William H. Prusoff

Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.

Abstract A theoretical analysis has been made of the relationship between the inhibition constant ( K I ) of a substance and the ( I 50 ) value which expresses the concentration of inhibitor required to produce 50 per cent inhibition of an enzymic reaction at a specific substrate concentration. A comparison has been made of the relationships between K I and I 50 for monosubstrate reactions when noncompetitive or uncompetitive inhibition kinetics apply, as well as for bisubstrate reactions under conditions of competitive, noncompetitive and uncompetitive inhibition kinetics. Precautions have been indicated against the indiscriminate use of I 50 values in agreement with the admonitions previously described in the literature. The analysis described shows K I does not equal I 50 when competitive inhibition kinetics apply; however, K I is equal to I 50 under conditions of either noncompetitive or uncompetitive kinetics.

Studies in the mouse of the pharmacology of 5-iododeoxyuridine, an analogue of thymidine

Abstract 5-Iododeoxyuridine labeled with I 131 or tritium was administered to normal and tumor-bearing mice, and a study was made of its metabolism, tissue distribution and excretion. The main pathway for degradation of IUdR involved a cleavage to 5-iodouracil and subsequent dehalogenation with the formation of uracil and inorganic iodide. Considerable iodination of non-nucleic acid derivatives was observed in the protein fractions of cells. The occurrence of iodinated derivatives of acyclic products of iodouracil catabolism was not demonstrated, although these probably were formed as a result of direct degradation. The acute LD 50 of IUdR in mice was 2–5 g/kg. The LD 50 for IUdR upon chronic administration intraperitoneally for 13 days was 318 mg/kg; this toxicity could be prevented completely by the prior administration of thymidine.

The pc12 cell as a model for studies of the mechanism of induction of peripheral neuropathy by anti-HIV-1 dideoxynucleoside analogs

The ability of DNA polymerase gamma to utilize ddNTPs+ as substrates, incorporating these chain terminators into DNA (see citations in[1]), has led us to propose that AZT-induced bone marrow suppression results from inhibition of mtDNA replication [1]. This could alter metabolism since mtDNA encodes proteins involved in ATP synthesis and mitochondrial ultrastructure. We have shown that AZT and some other ddNs used in AIDS therapy inhibit DNA replication by purified DNA polymerase gamma and by isolated mitochondria [1,2]. Moreover, proliferation of a hemopoietic cell, the Friend erythroleukemic cell, is strongly inhibited by AZT [2], and mitochondria from these cells show impairment of DNA replication++ [2]. Additional evidence comes from studies on the effect of ddC on the Molt-4F cell [3], and on the involvement of mitochondria in AZT-induced myopathy [4]. We propose that inhibition of mtDNA replication is also the primary step in ddC-induced [5], ddI-induced [6] and d4T-induced [7] peripheral neuropathy. We have found that a c-Ha-ras transformant of the PC12 cell, GS-ras-1+++, is a promising neuronal cell model for such studies. The PC12 cell line [8] is derived from a pheochromocytoma, an adrenal medullary tumor; cells of the adrenal medulla share their embryological origin with neuronal cells. When the PC12 cell is induced by NGF (or by dexamethasone in GS-ras-1) to differentiate, neurite outgrowth and increased synthesis of acetylcholine and other neuronal markers occur. We describe the effects of some ddNs on both the uninduced and the induced cell.

