Vishwanath R Lingappa

Prosetta Biosciences

We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all...

The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for innovative therapies with high genetic barriers to resistance. Therefore, there is pronounced interest in identifying new pharmacological targets in the SARS-CoV-2 viral life cycle. The small molecule PAV-104, identified through a cell-free protein synthe...

Assembly modulators are a new class of allosteric site-targeted therapeutic small molecules, some of which are effective at restoring nuclear localization of TDP-43 in ALS cellular models, and display efficacy in a variety of ALS animal models. One of these compounds has been shown to target a small subfraction of protein disulfide isomerase, a kno...

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease with a complex, multifactorial pathophysiology, most commonly manifest as loss of motor neurons. We introduce a new mechanism of ALS pathogenesis via a novel drug-like small molecule series that targets protein disulfide isomerase (PDI) within a previously unappreciated transient an...

23 Two structurally-unrelated small molecule chemotypes, represented by compounds PAV-617 and PAV-24 951 with antiviral activity in cell culture against monkeypox virus (MPXV) and human immunodeficiency 25 virus (HIV) respectively, were studied for anti-cancer efficacy. Each exhibited apparent pan-cancer 26 cytotoxicity, reasonable pharmacokinetics...

The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for novel therapies with high genetic barriers to resistance. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly...

Two structurally-unrelated small molecule chemotypes, represented by compounds PAV-617 and PAV-951, with antiviral activity in cell culture against monkeypox virus (MPXV) and human immunodeficiency virus (HIV) respectively, were studied for anti-cancer efficacy. Each exhibited apparent pan-cancer cytotoxicity, reasonable pharmacokinetics, and non-t...

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subty...

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomic tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtyp...

Herpes virus infections are endemic and ubiquitous. While only rarely leading to overt encephalitis, subchronic or latent infections have been associated to a variety of conditions, including Alzheimer’s disease (AD). The cellular consequences of herpes virus infection are determined by the host proteins recruited during virus replication and assem...

Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradien...

Decades after the eradication of smallpox and the discontinuation of routine smallpox vaccination, over half of the world’s population is immunologically naïve to variola virus and other orthopoxviruses (OPXVs). Even in those previously vaccinated against smallpox, protective immunity wanes over time. As such, there is a concomitant increase in the...

The concerning increase in HIV-1 resistance argues for prioritizing the development of host-targeting antiviral drugs because such drugs can offer high genetic barriers to the selection of drug-resistant viral variants. Targeting host proteins could also yield drugs that act on viral life cycle events that have proven elusive to inhibition, such as...

We present a small molecule chemotype, identified by an orthogonal drug screen, exhibiting nanomolar activity against members of all the six viral families causing most human respiratory viral disease, with a demonstrated barrier to resistance development. Antiviral activity is shown in mammalian cells, including human primary bronchial epithelial...

The success of antiretroviral treatment for HIV-1 is at risk of being undermined by the growing problem of drug resistance. Thus, there is a need to identify antiretrovirals that act on viral life cycle stages not targeted by drugs in use, such as the events of HIV-1 Gag assembly. To address this gap, we developed a compound screen that recapitulat...

The ability of viruses to evolve several orders of magnitude faster than their host cells has enabled them to exploit host cellular machinery by selectively recruiting multiprotein complexes (MPCs) for their catalyzed assembly and replication. This hijacking may depend on alternative, 'moonlighting' functions of host proteins that deviate from thei...

Given the projected increase in multidrug resistant HIV-1, there is an urgent need for development of antiretrovirals that act on virus life-cycle stages that are not targeted by antiretrovirals currently in use. Host-targeting drugs are of particular interest because they can offer a high barrier to resistance. Here we report identification of two...

Significance Synucleinopathies such as Parkinson’s disease feature deposition of misfolded α-synuclein. It is likely that cellular proteostasis compensates for misfolded α-synuclein to some extent, but, once exhausted, α-synuclein can form seeds for a prion-like spread in the brain. Here, we demonstrate that, in human dopaminergic neurons and in mo...

