A highly active heparin-mimetic HIV-1 entry inhibitor in vitro and free of anti-thrombin toxicity (PK II, SOLIS, is an outcome of pre-clinical studies generated in this project in the 1990s by our basic research findings and knowledge of the molecular and chemical glycobiology of the heparin/heparan sulfate class of polysaccharides (H/HS) and how its structural diversity provides ability to modulate functions of diverse proteins in normal and disease processes. [E.g., cell growth, secretion, development, blood coagulation, and infections by virus and other human pathogens.]. Due to the diversity of sequences within H/HS chains, libraries of unique H/HS oligoS have not been readily obtainenable for research. Pk II was isolated from a heparin-mimetic pharmaceutical comprised of a mixture of sulfated oligoxylans (S-oligoS). We devised enlarged procedures to enable the clinical preparation of PK II (SOLIS) and developed a macro combinational type strategy to obtain a library of heparin-mimetic S-oligoS for researchers. [HD 01315-01-08-10]. The latter enabled us to show that the in vitro inhibitions of HIV-1 cytotoxicity and syncytium-formation by PK II were governed by a degree of discrete structural specificities, as was its lack of anti-thrombin coagulation inhibitory activity, two essential indicators of usefulness for further drug development. SOLIS, A POTENTIAL HIV-1 BINDING/FUSION INHIBITOR: HIV-infection in Americans is reaching one million; morbidity and mortality of AIDS worldwide (estimated 50 million cases and16 million deaths since the 1980s) are increasing. There is no cure nor fully effective vaccine, despite global efforts for almost two decades. Constant daily treatments of AIDS patients with multiple anti-retroviral drugs has been successful in developed countries for long term control of viral load and deadly effects, but with high cost in toxicity, sensitivity, and mutation to multi-drug resistant strains (found in adults and children), which increasingly limit this armamentarium. FDA has approved 26 drugs; 16 vs genomic replication (reverse transcriptase), 9 vs protease, and one is vs entry (a peptide that prevents viral fusion to the CD4 cell membrane by blocking changes in conformations of the fusion subunit of the viral envelope protein. gp41). Broad consensus counsels that inhibitors vs viral components that have other functions in HIV-1 attack are critically needed. We are completing a clinical preparation of SOLIS for a small Phase I safety trial. SOLIS would be an adjunct drug vs binding and entry of virus into target cells (e.g., it inhibits CD4 cell adherence to gp120-coated t.c. plates and prevents syncytium- forming fusion of HIV-1 (gp41) with CD4 cell membrane. (See summary of our results on biological characteristics of SOLIS below). Heparin has long been known to modulate conformations of proteins and peptides. From our studies in this area, inhibition of the conformational changes in gp41 which are required for fusion progression, is a possible mechanism of action of SOLIS and if so would be relatively insensitive to virus mutation. The laboratory is in a unique position to test this idea given our library of S-oligoS. Pk II, however, also inhibited cytopathic effects of HIV-1, suggesting that such S-oligoS might interfere with other critical elements of its attack in addition to binding/entry.? SUMMARY OF PROPERTIES OF PK II (SOLIS):? TEST SYSTEMS: S-oligoS libraries; preparation samplings ? FUNCTIONS: cytopathology protection; syncytium/fusion inhibition; inhibition of binding? between gp120 and CD4 cell; anticoagulation capacity vs thrombin, endotoxin content.? MEASUREMENTS : cell-killing (formazan), EC50 ug/ml equals 0.2-0.3 (See note.); cell fusion count, EC50 ug/ml equals 0.05-0.1 (See note.); CD4-cell adherence to gp120-coated plates; Heparin Units/mg equals less than 0.05-0.3 (Rosenberg assay); FDA assayy (LAL), sampling during preps, less than 0.06 eu. MEASUREMENTS respectively with FUNCTIONS. ? OUTCOMES: differential capacity, positive protection vs.cytopathology; differential potency, highly active vs virus (gp41)-cell fusion; differential capacity, concentration-dependent prevention of cell (CD4) binding to gp120; differential potency, essentially negligable anti-thrombin toxicity; conforms to FDA laboratory monitoring level of pyrogen exclusion. Note: NIH laboratory data; this may be 7-10 times higher in some commercial labs. ? IN VIVO Efficacy of SOLIS will be tested in a SCID mouse model through the NIAID DAID program, if applicable. AN FDA LICENSED US heparin-mimetic, similar to the European made starting source of SOLIS (PK II) is currently investigated as an alternate and found to be suitable in preparative aspects. Second stage product, however, remain to be studied in further detail. Negotiations are underway to continue cooperation in a CDA with the pharmaceutical company and ultimately to obtain additional starting product for subsequent trials. VIRUS LIGANDS: Viral proteins that enable HIV to attack, survive, and replicate in target human cells have been elucidated in the field and are available for in vitro study. We have proposed to identify and isolate putative H/HS ligand(s) under conditions of HIV-1 tissue culture infection in the presence of a bifunctional SOLIS probe, and to achieve chemical bonding by flash illumination to endogenous viral/cell protein structures during virus attack. After cell membranes are purified, isolated proteins would be analyzed for fluorescent probe by gel electrophoresis. The probe would be synthesized by our method: synthesizing a bifunctional hydrazine derivative and subsequent reaction with the reducing aldehyde terminus of the inhibitor. Such ligand(s) might be protective antigens and/or a means of obtaining endogenous H/HS receptors. STRUCTURE-FUNCTION (SF): The mixture of S-oligoS remaining after chemical sulfation of the native xylan was shown previously to unexpectedly comprise a family of oligomers that increased in mass by additions which included a putative -D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3 motif. The SoligoS range from about 20 to less than 2 K on the average in mass, contain more or less sugars in alternate chair conformations, and a large proportion of GlcA lacking an 4-Ome substituent (FTIR, HMQC heteronuclear two-dimensional NMR proton-13C correlation spectra, GlcA analysis). Recent consideration of the SF underlying the above inhibitions of HIV-1 indicates that S-oligoS action against gp120 binding to the cell receptor(s) requires a minimum of 4 motifs to retain maximum potency. The requirement differs for the inhibition of gp41 helical change where the max number of motifs is 3. Capillary HPLC-mass spectroscopy will be used shortly to analyze and model more specifically the various structures of Pk II and other S-oligoS. This information might help define structures for potential inhibitors that might have simpler chemistry.
| Wright, T E; Milam, K A; Rougee, L et al. (2011) Agreement of umbilical cord drug and cotinine levels with maternal self-report of drug use and smoking during pregnancy. J Perinatol 31:324-9 |
