A series of phosphonate prodrugs were evaluated in an attempt to increase the oral bioavailability of the anti-HIV agent 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; 1). The majority of the bis(alkyl ester) and bis(alkyl amide) prodrugs were prepared by alcohol or amine displacement of dichlorophosphonate 2. Basic hydrolysis of the bis(esters) or bis(amides) provided the corresponding monoesters or monoamides. Synthesis of bis[(acyloxy)alkyl] phosphonates 10a-c was accomplished by alkylation of PMEA with the appropriate chloromethyl ether in the presence of N,N'-dicyclohexylmorpholinecarboxamidine. The systemic levels of PMEA following oral administration of a PMEA prodrug to rats were determined by measuring the concentration of PMEA in the urine for 48 h after administration of the prodrug. The oral bioavailability of PMEA employing this method was determined to be 7.8%. Oral dosing with bis(alkyl) phosphonates 3a,b resulted in apparent absorption of the prodrugs (> or = 40%), although neither of the esters were completely cleaved to liberate the parent phosphonate PMEA. The mono(alkyl esters) 7a-e and 8a,b exhibited poor oral bioavailability (< or = 5%). Phosphonamides 5, 6, and 9 were unstable under acidic conditions and provided levels of PMEA comparable to the parent compound after oral administration. Bis[(acyloxy)alkyl] phosphonates 10a-c demonstrated significantly improved oral bioavailabilities of 17.6%, 14.6%, and 15.4%, respectively. When evaluated in vitro against HSV-2, (acyloxy)alkyl phosphonates 10a-c were greater than 200-fold more active than PMEA.
A dynamical model for the structure of the human immunodeficiency virus 1 (HIV-1) protease dimer in aqueous solution has been developed on the basis of molecular dynamics simulation. The model provides an accurate account of the crystal geometry and also a prediction of the structural reorganization expected to occur in the protein in aqueous solution compared to the crystalline environment. Analysis of the results by means of dynamical cross-correlation coefficients for atomic displacements indicates that domain-domain communication is present in the protein in the form of a molecular "cantilever" and is likely to be involved in enzyme function at the molecular level. The dynamical structure also suggests information that may ultimately be useful in understanding and further development of specific inhibitors of HIV-1 protease.Human immunodeficiency virus 1 (HIV-1) protease is a dimer of 99-residue proteins which is vitally important to polyprotein processing in the life cycle of the AIDS virus (1). This enzyme is currently the only significant macromolecular component of the AIDS virus for which detailed structural information is available (2-4) and is a target of considerable pharmaceutical interest in the quest for AIDS therapies (5). The current structural model for HIV-1 protease developed from crystallography (3), depicted in Fig. 1, has established the essential secondary and tertiary structure of the enzyme. Homology with other aspartyl proteases (6) indicates that the protein dimer formed in the crystal is the native enzyme. The observed isotropic temperature factors indicate certain dynamical elements in the structure, particularly the flexibility in the flap regions opposite the active site Asp-Thr-Gly triads. The recent crystal structure of an HIV-1 protease-inhibitor complex (7) displays the binding site in detail and also provides evidence of substantial changes in the enzyme remote from the binding site. These changes emphasize the need for a consideration of structural dynamics in understanding HIV-1 protease action.We describe herein a theoretical model for HIV-1 protease dimer in dilute aqueous solution developed from molecular dynamics simulation. Analysis of the results provides knowledge about the structure and functional energetics that originates uniquely in the dynamical motions, and in particular evidence for through-space interactions between domains of well-defined secondary structure.
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