During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here...

Chronic mental illnesses such as schizophrenia or recurrent affective disorders are heterogeneous in their biological causes behind the clinical, syndromatic diagnosis. Viral infection has been linked to chronic mental illnesses both by direct mechanisms and indirect mechanisms through immune stimulation. For example, studies showed that intrauteri...

Background The toxic effects of α-synuclein (αSYN) pre-fibrillar aggregates on dopaminergic neurons are believed to be central to the pathogenesis of Parkinson´s Disease (PD). So far, therapeutics targeting these toxic αSYN forms have not been successful. PD patients generally receive L-DOPA substitution therapy, although there is evidence that L-D...

During immature capsid assembly in cells, the Gag protein of HIV-1 and other primate lentiviruses co-opts a host RNA granule, forming a pathway of assembly intermediates that contains host components, including two cellular enzymes shown to facilitate assembly, ABCE1 and DDX6. Here we asked whether a non-primate lentivirus, feline immunodeficiency...

Loss-of-function studies have identified Porcupine (PORCN) and Wntless (WLS) as essential mediators of Wnt secretion and signaling. Whereas PORCN is thought to palmitoylate Wnt proteins, WLS is believed to transport palmitoylated Wnt proteins to the cell surface. However, little is known about how these two proteins cooperate to regulate Wnt palmit...

Background Viral capsid assembly involves the oligomerization of the capsid nucleoprotein (NP), which is an essential step in viral replication and may represent a potential antiviral target. An in vitro transcription-translation reaction using a wheat germ (WG) extract in combination with a sandwich ELISA assay has recently been used to identify s...

Venezuelan (VEEV), eastern, and western equine encephalitis viruses, members of the genus Alphavirus, are causative agents of debilitative and sometimes fatal encephalitis. Although human cases are rare, these viruses pose a threat to military personnel, and to public health, due to their potential use as bioweapons. Currently, there are no license...

Background: In the course of exploring new concepts in antiviral disease therapeutics, namely the identification of small molecules that block aberrant host assembly machines catalytic for viral capsid formation, we have made observations with important implications for brain diseases. Our working hypothesis is that much of what is generally viewed...

We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a host-catalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiv...

The nucleoprotein of Rift Valley Fever Virus (RVFV NP), a member of family Bunyaviridae and of Lassa Fever Virus (LASV NP) a member of family Arenaviridae, both of which have helical capsids, have been expressed in a system for cell-free protein synthesis.

Wnts are secreted signal molecules that are important for patterning of tissues during embryogenesis. Wnt secretion is dependent on the lipidation of two conserved residues. Lipidation of the conserved serine is facilitated by Porcupine (Porcn), a membrane-bound O-acyltransferase. To better understand the role of Porcn in Wnt secretion, we sought t...

Background: Protein translation and translocation at the rough endoplasmic reticulum (RER) are the first steps in the secretory pathway. The translocon through which newly made proteins are translocated into or across the RER membrane consists of three main subunits: Sec61α, -β, and -γ. Sec61β facilitates translocation, and we and others have show...

In general, drug discovery in the therapeutic field of infectious disease has a stellar track record. And yet, subsets of pathogens, for example many classes of viruses other than HIV, HSV, influenza, and HCV have been poorly addressed. In addition, the development of resistance remains a specter of great concern for almost all current chemotherapy...

This chapter focuses on the emergence of small-molecule inhibitors of capsid assembly as potential antiviral therapeutics. There is a large degree of variation among viruses that cause human disease, but the general life cycle of viruses share common features that are potential targets for antiviral drugs. The features include viral attachment, vir...

We recently reported the presence of a novel 32 kDa protein immunoreactive to a copper, zinc superoxide dismutase (SOD1) antibody within the spinal cord of patients with amyotrophic lateral sclerosis (ALS). This unique protein species was generated by biotinylation of spinal cord tissue extracts to detect conformational changes of SOD1 specific to...

