Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific ...interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ∼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition.
N-Acyl ethanolamines (NAEs) constitute a large and diverse class of signaling lipids that includes the endogenous cannabinoid anandamide. Like other lipid transmitters, NAEs are thought to be ...biosynthesized and degraded on-demand rather than being stored in vesicles prior to signaling. The identification of enzymes involved in NAE metabolism is therefore imperative to achieve a complete understanding of this lipid signaling system and control it for potential therapeutic gain. Recently, an N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) was identified as a candidate enzyme involved in the biosynthesis of NAEs. Here, we describe the generation and characterization of mice with a targeted disruption in the NAPE-PLD gene NAPE-PLD(−/−) mice. Brain tissue from NAPE-PLD(−/−) mice showed more than a 5-fold reduction in the calcium-dependent conversion of NAPEs to NAEs bearing both saturated and polyunsaturated N-acyl chains. However, only the former group of NAEs was decreased in level in NAPE-PLD(−/−) brains, and these reductions were most dramatic for NAEs bearing very long acyl chains (≥C20). Further studies identified a calcium-independent PLD activity in brains from NAPE-PLD(−/−) mice that accepted multiple NAPEs as substrates, including the anandamide precursor C20:4 NAPE. The illumination of distinct enzymatic pathways for the biosynthesis of long chain saturated and polyunsaturated NAEs suggests a strategy to control the activity of specific subsets of these lipids without globally affecting the function of the NAE family as a whole.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
Monoclonal antibodies have evolved from research tools to powerful therapeutics in the past 30 years. Clinical success rates of antibodies have exceeded expectations, resulting in heavy investment in ...biologics discovery and development in addition to traditional small molecules across the industry. However, protein therapeutics cannot drug targets intracellularly and are limited to soluble and cell-surface antigens. Tremendous strides have been made in antibody discovery, protein engineering, formulation, and delivery devices. These advances continue to push the boundaries of biologics to enable antibody conjugates to take advantage of the target specificity and long half-life from an antibody, while delivering highly potent small molecule drugs. While the "magic bullet" concept produced the first wave of antibody conjugates, these entities were met with limited clinical success. This review summarizes the advances and challenges in the field to date with emphasis on antibody conjugation, linker-payload chemistry, novel payload classes, absorption, distribution, metabolism, and excretion (ADME), and product developability. We discuss lessons learned in the development of oncology antibody conjugates and look towards future innovations enabling other therapeutic indications.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The application of protein engineering technologies toward successfully improving antibody pharmacokinetics has been challenging due to the multiplicity of biochemical factors that influence ...monoclonal antibody (mAb) disposition in vivo. Physiological factors including interactions with the neonatal Fc receptor (FcRn) and specific antigen binding properties of mAbs, along with biophysical properties of the mAbs themselves play a critical role. It has become evident that applying an integrated approach to understand the relative contribution of these factors is critical to rationally guide and apply engineering strategies to optimize mAb pharmacokinetics. The study presented here evaluated the influence of unintended non-specific interactions on the disposition of mAbs whose clearance rates are governed predominantly by either non-specific (FcRn) or target-mediated processes. The pharmacokinetics of 8 mAbs representing a diverse range of these properties was evaluated in cynomolgus monkeys. Results revealed complementarity-determining region (CDR) charge patch engineering to decrease charge-related non-specific binding can have a significant impact on improving the clearance. In contrast, the influence of enhanced in vitro FcRn binding was mixed, and related to both the strength of charge interaction and the general mechanism predominant in governing the clearance of the particular mAb. Overall, improved pharmacokinetics through enhanced FcRn interactions were apparent for a CDR charge-patch normalized mAb which was affected by non-specific clearance. The findings in this report are an important demonstration that mAb pharmacokinetics requires optimization on a case-by-case basis to improve the design of molecules with increased therapeutic application.