Nucleoside analogs with antiviral activity

The fact that numerous nucleoside analogs have been synthesized as potential antiviral drugs demonstrates that there is no magic road to the development of an ideal antiviral agent. Many of the compounds tested evolved from programs centered on the preparation of antineoplastic agents, and several nucleoside derivatives have been obtained that are clinically useful as both antiviral and anticancer drugs. Is it, however, possible to prepare nucfeosides which exhibit highly specific antiviral activity? What are the desirable features of such an ideal antiviral drug’? How close do the nucleosides currently in clinical use or trial come to achieving these objectives? In this solitary. we will attempt to answer some of these questions. The first nucleoside approved for clinical use in the early 1960’s was 5-iodo-2’-deoxyuridine (IdUrd). It is of some historical interest that Johnson and Johns [l] reported the synthesis of 5-i~opyrimidines in 1905 from Yale University. It was not until 1945, however, that Hitchings et al. [2] initiated a systematic study of the biological activities of various analogs of purine and pyrimidine bases. Soon after. this group [3] documented the modest inhibition by 5bromouracil,5hydroxyuracil and 2,4-dithiothymine of the replication of vaccinia virus in cell culture. Visser et al. [4] in 1952 reported that various 5-substituted analogs of uridine (%chIoro, 5-diazo, 5-forrnamido, S-hydroxy and 5-amino) inhibited the replication of Theiler’s mouse encephalitis virus in mouse brain cultures. Because pyrimidine bases and ribonucleosides with substituents in the S-position are poorly utilized in the biosynthesis of DNA precursor nucleotides, it perhaps was predictable, retrospectively, that when the 5-chloro [5], 5-bromo [6] and 5-iodo [7] derivatives of deoxyuridine were synthesized, they would exhibit, relative to the free bases. si~ificant antiviral activity in t’itro [S]. The subsequent demonstration that IdUrd was effective in an established virus infection in man [g3 lo] was, therefore, of utmost importance. Other antiviral nucleosides were soon found; for example, 6-azauridine [tl] and l-P-D-arabinofuranosyl cytosine [12,13]. A representative example of the type of modifications which yield nucleosides with antiviral activity is depicted in Fig. 1 for the pyrimidine series. The rationale for the synthesis of many of the known antiviral nucleosides evolved logically from our know-

Anti-HIV-1 activity and cellular pharmacology of various analogs of gossypol

We previously reported that the racemic mixture and both enantiomers of gossypol inhibit the replication of human immunodeficiency virus-type 1 (HIV-1) (Lin et al., Antimicrob Agents Chemother 33: 2149-2151, 1989). The present study evaluates the activities of a variety of analogs of gossypol as well as a few non-gossypol analogs. Compounds 2, 3, 10, and 13 were slightly more inhibitory than (-)-gossypol to the replication of HIV-1 in cell culture. Compounds 4 and 8 were cytotoxic to human peripheral blood monocyte (PBM) cells, and compounds 2 and 3 were cytotoxic to Vero cells but not PBM cells. The effects of the two enantiomers of gossypol on the cell volume and migration of H9 cells through the cell cycle were evaluated during 72 hr of incubation. The (-)-enantiomer of gossypol was more toxic to H9 cells than the (+)-enantiomer of gossypol as evidenced by cell destruction. Prior to cell destruction, there appeared to be no significant effect on cell cycle distribution with either enantiomer.

Incorporation of iododeoxyuridine into the deoxyribonucleic acid of mouse Ehrlich-ascites-tumor cells in vivo

Abstract Evidence has been presented for the incorporation of radioactive iododeoxyuridine into the DNA of mouse Ehrlich ascites tumor cells in vivo . Although thymidine was utilized preferentially (to a 40-fold greater extent than iododeoxyuridine) for the biosynthesis of DNA, an almost identical incorporation of iododeoxyuridine occurred when the total amount of [ 131 I]iododeoxyuridine injected was increased by 12-fold and the frequency of administration of the analogue was doubled.

Deoxyribonucleotide metabolism in Herpes simplex virus infected HeLa cells.

The effect of Rolly No. 11 strain herpes simplex virus infection of HeLa cells in culture on deoxynucleotide metabolism and the level of various enzymes concerned with the biosynthesis of DNA has been investigated. Of 18 enzyme activities studied, thymidine kinase, DNA polymerase and deoxyribonuclease were markedly augmented, a finding in agreement with previous reports. Deoxycytidine kinase, ribonucleotide reductase, thymidylate kinase and deoxycytidylate deaminase activities, in contrast with previous reports, did not increase; the activities of the other enzymes studied, also did not increase. Whereas most of the radioactivity derived from [14-C] thymidine in the acid-soluble fraction of the uninfected cells was present as deoxythymidine triphosphate, that present in the infected cells was primarily in the form of deoxythymidine monophosphate. Thus, in the infected cell deoxythymidylate kinase is a rate-limiting enzyme in the biosynthesis of deoxythymidine triphosphate. A marked increase in the pools of the four naturally occurring deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP) was found. The rate of formation of the virus-induced enzymes was determined, as were the various nucleoside triphosphate pools and the other phosphorylated derivatives of thymidine; a maximum was reached for all these csmponents between 6 to 8 h post infection. Although an apparent greater synthesis of DNA occurred in the uninefected cells, when the specific activity of the radioactive deoxythymidine triphosphate was taken into account, there was actually a greater rate of DNA synthesis in the infected cells, with the peak at 8 h post infection.