The cellular prion protein (PrP(C)) is a GPI-anchored cell-surface protein. A small subset of PrP(C) molecules, however, can be integrated into the ER-membrane via a transmembrane domain (TM), which also harbors the most highly conserved regions of PrP(C), termed the hydrophobic core (HC). A mutation in HC is associated with prion disease resulting...

Post-translational modifications of the extracellular matrix receptor dystroglycan (DG) determine its functional state, and defects in these modifications are linked to muscular dystrophies and cancers. A prominent feature of DG biosynthesis is a precursor cleavage that segregates the ligand-binding and transmembrane domains into the noncovalently...

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron degenerative disease whose etiology and pathogenesis remain poorly understood. Most cases of ALS (≈90%) are sporadic (SALS), occurring in the absence of genetic associations. Approximately 20% of familial ALS (FALS) cases are due to known mutations in the copper, zinc superoxide dism...

The sequence of a transmembrane (TM) domain and the adjacent regions are important for recognition, orientation, and integration at the translocon during membrane protein biosynthesis. However, the sequences of individual TM domains vary considerably. Although some general effects of electrostatic and hydrophobic interactions have been observed, it...

Prions are composed solely of the disease-causing prion protein (PrPSc) that is formed from the cellular isoform PrPC by a posttranslational process. Here we report that short phosphorothioate DNA (PS-DNA) oligonucleotides diminished the levels of both PrPC and PrPSc in prion-infected neuroblastoma (ScN2a) cells. The effect of PS-DNA on PrP levels...

Signal sequences for the transfer of proteins across membranes are usually found at the NH2-terminus of nascent secretory and transmembrane proteins. The functionally equivalent signal sequence of chicken ovalbumin however, is localized in the middle of the molecule. The implication of this surprising finding for biogenesis of membrane proteins is...

The C-type lectins DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) bind to the ebolavirus glycoprotein (EBOV-GP) and augment viral infectivity. DC-SIGN/R strongly enhance infection driven by the GP of EBOV subspecies. Zaire (ZEBOV) but have a much less pronounced effect on infection mediated by the GP of EBOV subspecies. Sudan (SEBOV)....

We review the important role that cell-free protein-synthesizing systems (CFPSS) have played in the history of modern biology, and highlight two recent applications that illustrate their continued utility for the exploration of otherwise intractable aspects of gene expression and its regulation. Viral capsid assembly recreated in CFPSS reveals a ca...

Biosynthesis of the prion protein at the endoplasmic reticulum generates multiple topological forms. The topology of an individual chain is determined first by the localization of the N terminus and then by potential integration of the transmembrane domain into the lipid bilayer. Here, we provide the first evidence that signal sequences affect the...

An interaction between an N-terminal signal sequence and the translocon leads to the initiation of protein translocation into the endoplasmic reticulum lumen. Subsequently, folding and modification of the substrate rapidly ensue. The close temporal coordination of these processes suggests that they may be structurally and functionally coordinated a...

We previously showed that the exocyst complex specifically affected the synthesis and delivery of secretory and basolateral plasma membrane proteins. Significantly, the entire spectrum of secreted proteins was increased when the hSec10 (human Sec10) component of the exocyst complex was overexpressed, suggestive of post-transcriptional regulation (L...

The steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol into mitochondria to initiate steroidogenesis, but its site of action on the mitochondria has been uncertain. One model states that StAR has a fairly rigid structure and functions in the intramembranous space (IMS) where it transports cholesterol from the oute...

We suggest a new view of secretory and membrane protein folding that emphasizes the role of pathways of biogenesis in generating functional and conformational heterogeneity. In this view, heterogeneity results from action of accessory factors either directly binding specific sequences of the nascent chain, or indirectly, changing the environment in...