Fatty acid amide hydrolase (FAAH) is the primary catabolic regulator of several bioactive lipid amides in vivo, including the endogenous cannabinoid anandamide and the sleep-inducing substance ...oleamide. Inhibitors of FAAH are considered a potential therapeutic approach for the treatment of several nervous system disorders, including pain, anxiety, and insomnia. However, for FAAH inhibitors to achieve clinical utility, they must not only display efficacy in vivo but also selectivity for this enzyme relative to the numerous other serine hydrolases present in mammalian proteomes. Here, we report a general strategy for evaluating the pharmacological activity and target specificity of FAAH inhibitors and its implementation to develop the first class of selective reversible inhibitors of this enzyme that are highly efficacious in vivo. Using a series of functional proteomics, analytical chemistry, and behavioral pharmacology assays, we have identified a class of alpha-keto-heterocycles that show unprecedented selectivity for FAAH relative to other mammalian hydrolases, and, when administered to rodents, raise central nervous system levels of anandamide and promote cannabinoid receptor 1-dependent analgesia in several assays of pain sensation. These studies provide further evidence that FAAH may represent an attractive therapeutic target and describe a general route by which inhibitors of this enzyme can be optimized to achieve exceptional potency, selectivity, and efficacy in vivo.
To realize the promise of genomics-based therapeutics, new methods
are needed to accelerate the discovery of small molecules that selectively
modulate protein activity. Toward this end, advances in ...combinatorial synthesis
have provided unprecedented access to large compound libraries of considerable
structural complexity and diversity, shifting the bottleneck
in drug discovery to the development of efficient screens for protein
targets. Screening for reversible enzyme inhibitors typically
requires extensive target-specific work, including protein expression and
purification, as well as the development of specific substrate assays. Here we
report a proteomic method for the discovery of reversible enzyme inhibitors
that avoids these steps. We show that competitive profiling of a library of
candidate serine hydrolase inhibitors in complex proteomes with activity-based
chemical probes identifies nanomolar reversible inhibitors
of several enzymes simultaneously, including the endocannabinoid-degrading
enzyme fatty acid amide hydrolase (FAAH), triacylglycerol
hydrolase (TGH) and an uncharacterized membrane-associated
hydrolase that lacks known substrates. The strategy tests inhibitors against
numerous enzymes in parallel, assigning both potency and selectivity factors to
each agent. In this way, promiscuous inhibitors were readily rejected in favor
of equally potent compounds with 500-fold or greater selectivity for their
targets.
Full text
Available for:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against ...para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mmCHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4′) was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Display omitted
The concurrent implementation of a proteome-wide serine hydrolase selectivity screen with traditional efforts to optimize fatty acid amide hydrolase (FAAH) inhibition potency led to ...the expedited discovery of a new class of exceptionally potent (
K
i
<
300
pM) and unusually selective (>100-fold selective) inhibitors. The iterative inhibitor design and evaluation with assistance of the selectivity screen served to differentiate otherwise indistinguishable inhibitors permitting the simultaneous optimization of potency and selectivity. Significantly, the simultaneous assessment of all potential competitive enzymes with the selectivity screen does not require the use of expressed or purified enzymes or a competitive substrate, no modification of the inhibitors is required, and the relative potency for competitive enzymes can be quantified (IC
50’s) including those that lack known substrates or function.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Phosphoprotein phosphatase 2A (PP2A), a major serine-threonine protein phosphatase in eukaryotes, is an oligomeric protein comprised of structural (A) and catalytic (C) subunits to which a variable ...regulatory subunit (B) can associate. The C subunit contains a methyl ester post-translational modification on its C-terminal leucine residue, which is removed by a specific methylesterase (PME-1). Methylesterification is thought to control the binding of different B subunits to AC dimers, but little is known about its physiological significance in vivo.
Here, we show that targeted disruption of the PME-1 gene causes perinatal lethality in mice, a phenotype that correlates with a virtually complete loss of the demethylated form of PP2A in the nervous system and peripheral tissues. Interestingly, PP2A catalytic activity over a peptide substrate was dramatically reduced in PME-1(-/-) tissues, which also displayed alterations in phosphoproteome content.
These findings suggest a role for the demethylated form of PP2A in maintenance of enzyme function and phosphorylation networks in vivo.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
