Complex interplay of kinetic factors governs the synergistic properties of HIV-1 entry inhibitors.

The homotrimeric HIV-1 envelope glycoprotein (Env) undergoes receptor-triggered structural changes that mediate viral entry through membrane fusion. This process is inhibited by chemokine receptor antagonists (CoRAs) that block Env-receptor interactions and by fusion inhibitors (FIs) that disrupt Env conformational transitions. Synergy between CoRAs and FIs has been attributed to a CoRA-dependent decrease in the rate of viral membrane fusion that extends the lifetime of the intermediate state targeted by FIs. Here, we demonstrated that the magnitude of CoRA/FI synergy unexpectedly depends on FI-binding affinity and the stoichiometry of chemokine receptor binding to trimeric Env. For C-peptide FIs (clinically represented by enfuvirtide), synergy waned as binding strength decreased until inhibitor combinations behaved additively. Curiously, this affinity dependence on synergy was absent for 5-Helix-type FIs. We linked this complex behavior to the CoRA dependence of Env deactivation following FI binding. For both FI classes, reducing chemokine receptor levels on target cells or eliminating competent chemokine receptor-binding sites on Env trimers resulted in a loss of synergistic activity. These data imply that the stoichiometry required for CoRA/FI synergy exceeds that required for HIV-1 entry. Our analysis suggests two distinct roles for chemokine receptor binding, one to trigger formation of the FI-sensitive intermediate state and another to facilitate subsequent conformational transitions. Together, our results could explain the wide variety of previously reported activities for CoRA/FI combinations. These findings also have implications for the combined use of CoRAs and FIs in antiviral therapies and point to a multifaceted role for chemokine receptor binding in promoting HIV-1 entry

Dissecting the Complex Mechanisms Behind the Combinatorial Activities of HIV-1 Entry Inhibitors

For HIV-1 surface glycoprotein Envelope (Env) to mediate the viral entry process, it must interact with two receptors on the target cell, CD4 and coreceptor (CoR). These receptors trigger a series of conformational changes that result in the fusion of viral and target cell membranes. How receptor binding triggers these conformational changes is not fully understood. While there are three binding sites for each receptor in an Env trimer, neither the minimal receptor binding requirement for entry, nor the role of multivalent binding is known. To address these gaps in knowledge, we examined the combinatorial activity of three classes of HIV-1 entry inhibitors. The first two classes are CD4 (CD4A) and CoR antagonists (CoRA). These antagonists bind the cellular receptors and occlude them from Env. The third class, fusion inhibitors (FI), binds an intermediate conformation of Env during entry and stops membrane fusion. As FI bind a transient intermediate conformation, potency is dependent on the kinetics of entry. In our first project we examined CoRA and FI combinations. Previous studies have shown that these inhibitors are synergistic with one another and attributed synergy to CoRAs slowing the kinetics of entry. However, other studies have reported only additivity. By dissecting the synergistic process using a variety of FIs and mutant Env, we were able to determine that synergy can be lost through two factors, the affinity of FI, and the stoichiometry of CoR binding. These results explain the discrepancies observed in the literature, provide new understanding of the role of CoR binding stoichiometry in entry, and have implications for the combinatorial use of these inhibitors in the clinic. In a second study, we probed the coupling of CD4 and CoR binding by investigating the combinatorial activity of CD4A and CoRA. Unexpectedly, we observed that these two inhibitors were antagonistic with another. However, the combinatorial activity was highly HIV-1 strain dependent. By comparing the combinatorial activity of these inhibitors across multiple HIV-1 strains and dissecting the mechanism of antagonism, we determined that CD4 and CoR binding can drive competing processes in during HIV-1 entry

APC mutations in human colon lead to decreased neuroendocrine maturation of ALDH+ stem cells that alters GLP-2 and SST feedback signaling: Clue to a link between WNT and retinoic acid signalling in colon cancer development.

APC mutations drive human colorectal cancer (CRC) development. A major contributing factor is colonic stem cell (SC) overpopulation. But, the mechanism has not been fully identified. A possible mechanism is the dysregulation of neuroendocrine cell (NEC) maturation by APC mutations because SCs and NECs both reside together in the colonic crypt SC niche where SCs mature into NECs. So, we hypothesized that sequential inactivation of APC alleles in human colonic crypts leads to progressively delayed maturation of SCs into NECs and overpopulation of SCs. Accordingly, we used quantitative immunohistochemical mapping to measure indices and proportions of SCs and NECs in human colon tissues (normal, adenomatous, malignant), which have different APC-zygosity states. In normal crypts, many cells staining for the colonic SC marker ALDH1 co-stained for chromogranin-A (CGA) and other NEC markers. In contrast, in APC-mutant tissues from familial adenomatous polyposis (FAP) patients, the proportion of ALDH+ SCs progressively increased while NECs markedly decreased. To explain how these cell populations change in FAP tissues, we used mathematical modelling to identify kinetic mechanisms. Computational analyses indicated that APC mutations lead to: 1) decreased maturation of ALDH+ SCs into progenitor NECs (not progenitor NECs into mature NECs); 2) diminished feedback signaling by mature NECs. Biological experiments using human CRC cell lines to test model predictions showed that mature GLP-2R+ and SSTR1+ NECs produce, via their signaling peptides, opposing effects on rates of NEC maturation via feedback regulation of progenitor NECs. However, decrease in this feedback signaling wouldn't explain the delayed maturation because both progenitor and mature NECs are depleted in CRCs. So the mechanism for delayed maturation must explain how APC mutation causes the ALDH+ SCs to remain immature. Given that ALDH is a key component of the retinoic acid (RA) signaling pathway, that other components of the RA pathway are selectively expressed in ALDH+ SCs, and that exogenous RA ligands can induce ALDH+ cancer SCs to mature into NECs, RA signaling must be attenuated in ALDH+ SCs in CRC. Thus, attenuation of RA signaling explains why ALDH+ SCs remain immature in APC mutant tissues. Since APC mutation causes increased WNT signaling in FAP and we found that sequential inactivation of APC in FAP patient tissues leads to progressively delayed maturation of colonic ALDH+ SCs, the hypothesis is developed that human CRC evolves due to an imbalance between WNT and RA signaling