Most mitochondrial proteins are synthesized on cytoplasmic ribosomes and imported into mitochondria. The imported proteins are directed to one of four submitochondrial compartments--the outer mitochondrial membrane, the inner mitochondrial membrane, the intramembraneous space, or the matrix--where the protein then functions. Here we show that the s...

Integral membrane protein biogenesis requires the coordination of several events: accurate targeting of the nascent chain to the membrane; recognition, orientation and integration of transmembrane (TM) domains; and proper formation of tertiary and quaternary structure. Initially unanticipated inter- and intra-protein interactions probably mediate e...

Amino-terminal signal sequences target nascent secretory and membrane proteins to the endoplasmic reticulum for translocation. Subsequent interactions between the signal sequence and components of the translocation machinery at the endoplasmic reticulum are thought to be important for the productive engagement of the translocon by the ribosome-nasc...

Much attention has been focused on the unusual mode of propagation of the prion protein (PrP). However this is not the only pathophysiologically relevant aspect of PrP biology. Our studies suggest that the mechanism of PrP biogenesis plays a crucial role in the pathway leading to neurodegeneration in prion diseases. First, we have found that, unlik...

Prion diseases can be infectious, sporadic and genetic. The infectious forms of these diseases, including bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, are usually characterized by the accumulation in the brain of the transmissible pathogen, an abnormally folded isoform of the prion protein (PrP) termed PrPSc. However, certain inh...

To examine possible effects of the E323K mutation in the trabecular meshwork glucocorticoid response (TIGR) gene (also known as myocilin [MYOC]), using assays of translocational processing through the endoplasmic reticulum (ER). The E323K mutation was of particular interest, since the mutation shows a strong association with early onset open-angle...

The biogenesis of most secretory and membrane proteins involves targeting the nascent protein to the endoplasmic reticulum (ER), translocation across or integration into the ER membrane and maturation into a functional product. The essential machinery that directs these events for model secretory and membrane proteins has been identified, shifting...

In mammalian cells, the Sec61 complex and translocating chain-associated membrane protein (TRAM) are necessary and sufficient to direct the biogenesis, in the appropriate topology, of all secretory and membrane proteins examined thus far. We demonstrate here that the proper translocation of the prion protein (PrP), a substrate that can be synthesiz...

One potential mechanism by which apolipoprotein (apo) B secretion is regulated is via transient pausing during translocation across the endoplasmic reticulum membrane. We have previously shown that translocation and secretion of full-length and truncated variants of apoB 100 are impaired in hepatocytes in which microsomal membranes are enriched in...

Translocational pausing is a mechanism used by certain specialized secretory proteins whereby discrete domains of a nascent chain destined for the endoplasmic reticulum lumen are transiently exposed to the cytosol. Proteoliposomes reconstituted from total endoplasmic reticulum proteins properly assemble translocationally paused intermediates. The c...

At the endoplasmic reticulum membrane, the prion protein (PrP) can be synthesized in several topological forms. The role of these different forms was explored with transgenic mice expressing PrP mutations that alter the relative ratios of the topological forms. Expression of a particular transmembrane form (termedCtmPrP) produced neurodegenerative...

Lipoprotein assembly requires a complex and regulated set of events that includes apolipoprotein B (apoB) translocation across the endoplasmic reticulum (ER) membrane, folding, and association with lipids. Unlike simple secretory proteins which are cotranslationally translocated directly into the ER lumen, nascent apoB contains pause transfer (PT)...

Tight docking of the ribosome at the translocation channel ensures that nascent secretory proteins are shielded from the cytoplasm during transfer into the endoplasmic reticulum. Discrete pause transfer sequences mediate the transient stopping of translocation in certain proteins. Here we show that during a translocational pause, the junction betwe...

Proposition 186 was an initiative on the November 1994 California ballot which proposed to establish a state single-payer health care program. Although Prop 186 was overwhelmingly defeated in the November 1994 election (73% No, 27% Yes), it accomplished many things. Model legislation was developed showing the feasibility of a specific single-payer...

