levels of DAT mRNA and low TH mRNA levels within the SNpc of PD cases. There was no change in TH protein but an elevation of TH mRNA concentrations per neuron without any change in concentrations of DAT mRNA in the AD group. The AD/Park group did not exhibit changes in the level of TH protein, but showed a small loss (26%) of neurons in the SNpc and a greater loss in other regions of the midbrain (43-53%). Remaining DA neurons showed a marked shift to lower concentrations of DAT mRNA per neuron and a nonsignificant shift in cellular concentration of TH mRNA to higher levels. This is consistent with our previous work showing that with AD/Park there is a significant reduction in the number of DAT sites located on DA terminals in the striatum, but the midbrain neurons have not died. Our results indicate that the differential regulation of mRNAs encoding TH and DAT is similar in the parkinsonian disorders (PD and AD/Park) even though the degree of cell death is very different. This might suggest that compensatory events occur in these DA neurons in AD/Park that are similar to those in PD and that result in differential effects on mRNAs encoding TH and DAT proteins.     

Coevolutionary analysis of resistance-evading peptidomimetic inhibitors of HIV-1 protease

We have developed a coevolutionary method for the computational design of HIV-1 protease inhibitors selected for their ability to retain efficacy in the face of protease mutation. For HIV-1 protease, typical drug design techniques are shown to be ineffective for the design of resistance-evading inhibitors: An inhibitor that is a direct analogue of one of the natural substrates will be susceptible to resistance mutation, as will inhibitors designed to fill the active site of the wild-type or a mutant enzyme. Two design principles are demonstrated: (i) For enzymes with broad substrate specificity, such as HIV-1 protease, resistance-evading inhibitors are best designed against the immutable properties of the active site-the properties that must be conserved in any mutant protease to retain the ability to bind and cleave all of the native substrates. (ii) Robust resistance-evading inhibitors can be designed by optimizing activity simultaneously against a large set of mutant enzymes, incorporating as much of the mutational space as possible.     

Design, synthesis, and evaluation of tetrahydropyrimidinones as an example of a general approach to nonpeptide HIV protease inhibitors

Re-examination of the design of the cyclic urea class of HIV protease (HIVPR) inhibitors suggests a general approach to designing novel nonpeptide cyclic HIVPR inhibitors. This process involves the inversion of the stereochemical centers of the core transition-state isostere of the linear HIVPR inhibitors and cyclization of the resulting core using an appropriate cyclizing reagent. As an example, this process is applied to the diamino alcohol class of HIVPR inhibitors to give tetrahydropyrimidinones. Conformational analysis of the tetrahydropyrimidinones and modeling of its interaction with the active site of HIVPR suggested modifications which led to very potent inhibitors of HIVPR (24 with a Ki = 0.018 nM). The X-ray crystallographic structure of the complex of 24 with HIVPR confirms the analysis and modeling predictions. The example reported in this study and other examples that are cited indicate that this process may be generally applicable to other linear inhibitors.     

Tricyclic ureas: a new class of HIV-1 protease inhibitors

A new class of tricyclic ureas containing a conformationally constrained proline was designed with the aid of molecular modeling. Efficient stereoselective intermolecular pinacol coupling represented the highlight of the synthesis. These rigid cyclic ureas are active towards HIV-1 protease, with 9 being the most potent compound (Ki = 9 nM) despite interacting with only three side chain binding pockets of HIV protease.     

Peptide inhibitors of HIV-1 protease and viral infection of peripheral blood lymphocytes based on HIV-1 Vif

We recently reported that HIV-1 Vif (virion infectivity factor) inhibits HIV-1 protease in vitro and in bacteria, suggesting that it may serve as the basis for the design of new protease inhibitors and treatment for HIV-1 infection. To evaluate this possibility, we synthesized peptide derivatives from the region of Vif, which inhibits protease, and tested their activity on protease. In an assay of cleavage of virion-like particles composed of HIV-1 Gag precursor polyprotein, full-length recombinant Vif, and a peptide consisting of residues 21-65 of Vif, but not a control peptide or BSA, inhibited protease activity. Vif21-65 blocked protease at a molar ratio of two to one. We then tested this peptide and a smaller peptide, Vif41-65, for their effects on HIV-1 infection of peripheral blood lymphocytes. Both Vif peptides inhibited virus expression below the limit of detection, but control peptides had no effect. To investigate its site of action, Vif21-65 was tested for its effect on Gag cleavage by protease during HIV-1 infection. We found that commensurate with its reduction of virus expression, Vif21-65 inhibited the cleavage of the polyprotein p55 to mature p24. These results are similar to those obtained by using Ro 31-8959, a protease inhibitor in clinical use. We conclude that Vif-derived peptides inhibit protease during HIV-1 infection and may be useful for the development of new protease inhibitors.     

Tripeptide aldehyde inhibitors of human rhinovirus 3C protease: design, synthesis, biological evaluation, and cocrystal structure solution of P1 glutamine isosteric replacements

The investigation of tripeptide aldehydes as reversible covalent inhibitors of human rhinovirus (HRV) 3C protease (3CP) is reported. Molecular models based on the apo crystal structure of HRV-14 3CP and other trypsin-like serine proteases were constructed to approximate the binding of peptide substrates, generate transition state models of P1-P1' amide cleavage, and propose novel tripeptide aldehydes. Glutaminal derivatives have limitations since they exist predominantly in the cyclic hemiaminal form. Therefore, several isosteric replacements for the P1 carboxamide side chain were designed and incorporated into the tripeptide aldehydes. These compounds were found to be potent inhibitors of purified HRV-14 3CP with Kis ranging from 0.005 to 0.64 microM. Several have low micromolar antiviral activity when tested against HRV-14-infected H1-HeLa cells. The N-acetyl derivative 3 was also shown to be active against HRV serotypes 2, 16, and 89. High-resolution cocrystal structures of HRV-2 3CP, covalently bound to compounds 3, 15, and 16, were solved. These cocrystal structures were analyzed and compared with our original HRV-14 3CP-substrate and inhibitor models.