Early nucleoside reverse transcriptase inhibitors for the treatment of HIV: a brief history of stavudine (D4T) and its comparison with other dideoxynucleosides

The occasion of this 25th anniversary issue encouraged us to reminisce about the important history of the discovery of the dideoxynucleoside analogues for the treatment of HIV/AIDS and to chronicle our thoughts about a particular exciting and rewarding period of our scientific careers. Following the identification of the anti-HIV activity of zidovudine (AZT), we participated in the urgent quest to discover optimal treatments of HIV infection and AIDS. A number of previously synthesized nucleoside analogues were comparatively evaluated, and stavudine (D4T) emerged as a promising candidate for development. Following clinical evaluation, D4T became a mainstay of the initial antiretroviral combination therapy, prolonging and saving numerous lives. It has only recently been supplanted by better-tolerated treatments. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, vol. 85, issue 1, 2010.

Further studies on the inhibition of nucleic acid biosynthesis by azathymidine and by deoxyadenosine.

Abstract Azathymidine, an analogue of thymidine, inhibited reversibly the utilization of radioactive thymidine for the biosynthesis of DNA-thymine of Ehrlich ascites tumour cells in vitro . The thymidine analogue also inhibited the utilization of radioactive adenine for the biosynthesis of DNA-adenine, but an excess of thymidine did not completely prevent this inhibition. Deoxyadenosine, but not adenosine, adenine or deoxyguanosine, inhibited the utilization of formate and to a lesser extent that of thymidine for the biosynthesis of DNA-thymine.

CELLULAR AND ANTIVIRAL EFFECTS OF HALOGENATED DEOXYRIBONUCLEOSIDES

A study has been in progress of the effect of 5-iodo-2’-deoxyuridine (IUDR) and its phosphorylated derivatives on the enzymes concerned with the formation of precursors of DNA-thymine in a cell-free extract of herpes simplex virusinfected and noninfected cells. The problem is to determine whether the enzymes concerned with these biotransformations in the virus-infected cells are more sensitive to inhibition by the iodinated analog or its phosphorylated derivatives than are the corresponding enzymes of the noninfected cells. Such a difference in susceptibility to inhibition could account for the virus being inhibited under conditions that are noninhibitory to the host cell. That a difference might exist in the enzymes of the normal host cell and the cell infected with animal virus is supported by observations in phage-infected bacterial cells that indicate the formation of a new phage-specific DNA polymerase’ and a deoxynucleotide kinase.24 Whereas the polymerase present normally in Escherichiu coli utilizes native DNA as a primer, that induced by infection with Tz phage functions best with denatured DNA and is inert with native DNA. The formation of enzymes induced by bacteriophage in bacterial cells has been reviewed by Cohens and Schmidt.s Increases in the activities of enzymes concerned with the formation of precursors of DNA-thymine, as well as with the polymerization of have been reported to occur upon infection of mammalian cells with certain animal viruses. This, however, is neither a general finding nor applicable to all enzyme~.*,~J~ Physical differences between enzymes induced by animal viruses and their counterpart in the noninfected cell have been observed recently. Nohara and KaplanZ2 have demonstrated that thymidine monophosphate kinase induced by infection of rabbit kidney cells with pseudorabies virus has a markedly greater thermostability than the enzyme present in the noninfected cells. Rogers and Moore23 reported that the enzyme, arginase, induced by Shope papilloma virus differed from the normal enzyme of the host animal in several physicochemical characteristics as well as metal requirements. McAuslanZ4 found thymidine kinase induced by pox virus to have a higher thermostability and a lower Michaelis constant than the corresponding enzyme present normally. Such findings support the possibility that a difference between an enzyme present normally in mammalian cells and the corresponding virus-induced enzyme may be found that will permit preferential inhibition of the latter by a drug and hence selective toxicity. Indeed, IUDR has been reported to inhibit the replication of several DNA-containing viruses under conditions that exert no apparent inhibition of the host cells. The present report is an evaluation of the inhibitory effect of IUDR and its monophosphate derivative on the thymidine kinase and thymidylic * This investigation was supported by U.S. Public Health Research Grant CA-526245 and US. National Science Foundation Grant GB-2805. t Support from the James Hudson Brown Memorial Fund (1962-1963) and the Anna Fuller Fund (1963-1964) is gratefully acknowledged (Y. S. Bakhle).