Nascent polypeptides enter into high molecular weight complexes with other proteins during chain elongation in vitro and in vivo. The nature of these complexes was investigated using an in vitro translation system programmed with a single mRNA lacking a translational termination codon. Complexes containing nascent polypeptides (molecular mass < 20...

The prion protein (PrP) displays some unusual features in its biogenesis. In cell-free systems it can be synthesized as either an integral transmembrane protein spanning the membrane twice, with both amino and carboxyl domains in the lumen of the endoplasmic reticulum, or as a fully translocated polypeptide. A charged, extracytoplasmic region, term...

Understanding how the multidrug resistance phenotype is manifest in human cancer cells will require insight into the mechanism of assembly, transmembrane topology, and intracellular trafficking of human P-glycoprotein (MDR1). Previously, we showed that MDR1 amino terminus biogenesis occurred through an unexpected interaction between novel topogenic...

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The result...

We have established a system for assembly of hepatitis B virus capsid, a homomultimer of the viral core polypeptide, using cell-free transcription-linked translation. The mature particles that are produced are indistinguishable from authentic viral capsids by four criteria: velocity sedimentation, buoyant density, protease resistance, and electron...

Signal, stop transfer, and signal-anchor sequences direct a nascent polypeptide to a single topology with respect to the membrane of the endoplasmic reticulum. However, other types of sequences direct nascent proteins, either transiently or permanently, to more than one topologic form. Pause transfer sequences direct nascent apolipoprotein B to pau...

Transmembrane topology of polytopic integral membrane proteins is established during protein synthesis at the endoplasmic reticulum membrane. For some polytopic proteins, sequential and independent signal, stop transfer, and/or signal anchor sequences contained in the nascent chain direct this process. Here we define the topology of human P-glycopr...

In contrast to typical secretory proteins, apolipoprotein B pauses at distinct points along the nascent chain during its translocation into the lumen of the endoplasmic reticulum. Specific pause transfer sequences mediate such discrete pauses in the translocation of apolipoprotein B. These sequences have been shown to confer this translocational be...

We have studied the transmembrane topology of human P-glycoprotein (MDR1) using protein chimeras in Xenopus oocytes and full-length native protein in a cell-free translation system. We find both in vivo and in vitro, that the peptide region between putative transmembrane helices (TM) 8 and 9 resides in the endoplasmic reticulum lumen not in the cyt...

The cystic fibrosis gene product (CFTR) is a complex protein that functions as an adenosine 3,5-monophosphate (cAMP)-stimulated ion channel and possibly as a regulator of intracellular processes. In order to determine whether the CFTR molecule contains a functional aqueous pathway, anion, water, and urea transport were measured in Xenopus oocytes e...

Previously, we described the stepwise translocation of a large amino-terminal fragment of apolipoprotein B (apo B15) in which the nascent secretory chain translocates through a series of distinct, nonintegrated transmembrane intermediates with large domains exposed to the cytoplasm. Thus, apo B15 appears to stop and restart translocation at several...

I have described recent work that supports several conclusions that might not have been previously expected: first, that stop transfer, like the initiation of translocation, is receptor-mediated; second, that at least some of the topology-determining events at the ER membrane can be regulated (an example is provided where regulation may occur devel...

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Lipocortin I has been presumed to be synthesized and secreted in response to glucocorticoids yet the amino acid sequence of lipocortin I reveals no signal sequence typically necessary for proteins to enter the secretory pathway. The translocation of lipocortin I across membranes was analyzed in a cell-free system and in Xenopus oocytes. Based on th...

Apolipoprotein B (apo B) is crucial for the transport of cholesterol in humans. It is a large secretory protein that mediates the uptake of low-density lipoproteins and renders several forms of lipid droplets soluble in the blood. The binding of lipid by apo B also prevents this hydrophobic protein from precipitating in aqueous solution. In the end...