Model with two types of CTL regulation and experiments on CTL dynamics.

Recently, we developed a mathematical model of interaction between the HIV and the immune system to match various dynamic experiments carried out in HIV-infected humans and SIV-infected macaques. The model includes helper cell-dependent and helper cell-independent cytotoxic lymphocytes (CTLs) and predicts two stable steady states, a state with a high virus load and few helper cells, and another state with a low virus load and many helper cells. Here we upgrade the model to take into account recent reports on the link between the activation status of infected cells and their ability to produce virus, the effect of helper cells at the time of priming on CTL differentiation, and virus dynamics in unvaccinated macaques with a broad genetic background acutely infected with SIVmac251. We also discuss in detail the experimental justification of the CTL block and the robustness of model predictions with respect to the hypothesis of two CTL subtypes.

Interpreting the effect of vaccination on steady state infection in animals challenged with Simian immunodeficiency virus.

A representative vaccinated macaque challenged with SIVmac251 establishes a persistent infection with a lower virus load, higher CTL frequencies, and much higher helper cell frequencies, than a representative control animal. The reasons for the difference are not fully understood. Here we interpret this effect using a mathematical model we developed recently to explain results of various experiments on virus and CTL dynamics in SIV-infected macaques and HIV-infected humans. The model includes two types of cytotoxic lymphocytes (CTLs) regulated by antigen-activated helper cells and directly by infected cells, respectively, and predicts the existence of two steady states with different viremia, helper cell and CTL levels. Depending on the initial level of CTL memory cells and helper cells, a representative animal ends up in either the high-virus state or the low-virus state, which accounts for the observed differences between the two animal groups. Viremia in the low-virus state is proportional to the antigen sensitivity threshold of helper cells. Estimating the infectivity ratio of activated and resting CD4 T cells at 200-300, the correct range for the critical memory cell percentage and the viremia peak suppression is predicted. However, the model does not explain why viremia in the "low-virus state" is surprisingly high , relative to vaccinated animals infected with SHIV, and broadly distributed among challenged animals. We conclude that the model needs an update explaining extremely low sensitivity of uninfected helper cells to antigen in vaccinated animals.

Discovery of (R)-7-cyano-2,3,4, 5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3- (phenylmethyl)-4-(2-thienylsulfonyl)-1H-1,4-benzodiazepine (BMS-214662), a farnesyltransferase inhibitor with potent preclinical antitumor activity.

Continuing structure-activity studies were performed on the 2,3,4, 5-tetrahydro-1-(imidazol-4-ylalkyl)-1,4-benzodiazepine farnesyltransferase (FT) inhibitors. These studies demonstrated that a 3(R)-phenylmethyl group, a hydrophilic 7-cyano group, and a 4-sulfonyl group bearing a variety of substituents provide low-nanomolar FT inhibitors with cellular activity at concentrations below 100 nM. Maximal in vivo activity in the mutated K-Ras bearing HCT-116 human colon tumor model was achieved with analogues carrying hydrophobic side chains such as propyl, phenyl, or thienyl attached to the N-4 sulfonyl group. Several such compounds achieved curative efficacy when given orally in this model. On the basis of its excellent preclinical antitumor activity and promising pharmacokinetics, compound 20 (BMS-214662, (R)-7-cyano-2,3,4, 5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thie nyl sulfonyl)-1H-1,4-benzodiazepine) has been advanced into human clinical trials.

Discovery and structure-activity relationships of imidazole-containing tetrahydrobenzodiazepine inhibitors of farnesyltransferase.

2,3,4,5-Tetrahydro-1-(imidazol-4-ylalkyl)-1,4-benzodiazepines were found to be potent inhibitors of farnesyltransferase (FT). A hydrophobic substituent at the 4-position of the benzodiazepine, linked via a hydrogen bond acceptor, was important to enzyme inhibitory activity. An aryl ring at position 7 or a hydrophobic group linked to the 8-position through an amide, carbamate, or urea linkage was also important for potent inhibition. 2,3,4, 5-Tetrahydro-1-(1H-imidazol-4-ylmethyl)-7-(4-pyridinyl)-4-[2-(t rifluo romethoxy)benzoyl]-1H-1,4-benzodiazepine (36), with an FT IC(50) value of 24 nM, produced 85% phenotypic reversion of Ras transformed NIH 3T3 cells at 1.25 microM and had an EC(50) of 160 nM for inhibition of anchorage-independent growth in soft agar of H-Ras transformed Rat-1 cells. Selected analogues demonstrated ip antitumor activity against an ip Rat-1 tumor in mice.

Localization of osteogenic protein-1 (bone morphogenetic protein-7) during human embryonic development: high affinity binding to basement membranes.

Osteogenic protein-1 (OP-1) is a member of the bone morphogenetic protein subfamily of the transforming growth factor-beta (TGF-beta) superfamily. Since members of the TGF-beta superfamily have a role in tissue development the distribution of OP-1 in developing human embryos (gestational age 5-14 weeks) was examined by immunohistochemical methods. Positive staining for OP-1 was observed in: sclerotome, hypertrophied chondrocytes, osteoblasts, periosteum, epithelial cells of the adrenal "provisional cortex" and the convoluted tubules of developing kidneys. In the developing lungs, pancreas and skin, OP-1 was localized in basement membranes underlying the epithelium. In vitro binding studies of 125I-OP-1 to various extracellular matrix components revealed high affinity of OP-1 for type IV collagen and less for heparin, collagen types I and VI. Present findings suggest that, in addition to bone formation, OP-1 could have other important regulatory roles in human embryogenesis with high binding affinity to a basement membrane component.

Biosynthetic antibody binding sites: development of a single-chain Fv model based on antidinitrophenol IgA myeloma MOPC 315.

The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein in E. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values for Ka,app of 1.5 to 2.2 x 10(6) M-1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv315 fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.